BIO 1: CASPASE-8 LEVELS DETERMINE SIGNALLING PATHWAYS USED IN DEATH LIGAND-INDUCED GLIOMA CELL APOPTOSIS

David M. Ashley, Christopher D. Riffkin, Paul G. Ekert, Melissa J. Knight and Christine J. Hawkins

Department of Haematology and Oncology, Royal Children’s Hospital, Parkville 3052, Australia

The majority of high-grade glioma patients die within a several years of diagnosis. Elucidating apoptosis signaling pathways may assist in designing better adjuvant therapies. Our earlier work has demonstrated that glioma cells may either employ mitochondrial independent or dependent death receptor-induced apoptotic pathways, characteristic of cells termed type I and type II respectively. In the present study we generated panels of clonal transfectants overexpressing various levels of Bcl-2, in two parental glioma cell lines. These cells were used to explore molecular factors determining the necessity for mitochondrial amplification of death receptor signaling. Our studies have confirmed that Caspase-8 levels can influence the requirement for mitochondrial involvement in death receptor apoptotic signaling in glioma cells. Supporting the significance of these findings are observations that many primary patient glioblastoma samples have low or absent Caspase-8 expression levels.

BIO 2: THE HYPOXIA INDUCIBLE CELL DEATH GENE BNIP3 IS MUTATED IN MALIGNANT ASTROCYTIC TUMORS

Priti Baijal, Teralee Burton, Shunzhen Zhang, Elizabeth Henson, Nicolle Bristow, Mario Fonseca, Spencer Gibson and David D Eisenstat

Manitoba Institute of Cell Biology and Departments of Pediatrics & Child Health, Human Anatomy & Cell Science, Physiology and Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, MB Canada

Astrocytic tumors are the most common brain tumor diagnosed in children and adults. Glioblastoma multiforme (GBM, WHO grade IV) represents the most malignant form of astrocytoma with a time to progression of 12 weeks without intervention and 12-15 months survival with combined modality therapy, including surgery, radiation and chemotherapy. Response to therapy fails, in part, due to tumor hypoxia facilitating resistance to radiation and chemotherapy. The BCL2 Nineteen Kilodalton Interacting protein, BNIP3, is a Bcl-2 family member that is up-regulated in hypoxic regions in many solid tumors. BNIP3 is directly activated by the transcription factor HIF1a and mediates cell death in a caspase-independent manner through the interaction of the transmembrane (TM) domain of BNIP3 with the mitochondrial membrane. We have determined that BNIP3 is up-regulated in GBM compared to normal brain and is expressed in malignant astrocytes. This increased BNIP3 expression correlates with increased HIF1a and glut-1 that indicate hypoxic regions within these tumors. In glioma cell lines, BNIP3 expression is increased under hypoxia. In 33% of primary (de novo) GBM we have detected mutations in the PEST and conserved (CD) domains of BNIP3 that result in a truncated protein lacking a functional TM domain. The cDNA mutations have been confirmed by SSCP or by allele-specific PCR and DNA sequencing. Over-expression of BNIP3 into malignant glioma cells induces cell death whereas treatment with antisense, dominant negative or mutant BNIP3 constructs blocks hypoxia induced cell death. This blockage of cell death is due to failure of BNIP3 to localize to the mitochondria and inhibition of BNIP3-mediated mitochondrial dysfunction. Our discovery suggests that BNIP3 acts as a tumor suppressor and selective pressure within the tumor generates BNIP3 mutations providing a survival advantage. This could explain why treatments for malignant gliomas are often ineffective in hypoxic regions of these tumors.

BIO 3: MICROVESSEL DENSITY PREDICTS BEHAVIOR IN PEDIATRIC OPTIC PATHWAY/HYPOTHALAMIC GLIOMAS

Ute K. Bartels, Cynthia Hawkins, Jing Ma, Amit Ray, Peter Dirks, James Rutka, Eric Bouffet; The Hospital for Sick Children, Paediatric Brain Tumour Program, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8

Background: Optic pathway/hypothalamic gliomas (OPHG) are predominately

low-grade tumors. However, OPHGs show an unpredictable behavior and there is no recognized histologic or molecular marker anticipating

this behavior. This study was performed to investigate angiogenic features as possible independent prognostic factors.

Methods: We searched the databases of the Hospital for Sick Children for patients with pathologically diagnosed OPHGs between 1985 and 2002. Tumor specimens were reviewed and reconfirmed as low-grade gliomas. Those with sufficient tissue for staining were included in the study. Sections were immunostained with factor VIII (F8) and counted for microvessel density (MD). A ratio of alpha-smooth muscle actin to F8 immunostaining was calculated to give a vascular maturity index. Vascular endothelial growth factor (VEGF) and VEGF-receptor immunostaining was performed to assess angiogenic features and MIB-1 labeling index (LI) to assess proliferation. These factors were evaluated with respect to progression-free survival (PFS) and outcome of treatment.

Results: 60 patients were identified and 41 had sufficient material for further analysis. All patients underwent surgery. 30 required additional treatment (16 chemotherapy, 8 radiation, 6 chemotherapy + radiation). 8 patients had NF1. 38 are alive. MD showed a wide range of variation between tumors (4.8-73.6, median: 28 vessels/1.2 mm²). The absolute number of F8 stained vessels was significantly higher in infants (p<0.01). A high MD (>20 vessels/1.2mm²) was associated with a significantly shorter PFS compared to tumors with a low MD (<20 vessels/1.2 mm²)(p=0.025). MIB-1 values ranged from 0-10 (median 2.5). Intensity and distribution of VEGF and VEGFR staining and MIB-1 LI were not significantly associated with outcome.

Conclusions: Our findings suggest that angiogenesis is important in low-grade glioma and MD rather than MIB-1 has a prognostic value in OPHGs.

BIO 4: p38 MITOGEN-ACTIVATED PROTEIN KINASE IS A NEGATIVE REGULATOR OF retinoic acid receptor transcriptional activity IN PRIMITIVE NEUROECTODERMAL TUMOR CELLS

Sandeep Batra, Heather Mears, Stewart Goldman, Jessica Smith, Antonella Sassano, Lakhvir Lal, Yongzhong Li, and Leonidas C. Platanias

Robert H. Lurie Comprehensive Cancer Center and Departments of Pediatrics and Medicine, Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60611

13-cis-Retinoic acid (RA), a synthetic retinoid has recently been shown to inhibit the growth, and induce differentiation of central nervous system primitive neuroectodermal tumors of childhood (PNET) cells. The RA-retinoic acid nuclear receptor complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription. Since the p38 Mitogen-activated protein kinase (MAPK) is known to play a key role in cell proliferation, apoptosis, and differentiation, we hypothesized that both upstream and downstream effectors of p38 MAPK are activatred by RA, and that p38 MAPK may regulate RA-induced gene transcription. We found that p38 is phosphorylated, accompanied by the induction of its kinase activity (a and g isoforms), in a time-dependent manner, following RA treatment. Our data also demonstrate that mitogen activated kinase kinase 3/6 (MKK3/6) is activated by RA and may function as an upstream regulator of p38 activation. Using in vitro kinase assays, we determined that RA also induces activation of MapKapK2 and of mitogen- and stress-activated protein kinase 1 (MSK1) kinase activity in DAOY cells. Luciferase reporter assays were done to measure RARE transcriptional activity in DAOY cells. Pharmacological inhibition of p38 (a and b isoforms) with SB 202190 (5-10 mM), increased RA-dependent gene transcription via RARE elementss (P<0.01). Altogether, our data provide evidence that p38 MAPK is activated by RA and exhibits negative regulatory effects on RA-induced gene transcription in PNET cells.

BIO 5: MEDULLOBLASTOMA NEOPLASTIC MENINGITIS TARGETED RADIOTHERAPY WITH INTRATHECAL a-EMITTER ²¹¹AT-LABELED THYMIDINE ANALOGUE

Abraham Boskovitz, Ganesan Vaidyanathan, Gary E. Archer, Hidenobu Ochiai, Tatsunori Okamura, John H. Sampson, Darell D. Bigner, and Michael R. Zalutsky

Brain Tumor Program, Duke University Medical Center, Durham, NC, USA

Medulloblastoma is the most frequent pediatric malignant brain tumor and accounts for up to 25% of all intracranial tumors in children. Because of a strong tendency to disseminate into the CSF and metastasize along the leptomeninges, surgical resection is followed by radiotherapy of the entire neuroaxis. To avoid or reduce radiotoxicity to normal tissues of the CNS, targeted radiotherapy was explored in an animal model of medulloblastoma neoplastic meningitis.

Targeted radiotherapy is potentially ideal for disseminated, thin-sheeted tumors if the radioisotope and its specific carrier are carefully selected according to the tumor characteristics. The thymidine analogue iododeoxyuridine (IUdR) is incorporated into the cellular DNA of cells in mitotic S-phase, conferring on it specificity for proliferating tumor cells within the intrathecal space. Astatine-211 ²¹¹At is an a-emitter with a high linear energy transfer (97 keV/mm), a short range in tissues (73 mm), and a half-life of 7.2 h. The astatinated analogue of IUdR, 5-[²¹¹At] astato-2’-deoxyuridine (AUdR), has demonstrated an exquisite cytotoxicity in vitro to malignant cell populations.

We inoculated athymic male rats intrathecally with D341 medulloblastoma cells through a previously surgically implanted intrathecal catheter. In experiment 1, animals were treated intrathecally with 43 mCi of AUdR (n=10), 45 mCi of free [²¹¹At] astatide (n=9), or saline (n=9). Median survival was improved from 18 days for the saline group to 21.5 days with AUdR (p=0.02) and 20 days with [²¹¹At] astatide (p=0.56). In experiment 2, groups of animals received three doses, on a daily basis, of 30 mCi of AUdR (n=9), 57 mCi of AUdR (n=7), or saline (n=9). Median survival was increased from 22 days for the saline group to 27 and 31 days for the low and high AUdR regimens, respectively. These results suggest that targeted radiotherapy with ²¹¹At-labeled thymidine analogues may warrant further investigation as a targeted radiotherapeutic for medulloblastoma neoplastic meningitis.

BIO 6: GENE EXPRESSION PROFILING OF PROGRESSIVE AND STABLE CHILDHOOD PILOCYTIC ASTROCYTOMAS

Daniel C. Bowers, Luc Girard, John Minna, Tina Chen, and Gail Tomlinson

University of Texas Southwestern Medical Center, Dallas, TX USA.

Children with Pilocytic Astrocytomas (PAs), the most common CNS tumor in childhood, may have either a quiescent or progressive clinical course. At present, extent of tumor resection and tumor location are the most important prognostic factors; biological prognostic factors are unknown. The identification of new prognostic factors for PAs may have important clinical and therapeutic implications.

Ten mcg of purified mRNA was extracted from archival flash frozen tumor tissue from five completely resected recurrent PAs and six incompletely resected quiescent PAs. Samples were hybridized to the Affymetrix GeneChip arrays HG-U133A (22,283 probe sets) and HG-U133B (22,645 probe sets). The two arrays together evaluate the expression of 24,698 unique genes. Images from scanned chips were processed using Affymetrix software GCOS 1.0 to get the raw signals and p-values for each probe set. This data was then analyzed using in-house software (MATRIX): Array data were median-normalized and replicate genes were combined by averaging their signals. Samples were then grouped according to tumor recurrence or non-recurrence. Signals were averaged within each group and then compared (by calculating log ratios of recurrent vs non-recurrent PAs) and sets of genes showing differential RNA expression in the two groups were derived. Unsupervised hierarchical clustering was performed.

211 genes were found to be overexpressed at least 2-fold in the subset of progressive PAs compared to the subset of quiescent PAs while 147 genes were found to be underexpressed more than 2-fold. Several of these genes were found to have a neuronal function (e.g. synaptoporin, synaptotagmin IV). Hierarchical clustering did not reveal any association between recurrence and global gene expression.

These results demonstrate the ability of gene expression profiling to identify PAs with progressive clinical courses. Children with PAs with such profiling may benefit from closer monitoring and/or earlier adjuvant therapy.

BIO 7: AUTOFLUORESCENT MOUSE MODELS OF HUMAN MEDULLOBLASTOMA

Christopher Calabrese, Waleed Gaber, John Killmar, Christine Fuller, Meredith Allen, Richard Gilbertson¹

Department of Developmental Neurobiology, St Jude Children’s Research Hospital, Memphis, TN

¹Corresponding Author: R.J.G Dept. Developmental Neurobiology, D2006G,

St Jude Childrens Research Hospital,Memphis, TN 38105

We have developed a new in vivo model of medulloblastoma that recapitulates the behavior of classic and large cell anaplastic (LCA) forms of the disease and allows for serial measurement of tumor growth. To do this we generated green fluorescent human medulloblastoma cells (Daoy, MEB-MED-8A and MHH-MED-1) by in vitro infection using an MSCV-Green Fluorescent Protein (GFP) retrovirus. Under stereotactic control, 10⁶ GFP-tagged medulloblastoma cells were inoculated into the superficial cerebral cortex of CD1 nude mice and the cranium sealed with a glass plate. Serial tumor measurements can then be made through the cranial window using intravital fluorescence microscopy. By comparing in vivo total fluorescence measurements with 3D tumor reconstruction we show that the measurement of in vivo fluorescence provides a highly accurate measure of tumor burden. MEB-MED-8A tumors grow very rapidly and result in the death of animals within two weeks: histopathologically these tumors are LCA, invasive and contain an isochromosome of 17q and amplification of MYCC. In contrast, MHH-MED-1 and Daoy tumors display classic morphology with focal anaplasia and are much less aggressive in behavior taking more than 7 weeks to become symptomatic. We are now using these models to test a number of molecular targeted therapies for medulloblastoma. We previously reported ERBB2 to be an independent marker of poor prognosis in medulloblastoma. Daoy.2 and MEB-MED-8A tumors express high levels of the ERBB2 receptor while MHH-MED-1 cells express ERBB4 and low levels of ERBB2. Using our models we now show that well tolerated doses (50mg/kg/bd) of the oral ERBB1 and ERBB2 kinase inhibitor Erlotinib (Tarceva, OSI774) abolish ERBB2 signaling, induce cell cycle arrest and generate marked inhibition of medulloblastoma growth in the CNS. Our model system provides a useful new tool for studying the biology of signal transduction systems in medulloblastomas in the brain, and affords an efficient system for pre-clinical testing of novel therapies for this disease.

BIO 8: TRANSGENIC MOUSE MODEL OF ERBB2 OVEREXPRESSION IN CEREBELLAR GRANULE NEURON PRECURSOR CELLS

Christopher Calabrese, Roberto Hernan, Charles Sherr, Martine Roussel, Richard Gilbertson¹

Department of Developmental Neurobiology, St Jude Children’s Research Hospital, Memphis, TN

¹Corresponding Author: R.J.G Dept. Developmental Neurobiology, D2006G,

St Jude Childrens Research Hospital,Memphis, TN 38105

ERBB2 is undetectable in developing and adult human cerebellum (Gilbertson et al., Cancer Res, 1998) but is expressed in 40% of medulloblastomas and is associated with a poor clinical outcome (Gajjar et al., JCO, in press). Therefore aberrant expression of ERBB2 in granule neuron precursor cells (GNPC) might play a role in the initiation and/or progression of medulloblastoma. To test this hypothesis we have generated transgenic mice in which ERBB2, placed under the control of a 1.7 Kb enhancer element of the Math1 promoter, is expressed at high levels in mitotic GNPC. Mice bearing the Math1-ERBB2 transgene display high level expression of ERBB2 throughout the neural tube from embryonic (E) day 14.5. High levels of membrane associated ERBB2 are also detected in the rhombic lip and throughout the mitotic zone of the cerebellar external germinal layer (EGL) from mid gestation through to postnatal day 20. Western blot analysis of whole postnatal (P) day 5 cerebellum taken from transgenic mice identified high level expression of phospho-Y1248 ERBB2 and phospho-Ser473 AKT1 relative to transgene negative littermate controls, indicating ERBB2 is actively signaling in mice bearing the transgene. Further, using Affymetrix expression array analysis of P5 cerebellum we have identified a group of genes that are significantly and differentially expressed in transgene positive compared to transgene negative littermate controls. We are now using this model to study the role of ERBB2 in tumorigenesis in the cerebellum. To do this we are expanding our colonies of Math1-ERBB2 animals for tumor surveillance studies. Further, we recently reported that the TP53-ARF pathway is disrupted in large cell anaplastic medulloblastoma, a tumor subtype that frequently contains high levels of ERBB2. Therefore, to determine whether ERBB2 expression and TP53-ARF defects are interdependent during medulloblastoma formation we are breeding Math1-ERBB2 mice with p53 null and Arf null animals. In addition to reporting the characterization of this new mouse model at the meeting, we will also provide a full update on tumor surveillance data.

BIO 9: IDENTIFICATION OF TUMOUR SUPPRESSOR GENES IN CHILDHOOD MEDULLOBLASTOMA BY PROMOTER HYPERMETHYLATION PROFILING

Steven C. Clifford, Janet C. Lindsey, Meryl E. Lusher, Richard J. Gilbertson, Andrew D.J. Pearson and David W. Ellison.

Northern Institute for Cancer Research, University of Newcastle, Newcastle upon Tyne, UK and Dept. Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA.

Medulloblastoma (MB) is the most common malignant brain tumour of childhood, however its molecular basis is not well understood. The epigenetic silencing of tumour suppressor gene (TSG) expression by promoter hypermethylation has emerged recently as a major mechanism of TSG inactivation. To assess the role of epigenetic events in MB and identify critical genes in its development, we profiled the promoter methylation status of ten candidate TSGs (p14^ARF, p15^INK4b, p16^INK4a, CASP8, HIC1, TIMP3, TP73, TSLC1, RIZ1 and RASSF1A) in MB and the normal cerebellum.

Extensive hypermethylation of RASSF1A was detected in nearly all MBs, but not in normal cerebellum (41/44 primary tumours vs. 0/5 normal cerebella). In contrast, complete methylation of HIC1 and CASP8 in a subset of primary tumours (17/44 and 14/39) occurred against a background of partial methylation in the normal cerebellum. For all three genes, methylation in MB cell lines was associated with their epigenetic transcriptional silencing and methylation-dependent re-expression following treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. Further investigation of RASSF1A revealed that complete methylation occurred in the absence of gene deletion or mutation, indicating that its inactivation occurs by bi-allelic promoter hypermethylation alone. The remaining genes showed either low frequency methylation (p14^ARF, p16^INK4a, RIZ1; <7% of cases) or no evidence of methylation (p15^INK4b, TIMP3, TP73, TSLC1), suggesting that their hypermethylation does not play a major role in MB. No evidence was found to support a concordant methylation phenotype affecting multiple genes in this disease.

These findings indicate that tumour-specific epigenetic inactivation of RASSF1A, HIC1 and CASP8 are frequent events in MB, and identify these as potentially critical TSGs in its pathogenesis. Epigenetic RASSF1A inactivation represents the most common defect detected to date in MB, emphasising the importance of its further investigation. Importantly, our data demonstrate that the comprehensive identification of MB genes will require systematic screening of both the genome and epigenome.

BIO 10: Gene expression levels assessed by microarray analysis and quantitative real-time RT-PCR – How well do they correlate?

Peter Dallas, Nick Gottardo, Martin Firth, Alex Beesley, Katrin Hoffmann, Philippa Terry, Joseph Freitas, Aaron Cummings, and Ursula Kees,

Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western Australia, Perth, Western Australia.

The use of microarray technology to assess gene expression levels in living cells is now widespread in biological research, and the methodology is likely to receive even broader application as the technology evolves, data analysis procedures improve, and costs decline. The validation of microarray results using independent mRNA quantitation techniques including Northern blotting, ribonuclease protection, in-situ hybridisation, or quantitative real-time RT-PCR (qRT-PCR) remains a critical element of any microarray experiment. Despite this, there have been few systematic validation studies of cDNA, or more noticeably, oligonucleotide microarray data using these independent approaches. For researchers to be confident with the interpretation of microarray results it is important that this issue is addressed. We have undertaken an extensive series of experiments examining gene expression profiles in paediatric cancer specimens and normal tissues using HG-U133A GeneChips (Affymetrix). We processed the microarray data using Affymetrix MAS 5.0 software, or as an alternative, the robust multichip average (RMA) method based on the R statistical package. We selected 34 genes for follow-up verification experiments using qRT-PCR (TaqMan Assays On Demand, Applied-Biosystems). We found that in the majority of cases (29/34; 85%) there was a statistically significant correlation (p<0.05) between expression levels determined for specific genes by microarray analysis and those determined for the same genes by qRT-PCR. Although our data indicate that generally Affymetrix microarray scores correlate well with qRT-PCR results, we observed a poor correlation (p>0.05) for 6/34 (18%) of the genes that we examined, emphasising the need for caution when interpreting microarray results and highlighting the importance of independent validation of gene expression levels for any gene of interest selected on the basis of microarray expression scores.

BIO 11: IDENTIFICATION OF PROGNOSTIC MARKERS IN MEDULLOBLASTOMA BY INTEGRATING COMPARATIVE GENOMIC HYBRIDIZATION AND EXPRESSION PROFILES

Jeffrey Deyo, Massimiliano DeBortoli, Tsz-Kwong Man, Pulivarthi H. Rao, John Y. H. Kim, Scott L. Pomeroy, Ching C. Lau.

Texas Children’s Cancer Center, Texas Children's Hospital and Baylor College of Medicine, Houston, TX, USA

Improving the management of medulloblastoma (MB), the most common malignant brain tumor of childhood, depends upon better understanding its biology. This requires more comprehensive investigation of the genetic alterations underlying tumorigenesis and progression. Genomic methods present the most efficient means of characterizing these changes. By integrating multiple platforms, we attempt to compensate for intrinsic limitations of each to identify genes associated with outcomes and biological characteristics, but would otherwise be overlooked by using any single approach. We describe a more rigorous approach than simply mapping expression profiles with cytogenetic changes to uncover statistically significant alterations in MB that may arise through more complex mechanisms.

With comparative genomic hybridization (CGH), we analyzed twenty-seven samples MB samples that were among 65 previously analyzed with oligonucleotide microarrays [Pomeroy et al. Nature, 2002]. We compared these twenty-seven samples to normal cerebellum samples to generate a “global” list of differentially expressed genes (based on fold change) and then partitioned the dataset for each of the most common chromosomal lesions detected (iso17q, +7q, +2p, -10q, -16q) to identify candidate genes. We selected those that were uniquely identified in the partitioned list and associated with chromosomal lesions, but not found in the original “global” list for further analysis. To strengthen outcome prediction, we divided all 65 samples originally analyzed for gene expression into three groups based on the expression level for each candidate gene (unchanged, low, and high). Twenty-five genes identified by partitioning are predictive for survival by Kaplan-Meier analysis (P £ 0.01).

Our algorithm exploits cytogenetic changes to guide the detection of novel candidate genes from expression profiles. With this strategy, we have identified candidate genes that are strongly associated with survival, but have previously eluded detection by either method alone.

BIO 12: TAMOXIFEN ACTIVATED CASPASE PATHWAYS IN ASTROCYTOMA

Andrew M. Donson, Joy Zartman, Julie Fleitz, Nicholas K. Foreman.

Department of Neuro-Oncology, University of Colorado Health Sciences Center, Denver, Colorado, USA.

Introduction: Despite promising pre-clinical results supporting the use of tamoxifen to treat astrocytoma, clinical studies have proved disappointing. In order to improve the clinical benefit of this drug, it is necessary to better understand the mechanism(s) of its anticancer effects. The object of our study was to address this paucity of data by filling in the missing links in the tamoxifen-induced apoptotic signaling pathways in astrocytoma.

Methods: Pre-clinical trials of novel drugs for the treatment of astrocytoma often use apoptosis as a measure of anti-tumor effect. Presently, there is no single reliable method to determine whether a cell is apoptotic in astrocytoma. Traditional methods for detecting apoptosis, including terminal deoxynucleotidyl transferase nick-end-labeling, nuclear morphology, DNA laddering, Annexin-V binding, and Western blotting for active caspase-3, are subjective, difficult to perform or difficult to quantify in astrocytoma. In this study we used recently developed cytometric bead array analysis for measurement active caspase-3 in cell lysates. This technique allowed rapid quantitation of apoptosis in astrocytoma.

Results: This study showed that tamoxifen induced apoptosis via caspase-3 activation. The results also revealed a time- and dose-dependent response of tamoxifen induced caspase-3 activity in astrocytoma. Using peptide inhibitors selective to individual caspases, we also demonstrated that both Caspase-8, and caspase-9 are involved in activation of caspase-3.

Conclusion: The results suggest that tamoxifen induces apoptosis in human astrocytoma cell lines primarily via caspase-3. Further elucidation of these apoptotic pathways will determine whether tamoxifen-triggered caspase-3 activation is occurring via the death receptor pathway or the mitochondrial pathway. Elucidation of these apoptotic pathways will aid the design of novel tamoxifen analogs that retain all of the beneficial effects but are devoid of toxicity.

BIO 13: Genomic Imbalances in HIGH AND LOW-GRADE Paediatric Astrocytomas

Sara Dyer^1,2, Emma Prebble¹_, Marie-Anne Brundler³, David Ellison⁴, Val Davison² & Richard Grundy¹

¹Institute of Cancer Studies, University of Birmingham, B15 2TT U.K. ²Regional Genetics Lab. BWH, Birmingham, B15 2TG U.K. ³Department of Pathology, Birmingham Children’s Hospital. ⁴Department of Neuropathology, Newcastle General Hospital

Astrocytomas are the most common brain tumours observed in adults and children. However, the majority of genetic analyses performed on astrocytomas have used tumours derived from adults and there are few reports of non-random copy number aberrations in their childhood counterparts. To address this, we have analysed 15 high-grade and 15 low-grade paediatric astrocytomas by high-resolution comparative genomic hybridisation (HR-CGH). Only 5 out of 15 (33%) low-grade tumours showed genomic imbalances and the only consistent abnormality detected was gain of chromosome 5 in 2 cases. However, 12 out of 15 (80%) high-grade tumours had detectable copy number aberrations. The median number of abnormalities detected in the high-grade tumours was 3 (range 0-16). Out of a total of 59 imbalances, 19 involved whole chromosomes and 40 involved chromosomal regions or arms. The most common gains were of 1q in 7 cases (47%) and of 2 or 2q in 4 (27%) cases. The most common losses were of 9 or 9p in 4 (27%) cases, loss of 10 or 10q in 4 (27%) cases and loss of 13 in 4 (27%) cases. Seven high-level gains were detected, three involved chromosome 7, two were gains of 1q, one high-level gain of chromosome 2 was observed and one high-level gain was observed on 8q. Gains of chromosome 7 and losses of 9 and 10 have been reported in adult high-grade astrocytomas however, gains involving chromosomes 1q and 2 have not been commonly reported to-date. This study therefore provides evidence of novel chromosomal imbalances in paediatric astrocytomas.

BIO 14: PDGFR INHIBITION IN MEDULLOBLASTOMA CELLS INDUCES APOPTOSIS AND ALTERS GENE EXPRESSION

Meliha Dzirlo, Katia M. Peterson, Rebekah Kushner, Thamara Abouantoun, Tobey MacDonald

Center for Cancer Research, Children’s Research Institute, Washington D.C. 20010

We previously showed that overexpression of platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma. PDGFR is a receptor tyrosine kinase (RTK) that undergoes autophosphorylation upon binding to platelet-derived growth factor (PDGF). In other cell types, PDGFR autophosphorylation has been shown to initiate signal transduction through RAS-MAPK and PI3K-AKT, which in turn can promote proliferation, migration, survival and gene transcription. To determine whether PDGFR plays a similar role in medulloblastoma cells, we inhibited PDGFR expression and activity in the Daoy human medulloblastoma cells using three separate methods: i) small interfering RNA (siRNA) to PDGFR, ii) RTK inhibition of PDGFR (Gleevecä), and iii) anti-PDGF neutralizing antibodies, and then measured the resultant changes in gene expression and survival following PDGF stimulation. These changes were compared to baseline serum-depleted cells, with and without PDGFR inhibition, stimulated with PDGF.

PDGFR-active cells stimulated with PDGF demonstrated enhanced proliferation and showed gene expression changes at 24h, as measured by Affymetrix microarray profiling, that were specific to the subtype of ligand (PDGFAA or PDGFBB) used for stimulation. PDGFBB, which activates both receptor subtypes (alpha and beta), was more potent than PDGFAA, which only activates the alpha subtype of PDGFR. PDGFBB induced gene expression encoding effectors that primarily regulate signal transduction and cell-cycle control, while PDGFAA induced gene expression encoding effectors that predominantly affect chemotaxis. Inhibition of PDGFR activity, as measured by loss of PDGFR autophosphorylation, resulted in a doubling of baseline apoptosis (8 to 17%) at 24h and loss of cell-cell contacts and cell death at 72h-96h. These cell responses corresponded to alterations in the PDGF-induced gene expression profiles promoting survival.

Together, these results suggest that PDGFR is important for medulloblastoma survival and that the mechanism by which this occurs is through signal transduction that promotes pro-survival gene transcription.

BIO 15: COMBINED HISTOPATHOLOGICAL AND MOLECULAR CYTOGENETIC STRATIFICATION DEFINES A HIGH-RISK SUBSET OF PATIENTS WITH MEDULLOBLASTOMA – A UNITED KINGDOM CHILDREN'S CANCER GROUP STUDY

David W. Ellison, Jayne M. Lamont, Charles S. McManamy, Andrew D.J. Pearson, and Steven C. Clifford

Northern Institute for Cancer Research, University of Newcastle Medical School, Newcastle-upon-Tyne, England.

We examined the utility of stratifying medulloblastomas by a combination of refined histopathological classification and molecular cytogenetic evaluation. Detailed histopathological classification of tumors from a cohort of patients (n=87) composed mainly of children entered into the International Society of Pediatric Oncology / United Kingdom Children’s Cancer Study Group PNET3 trial (n=65), included identification of the recently described large cell / anaplastic phenotype. Fluorescence in situ hybridization was used to detect chromosomal abnormalities previously associated with medulloblastoma pathogenesis (chromosome 17 abnormalities, losses of 9q22 and 10q24, and amplification of the MYCC and MYCN oncogenes) in formalin-fixed paraffin wax embedded tumor biopsies.

The large cell / anaplastic phenotype, which was present in 20% of medulloblastomas, emerged as an independent prognostic indicator (P=0.0005). Loss of 17p13.3 (38% of medulloblastomas) was found across all histopathological variants, whereas MYCC / MYCN amplification (6% / 8% of medulloblastomas) was significantly associated with the large cell / anaplastic phenotype (P=0.034). Both loss of 17p13.3 and high-frequency MYCC / MYCN amplification (present in >50% of tumor cells) emerged as prognostic indicators (P=0.003; P<0.0001 respectively). Loss of 9q22 was associated with the nodular / desmoplastic medulloblastoma variant, while loss of 10q24 loss was found in all variants. Together with metastatic disease at presentation, the following characteristics: large cell / anaplastic phenotype, 17p13.3 loss, or high-frequency MYC amplification, defined a high-risk group of children whose outcome was significantly (P=0.0002) poorer than a low-risk group without these tumor characteristics.

These data provide a scheme for the stratification of patients with medulloblastoma based on the combined evaluation of novel histopathological features and molecular cytogenetic abnormalities, which is assessable in routinely processed formalin-fixed tumor tissue. Using such schemes, patients likely to be cured could be spared the side effects of maximal therapy, which can be targeted at those with aggressive disease.

BIO 16: Survivin and Survivin Isoforms in Medulloblastoma Show Unique Patterns of Expression and Prognostic Value

Jason Fangusaro MD, Yuying Jiang MD PhD, Vivekanand Singh MD, Daniel Boué MD PhD, and Rachel Altura MD.

The Center for Cancer Research, Columbus Children’s Research Institute and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, 700 Children’s Drive, Columbus, Ohio 43205

Survivin is an Inhibitor of Apoptosis Protein (IAP) that has been identified as a marker for tumor aggression in a variety of pediatric and adult malignancies including lymphoma, neuroblastoma, Wilm’s tumor, breast cancer, colon cancer, gastric cancer, and CNS tumors. Survivin has three known splice variants: Survivin, Survivin-2B and Survivin Delta-3, and recent literature has suggested that each isoform may have unique functions and sub-cellular localization. In this study, we investigated the use of Survivin as a prognostic marker in medulloblastoma. We analyzed the levels of Survivin in 40 pediatric medulloblastomas via immunohistochemistry. The percentage of cells positive for Survivin was < 5% in 3 samples (7.5%), 5-10% in 5 samples (12.5%), 11-20% in 17 samples (42.5%), and 21-50% in 15 samples (37.5%). Staining patterns were evaluated using two different polyclonal antibodies to Survivin, and positivity localized to the nucleus in all tumors. There was a statistically significant association between the percentage of Survivin-positive cells and outcome, suggesting that higher percentages of positivity correlated with a poorer clinical outcome. We also evaluated 20 freshly frozen pediatric medulloblastomas for the presence of the Survivin splice variants by real-time PCR. All of the tumors expressed Survivin and Survivin-2B, but only a fraction of the tumors expressed Survivin Delta-3. Survivin-2B was the highest expressed isoform in all tumors tested. In summary, our study demonstrates a potential role for Survivin as a prognostic marker in medulloblastoma. This is the first report evaluating the expression of Survivin and Survivin isoforms in a series of medulloblastoma tumors.

BIO 17: GENE SPECIFIC ABERRANT METHYLATION IN MEDULLOBLASTOMAS (MB), ANAPLASTIC EPENDYMOMAS (AE) AND stPNET OF CHILDHOOD

Michael C. Frühwald, Jörg Mühlisch, Niels Sörensen, Werner Paulus, Heribert Jürgens, Carsten Hagemann, Giles Vince, University Children's Hospital Muenster, Department of Pediatric Hematology and Oncology; Albert-Schweitzer-Straße 33; 48149 Münster / Germany

Epigenetic modifications of DNA such as promoter methylation and histone deacetylation contribute to oncogenesis by influencing gene transcription. Recent data suggest methylation of a limited number of genes in medulloblastoma, the most common malignant brain tumor of childhood. Discrepant findings derived from screening studies suggest that up to 800 gene loci may become aberrantly methylated in these tumors (Oncogene 2002).

Methods: Using methylation specific PCR (MS-PCR) we screened 6 MB and 2 stPNET cell lines for aberrant methylation of the genes p14^ARF, p15^INK4B, p16^INK4A, DUTT1, DAPK1, SOCS1, TIMP3, CDH1 and MGMT. Results were verified by bisulfite sequencing and COBRA (combined bisulfite restriction analysis). Gene promoters that showed methylation were analyzed in 9 normal cerebella, 6 stPNET, up to 59 MB and 13 AE. Threshold levels for methylation detected by these methods were quantified using artificially methylated DNA with defined ratios of methylated to unmethylated DNA.

Results: We detected tumor type specific and overlapping methylation patterns. Methylation in normal cerebellar tissue was restricted to low level methylation and tissues from older patients. Promoter methylation was detected for CDH1 (3/6 stPNET , 39/57 MB, 7/12 AE), MGMT (1/6 stPNET, 25/59 MB, 5/11 AE), p16^INK4A (17/59 MB, 1/13 AE), p14^ARF (9/58 MB) and TIMP3 (1/6 stPNET, 6/59 MB, 1/13 AE). MS-PCR proved a rather sensitive methodology for detecting aberrant methylation while COBRA and bisulfite sequencing required higher levels of methylation.

Conclusion: Aberrant methylation in common CNS malignancies of childhood affects genes with diverse biological functions. Tumor type specific methylation was detected for p14^ARF, which was only methylated in MB. CDH1 on the contrary was methylated in almost 50% of the examined tumors. Methylation may predispose cells for invasion and metastasis. Furthermore aberrant methylation may along with other mechanisms represent a field defect rendering cells susceptible for genomic instability.

Supported by Deutsche Krebshilfe 10-1699-FR1, DFG FR 1516/1-1 and Karl Bröcker Stiftung

BIO 18: p53 pathway dysfunction in primary childhood ependymomas and xenografts

Nathalie Gaspar, Birgit Geoerger, Jacques Grill, Paule Opolon, Marie-José Terrier-Lacombe, Arielle Lellouch-Tubiana, Lysiane Laudani, Alain Pierre-Kahn and Gilles Vassal. Department of Pediatrics and «Pharmacology and New Treatments in Cancer» UPRES-EA 3535, Institut Gustave Roussy, Villejuif, France

Childhood ependymoma is a therapeutic challenge. We hypothesized that p53 function might be abrogated in childhood ependymoma and implicated in resistance to anticancer therapy. 
We analyzed 21 primary ependymomas at diagnosis or relapse from 18 children. Using a functional assay in yeast, p53 gene mutations/deletions were excluded. Neither MDM2 overexpression nor p14ARF deletions were observed in 6 and 5 tumors, using western blot analysis and quantitative RT-PCR, respectively. Pax5 mRNA expression was shown by RT-PCR in 5/13 primary tumors at diagnosis, 2/3 at relapses. 
We studied two xenografts, IGREP37 and IGREP83, derived from primary childhood ependymomas that presented anaplastic ependymoma features and monosomy 22. The tumor doubling times were 12 and 22 days, respectively. Consistent with our findings in primary tumors, xenografts revealed no p53 mutation/deletion, mdm2 overexpression or p14 deletion. We accessed the p53 pathway functionality in these xenografts using radiotherapy, and found an abrogated p53 pathway in IGREP37. Radiotherapy did not induce p53 stabilization, p21 expression (western blot), or G1/0 growth arrest (FACS analysis). In addition, IGREP37 was only moderately radiosensitive. In contrast, p53 appeared functional in IGREP83 after radiotherapy, with stabilization of p53, induction of p21-mediated G1/0 growth arrest and apoptotic cell death. This xenograft was highly radiosensitive. Interestingly, Pax5 mRNA was expressed in IGREP37, but not in IGREP83, suggesting its potential role in the abrogation of cellular p53 function. However, using western blot and immunohistochemistry, pax5 protein was not expressed in both xenografts. To strengthen our findings, we are evaluating pax5 expression in primary tumors using immunohistochemistry.
In conclusion, we demonstrated that p53 pathway in ependymoma might be abrogated without p53 mutation, mdm2 overexpression, p14ARF deletion or increased pax5 protein expression, and needs further investigation. The two xenografts represent valuable in vivo models of ependymomas with aggressive behavior for preclinical studies.

BIO 19: Potential targets in pediatric brain tumors for the tyrosine kinase inhibitor GLIVEC^®

Birgit Geoerger, Marie-Jose Terrier-Lacombe, Valerie Velasco, Jean-Francois Emile, Lysiane Laudani, Jacques Grill, Arielle Lellouch-Tubiana, Jean-Christophe Sabourin, Christian Sainte-Rose, Chantal Kalifa and Gilles Vassal

Department of Pediatrics and «Pharmacology and New Treatments in Cancer»

UPRES-EA 3535, Institut Gustave-Roussy, Villejuif, France.

Glivec^® inhibits specific activations of the platelet-derived growth factor receptor (PDGFR), c-kit and BCR/Abl tyrosine kinases and has significantly changed the treatment for chronic myeloic leukemia and gastrointestinal stromal tumors. To investigate its potential in the treatment in pediatric brain tumors we determined expression of c-kit, and PDGFRa and b in 39 primary pediatric brain tumors (11 brain stem glioma, 2 medulloblastoma, 4 PNET, 2 atypical teratoid/rhabdoid tumors (AT/RT), 2 ependymoma, 3 low grade glioma, 2 high grade glioma, 4 oligodendroglioma, 3 chordoma, and 5 others) and 18 xenograft tumors derived from primary brain tumors (4 malignant oligodendroglioma, 4 glioblastoma multiforme (GBM), 5 medulloblastoma, 4 PNET, and 2 ependymoma). Immunohistochemistry on formalin-fixed paraffin-embedded tissue sections was performed using the polyclonal antibodies anti-CD117 A4520 and anti-PDGFRa AF-307-NA, and the monoclonal anti-PDGFRb P-20.

C-kit was expressed in 3 primary tumors, 1 medulloblastoma, 1 pinealoblastoma, and 1 AT/RT. The expression was however significantly weaker compared with those observed in GISTs with a positive cytoplasmic staining of 20-30% tumors cells. None of the xenograft models expressed c-kit. PDGFRa was expressed in 7 primary tumors: 1 brain stem glioma, 1 AT/RT, 1 ependymoma with GBM transformation, 1 anaplastic astrocytoma, 2/4 oligodendroglioma, and 1 chordoma. The positivity of tumor cells ranged between 20 and 80%, latter observed in oligodendroglioma and chordoma. In addition, PDGFRa was expressed in 1 medulloblastoma xenograft, 1 malignant oligodendroglioma xenograft derived from a child, and 2 glioblastoma xenografts. Positivity of the xenograft tumors was confirmed by western blot analysis. PDGFRb was expressed in 1 brain stem glioma, 1 AT/RT, 2 ependymomas, 1 anaplastic astrocytoma, and 2 chordomas. We are currently evaluating c-kit and PDGFRa mutations in these tumors.

Glivec^® might play a role in the treatment of some pediatric brain tumors through targeting of the PDGFR, however c-kit expression is rare. Further investigations are necessary to determine the efficacy of this treatment. (supported by the ARC)

BIO 20: Conditionally Replicating Adenoviruses Expressing P53 or Targeted towards EGFR Exhibit Enhanced Oncolytic Potency in Malignant Gliomas

Birgit Geoerger, Victor W. van Beusechem, Paule Opolon, Jacques Grill, Martine L.M. Lamfers, Clemens M.F. Dirven, Winald R. Gerritsen and Gilles Vassal

Department of Pediatrics and Pharmacology and New Treatments in Cancer UPRES EA3535, Institute Gustave-Roussy, Villejuif, France

Prognosis of malignant glioma is poor and treatment remains a major challenge. Conditionally replicative adenoviruses (CRAds) are novel anti-cancer agents, designed to selectively replicate in and to destroy cancer cells. Critical determinants for the oncolytic potency of CRAds are their capacity to infect tumor cells and to induce cell lysis. Lack of adenovirus receptor CAR expression and dysfunctional cell death pathways, both often observed in malignant glioma, reduce CRAd efficacy. To overcome these limitations, we constructed two new CRAds based on AdD24 with specificity for cells dysfunctional in cell-cycle checkpoint control due to a mutated Rb-pathway. AdD24-425S11 produces a bispecific scFv adapter molecule to redirect infection via the Epidermal Growth Factor Receptor (EGFR), commonly expressed in malignant gliomas; AdD24-p53 expresses p53 in order to restore p53 mediated cell death mechanisms.

Compared to the parent AdD24, AdD24-425S11 exhibited enhanced oncolysis on CAR-deficient cell lines and 7/8 primary glioblastoma multiforme short-term cultures in vitro, as measured by cell viability quantified by WST-1 conversion assay after infection at low MOIs. AdD24-p53 killed almost all malignant glioma cell lines and 6/8 primary glioblastoma multiforme short-term cultures more effectively, independent of cellular p53 status as determined by p53-specific transactivation assay.

Both viruses caused superior antitumoral effects in subcutaneous human xenografts in nude mice derived from primary tumors in vivo. The effect of targeting was studied in the neuroblastoma IGR-NB8 due to the lack of an adequate CAR-deficient and EGFR-expressing glioma model. Five intratumoral injections of 10⁸ pfu AdD24-425S11 yielded significant tumor growth delay of 27 days (p<0.01) vs 13 days (n.s.) for AdD24 compared to PBS controls. The AdD24-p53 caused more frequent regression and more delayed growth of IGRG121 glioblastoma xenografts with 70% tumor regressions and 60% animals surviving more than 120 days tumor-free or with a minimal tumor residual.

Thus, CRAds with enhanced oncolytic capacity due to CAR-independent infectivity and/or restoration of p53-dependent cell death are promising agents for treatment of malignant gliomas. (supported by La Ligue contre le Cancer)

BIO 21: Transcription factors expressed by neural crest cells correlate with phenotype in development and in neuroectodermal tumors

Timothy R. Gershon, M.D., Ph.D., Steven S. Chin, M.D., Ph.D. Children’s Hospital of New York, Columbia University, Division of Child Neurology, HP540, Columbia Presbyterian Medical Center, New York, NY 10032

Embryonic cells derived from the neural crest (NC) have features in common with neuroectodermal tumor cells: both are pluripotent, proliferative cells that migrate from their sites of origin. NC cells and neuroectodermal tumor cells both derive from neuroectoderm and in acquiring proliferative, migratory behavior, neuroectodermal tumor cells resemble their undifferentiated ancestors. We tested the hypothesis that genes that direct NC development are expressed in neuroectodermal tumor cells. Using immunocytochemistry on human embryonic tissue, we developed an array of markers expressed sequentially as neuroectodermal precursors give rise to migrating NC cells. We then determined the patterns of marker expression in tumors of NC derived tissues and in tumors with primitive neuroectodermal histology.

The markers selected were PAX3, AP-2, SOX10, and PAX7. PAX3 was expressed by cells of the dorsal neural tube, migrating NC cells, and mesodermal cells. AP-2 was expressed by NC cells as they exited the neural tube and as they migrated throughout the embryo, as well as by epithelial cells. SOX10, in contrast, was expressed later in NC migration, after cells had exited the neural tube. PAX7 was not detected in extracranial neuroectodermal tissue, but rather served as a mesodermal marker.

Analyzing neurofibroma, benign and malignant Schwannoma, neuroblastoma, melanoma, Ewing’s sarcoma, medulloblastoma, and supratentorial PNET, we found these markers were expressed in tumor specific combinations. In a manner that echoed the progression of marker expression by NC-derived cells during embryogenesis, the more malignant, primitive tumors tended to express PAX3, while the less dysregulated tumors, with greater capacity for differentiation, tended to express SOX10. Our findings suggest that patterns of NC transcription factor expression may direct and predict neuroectodermal tumor behavior.

BIO 22: WHAT’S IN A NAME?

Floyd H Gilles, C Jane Tavarè, and Ignacio Gonzalez-Gomez (For the Childhood Brain Tumor Consortium)

Pediatric Neuropathology, Childrens Hospital Los Angeles and University of Southern California, Los Angeles CA, USA

Background: Most pediatric neuroglial tumors are histologically heterogeneous. Low intraobserver diagnostic reproducibility, histologic feature recognition reliability and inadequate diagnostic criteria result in subsets within diagnoses and grades with markedly varying survival expectation. This confounds accurate prognoses and evaluation of treatment regimes of childhood neuroglial tumors.

Materials and Methods: 1771 intracranial neuroglial tumors amongst 3291 children in the Childhood Brain Tumor Consortium (CBTC) database. Reliability was measured by Kappa and Jaccard (Dice) statistics. Survival distributions of histologic subsets of frequent WHO diagnoses or Daumas-Duport (DDGS) grades were compared.

Results: Only 8 WHO diagnoses had acceptable reproducibility and only 26 of 57 commonly used histologic features had acceptable reliability. Overall, the WHO classification separated only two prognostically distinct classes of supratentorial astrocytomas. Among infratentorial astrocytomas, there were 2 subsets with five year survival estimates of 0.70 and 0.27, and among supratentorial anaplastic astrocytomas there were 4 subsets with five year survival estimates of 0.57, 0.49, 0.21 and 0.15. The DDGS fared no better. For instance, in DDGS grade 3 (any 2 DDGS criteria), those neuroglial tumors with nuclear atypia and endothelial prominence had a significantly better survival distribution than other possible pairs, and survival was not statistically different from those in DDGS grade 1 or DDGS grade 2 with endothelial prominence only. Thus DDGS grades do not provide unique survival information. WHO classification and DDGS grades are inconsistent: e.g., half of infratentorial astrocytomas nos had DDGS grades of 3 and 4.

Conclusions: Current classification schemes result in subsets of diagnoses and grades with drastically different survival expectation. To better evaluate tumor survival and treatment regimes, tumors must be separated using complete reliable histology in statistical models that recognize tumor patterns in a reproducible and unbiased manner. This system is available (Pediatric Dev Pathol 2000, 3:126-139).

BIO 23: INHIBITION OF RECEPTOR TYROSINE KINASES AND MAMMALIAN TARGET OF RAPAMYCIN OFFERS COMBINATORIAL BENEFIT IN TUMOR CONTROL

Ranjit K. Goudar, Mark D. Hjelmeland, Stephen T. Keir, Charles A. Conrad, Roger E. McLendon, Carol J. Wikstrand, Peter Traxler, Heidi A. Lane, David A. Reardon, Webster K. Cavenee, Xiao-Fan Wang, Darell D. Bigner, Henry S. Friedman, Jeremy N. Rich

Division of Neurology and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710.

Pediatric malignant gliomas are highly lethal tumors that display striking genetic heterogeneity. Novel therapies that inhibit a specific molecular target may slow tumor progression, but tumors are likely not dependent on a single gene product. Rather, gliomas exhibit sustained mitogenesis and cell growth mediated, in part, through the effects of receptor tyrosine kinases and the mammalian target of rapamycin (mTOR). AEE788 is a novel orally active tyrosine kinase inhibitor that decreases the kinase activity associated with the ErbB family members and, at higher doses, the VEGF receptor 2 (KDR). RAD001 [everolimus] is an orally available mTOR inhibitor structurally related to rapamycin. We hypothesized that combined inhibition of upstream ErbB/KDR receptors with AEE788 and inhibition of the downstream mTOR pathway with RAD001 would result in increased efficacy against gliomas compared to single-agent therapy. In vitro experiments with glioma cultures showed that the combination of AEE788 and RAD001 resulted in increased rates of cell cycle arrest and apoptosis, and reduced proliferation more than either agent alone. Combined AEE788 and RAD001 administered orally to athymic mice bearing established human malignant glioma tumor xenografts resulted in greater tumor growth inhibition and greater increases in median survival than monotherapy. These studies suggest that simultaneous inhibition of the ErbB and mTOR pathways may offer increased benefit in glioma therapy.

BIO 24: BRAIN TUMORS IN CHILDREN WITH TUBEROUS SCLEROSIS COMPLEX- STUDY OF TUBERIN AND HAMARTIN EXPRESSION

Wieslawa.Grajkowska¹, Sergiusz.Jóźwiak², Katarzyna.Kotulska ³, Marcin.Roszkowski^4, Danuta Perek⁵

¹Pathology Departament, The Children’s Memorial Health Institute, Warsaw, Poland, ²Neurology Department, The Children’s Memorial Health Institute, Warsaw, Poland, ³Neurology Department, Medical University of Silesia, Katowice, Poland, ⁴Neurosurgery Department, The Children’s Memorial Health Institute, Warsaw, Poland, ⁵Oncology Department, The Children’s Memorial Health Institute, Warsaw, Poland, Pathology Department The Children’s Memorial Health Institute, Aleja Dzieci Polskich 20, 04-730 Warsaw, Poland

Objectives: Subependymal Giant Cell Astrocytomas (SEGAs) are rare, histologically benign brain tumors associated with tuberous sclerosis complex (TSC). Due to the production of hydrocephalus, they have usually poor prognosis. TSC is a neurocutaneus syndrome resulting from mutations in two genes, TSC1 and TSC2, encoding hamartin and tuberin, respectively.The exact pathway of hamartomas and neoplasms formation in TSC is not known, but recent studies emphasize the common role of tuberin and hamartin in cell cycle and cell size regulation control.

Aim: to elucidate the tuberin and hamartin expression in SEGAs

Materials and Methods: 10 specimens of SEGAs were analyzed using double-staining with specific anti-tuberin and/or anti-hamartin antibodies under confocal microscopy. The results were attributed to the genotype analysis performed earlier.

Results: We found loss of hamartin immunolabeling in all specimens. Three SEGAs were immunolabeled for tuberin.. Interestingly, one of them was obtained from a patient with TSC2 mutation ascertained.

Conclusions: These results support relationship between TSC1 and TSC2 genes products as well as suggest that tuberin and hamartin may somehow reciprocally regulate their expression.

BIO 25: TELOMERE MAINTENANCE IN CHILDHOOD PRIMITIVE NEUROECTODERMAL BRAIN TUMORS

Michael Grotzer, Domenico Didiano, Doris Lang, Tarek Shalaby

Department of Oncology, University Children’s Hospital of Zurich, Switzerland

Primitive neuroectodermal tumors (PNET), including medulloblastoma (PNET/MB) and supratentorial PNET (sPNET), are the most common malignant brain tumors of childhood. The stabilization of telomere lengths by telomerase activation is an important step in carcinogenesis and cell immortalization. Epigallocatechin-gallate (EGCG), the major polyphenol in green tea, is a telomerase inhibitor with antiproliferative and anticarcinogenic effects against different types of cancer.

In this study, we used real time RT-PCR to measure the mRNA expression of the human telomerase reverse transcriptase (hTERT) in 50 primary PNET (43 PNET/MB, 7 sPNET), 14 normal human brain samples and 6 human PNET cell lines. Compared to normal human cerebellum, 38/50 (76%) primary PNET had ³ 5x upregulated hTERT mRNA expression. While a positive correlation between hTERT mRNA expression and telomerase activity was detected in PNET cell lines, no correlation was found between telomerase activity and telomere length. Treatment of PNET cell lines with EGCG resulted in a dose-dependent inhibition of telomerase activity at µM levels. Although EGCG displayed strong proliferation inhibitory effects against TRAP-positive PNET cell lines, it had no significant effect against TRAP-negative D425 cells.

In summary, these results provide evidence for a possible role of telomerase in the pathogenesis of most PNET, and indicate that subsets of PNET maintain telomere length by alternative mechanisms. Inhibition of telomerase function represents a novel experimental therapeutic strategy in childhood PNET that warrants further investigation.

BIO 26: IGF-II is a growth factor for medulloblastoma cells and cerebellar progenitor cells.

Wolfgang Hartmann (1), Otmar D. Wiestler (1), Karl Schilling (2), and Torsten Pietsch (1)

(1) Department of Neuropathology, (2) Department of Anatomy, University of Bonn, Germany

Medulloblastomas (MB) represent the most frequent malignant brain tumors of childhood. They develop from progenitor cells of the cerebellum. The insulin-like growth factor II (IGF-II) is an important fetal mitogen which is known to be involved in the molecular pathogenesis of various embryonal tumors. Its gene has four developmentally regulated promoter elements P1-P4, of which P2, P3 and P4 are regulated by genomic imprinting.

To study the role of IGF-II in the pathogenesis of MB we analysed the proliferation of murine cerebellar neural progenitor cells (P8) of the external granule cell layer as well as of medulloblastoma cells by 3H-thymidine incorporation assays. In addition, we determined the IGF2 mRNA expression and imprinting status in a series of 16 MB of classic histology and 10 MB of the desmoplastic subtype by quantitative RT-PCR.

We identified IGF-II as a potent mitogen of cerebellar neural progenitor cells which acted synergistically with sonic hedgehog which is an essential growth factor of these cells. In human medulloblastoma cells growth promoting activity of IGF-II was identified in 8 of 10 MB cell lines. This activity could be blocked in most cases by anti IGF-I-receptor antibodies. In both classic and desmoplastic MB IGF2 mRNA expression was detectable. Similar to other embryonal tumors the IGF2 promoter P3 represented the predominant source of IGF2 transcripts. Interestingly, the activity of the promoters P3 and also P2 was significantly upregulated in MB of the desmoplastic subtype as compared to the classic subtype. This was not a result of alterations of imprinting since these occured in both subtypes.

These data suggest that IGF-II plays an important role in the proliferation control of cerebellar progenitors and medulloblastoma cells.

(Supported by the Deutsche Forschungsgemeinschaft (SFB400) and the BONFOR programme of the Medical Faculty of the University of Bonn)

BIO 27: OLIGOASTROCYTOMA/OLIGODENDROGLIOMA IN ASSOCIATION WITH DYSEMBRYOPLASTIC NEUROEPITHELIAL TUMOR (DNET)

John A. Heath¹, Wai Hoe Ng¹, Wirginia Maixner², Chung Wo Chow³, John Laidlaw⁴, Michael Gonzales⁵, and David M. Ashley¹

Departments of Haematology and Oncology¹, Neurosurgery², and Pathology³, Royal Children’s Hospital, Melbourne; Departments of Neurosurgery⁴ and Pathology⁵, Royal Melbourne Hospital, Melbourne; Australia.

Case 1: A young woman presented with right temporal headache and left arm numbness. MR brain imaging demonstrated a left frontal mass lesion. Craniotomy and subtotal excision was performed. Histopathological features were typical of simple DNET. Small oligodendroglia-like cells (synaptophysin positive processes) were seen forming a glioneuronal element and focally enclosing large neurons (NFP positive processes) within microcystic spaces. No glial nodules were identified. Nuclear staining for Ki-67 was not seen.

Three years later, she re-presented with headache and an expressive dysphasia. MRI showed enlargement of the left frontal tumor (4x3x3cm) with areas of contrast enhancement. Sections of the debulked tumor again showed features of simple DNET. In addition a moderately hypercellular (WHO grade 2) oligoastrocytoma (synaptophysin negative) was present in the underlying white matter with infiltration of the DNET and adjacent cortex. Nuclear Ki-67 staining, not apparent in the DNET, was seen in 8% of nuclei.

Case 2: A young man presented with a generalised tonic-clonic seizure. An EEG showed epileptiform activity and MRI a focal area of increased signal in the right antero-lateral aspect of the right temporal lobe. He underwent a craniotomy and resection of the anterior 4cm of the temporal lobe. Histopathology showed nodules with typical histological features of the glioneuronal element of DNET. The white matter was expanded by a moderately hypercellular (WHO grade 2) oligodendroglioma (synaptophysin negative). Ki-67 staining, not seen in nuclei in the cortical nodules of DNET, was seen in 6% of nuclei in the oligodendroglioma.

LOH analysis (1p and 19q) will be presented.

In both cases, the glial tumour was in excess of the nodular glial component frequently seen in complex DNET. These cases challenge present concepts about the biology of DNET. In particular, the occurrence of glioma and DNET together should be considered where complete tumor resection is not possible.

BIO 28: SB-431542, A SMALL MOLECULE TRANSFORMING GROWTH FACTOR-b-RECEPTOR ANTAGONIST, INHIBITS HUMAN GLIOMA CELL LINE PROLIFERATION AND MOTILITY

Mark Hjelmeland, Anita Hjelmeland, Sith Sathornsumetee, Michael H. Herbstreith, Nicholas J. Laping, Darell D. Bigner, Xiao-Fan Wang, Jeremy N. Rich

Division of Neurology and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710.

Transforming growth factor-b (TGFb) is a multifunctional cytokine that promotes tumor invasion, angiogenesis, and immunosuppression expressed in many pediatric central nervous system cancers. Antisense oligonucleotide suppression of TGFb₂ ligand expression has shown promise in preclinical and clinical studies of adult glioblastoma patients but at least two ligands mediate the effects of TGFb in gliomas. Therefore, we examined the effects of SB-431542, a novel, small molecule inhibitor of the type I TGFb-receptor, on a panel of human malignant glioma cell lines, including pediatric glioma lines. SB-431542 blocked the phosphorylation and nuclear translocation of the SMADs, intracellular mediators of TGFb signaling, with decreased TGFb-mediated transcription. Further, SB-431542 inhibited the expression of two critical effectors of TGFb – vascular endothelial growth factor and plasminogen activator inhibitor-1. SB-431542 treatment of glioma cultures inhibited proliferation, TGFb-mediated morphological changes, and cellular motility. Together, our results suggest that small molecule inhibitors of TGFb receptors may offer a novel therapy for malignant gliomas by reducing cell proliferation, angiogenesis, and motility.

BIO 29: A COMPARISON OF GENOMIC PROFILES OF INFANT AND NON-INFANT MEDULLOBLASTOMA BY ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION (CGH)

A. Huang, Ben Behesti, Maria Zielenska, Cynthia Hawkins, Eric Bouffet, Darren Hargrave and Jeremy Squire

Pediatric Brain Tumor Program, 555 University Avenue, Hospital for Sick Children, Toronto, Ontario. M5G 1X8

Medulloblastoma (MB) in very young children are associated with a higher incidence of metastatic disease and poorer outcome. It is unclear whether distinct genetic features of infant MB underlie these observations. Clearly, a better understanding of the genetic features of these tumours will be important for future advances in infant MB therapy.

Genomic changes in MB associated with DNA gain or loss has been well documented by many conventional cytogenetic and comparative genomic hybridization (CGH) studies which have included varying number of infant MB. We undertook a pilot study to compare the genomic profiles of infant MB to MB presenting in older children by array based CGH (A-CGH). 13 tumours were analysed: 5 infant tumours diagnosed at less than or equal to 3 years of age and 7 MB diagnosed at greater than 3 years of age were compared. DNA isolated from frozen and paraffin embedded tissue were hybridized to commercial genomic arrays (Human BAC array, Spectral Genomics, USA) containing 1000 or 2000 non-overlapping genomic clones that provide an average 2-4 MB genome wide resolution for detecting DNA copy number imbalances. A-CGH detected copy number gains at chr 5p15: a region encompassing the hTERT gene, at chr 8q24.2 associated with c-myc oncogene amplification, 1p36.33, 7p22.3, 7q31.2, 9q34.3 and 12p13.32. Infant and non-infant tumours generally showed similar changes but differed in the frequency and type of imbalances on chr 10 and 17. Infant Mb had a higher frequency of chr 10 loss; a profile consistent with isochromosome 17 was seen in all non-infant and only 2/7 infant MB. Two amplicons at chr 12q21-22 and 14q11.2 were seen in the majority of non-infant MB, and absent in the infant tumours. Validation of these observations in future studies with larger numbers of tumours will be important.

BIO 30 : INHIBITION OF PROTEIN KINASE C ACTIVITY AND INSULIN-LIKE GROWTH FACTOR II EXPRESSION ASSOCIATED WITH TAMOXIFEN MODULATION OF ETOPOSIDE CYTOTOXICITY IN BRAIN TUMOR CELLS

Ziad Khatib¹, Cheppail Ramachandran², Athena Pefkarou¹, John Fort¹, Hugo B. Fonseca¹, Steven J. Melnick³, and Enrique Escalon¹,

¹Division of Hematology/Oncology, ²Research Institute, ³Department of Pathology, Miami Children’s Hospital, Miami, FL 33155

Tamoxifen, a non-steroidal anti-estrogen widely used against breast cancer, is also useful for treatment of other malignancies, due to its sensitizing effect on other chemotherapeutic agents and radiation. We have investigated the advantages of combining tamoxifen with one of the commonly used cancer chemotherapeutic drug, etoposide (VP-16) in brain tumor cell lines. Tamoxifen (10 µM) increased etoposide cytotoxicity 8.3-fold in the human glioma cell line (HTB-14), 47.5- and 40-fold in two primary medulloblastoma cell lines (MCH-BT-31 and MCH-BT-39), 6- and 2.7-fold in two primary ependymoma cell lines (MCH-BT-30 and MCH-BT-52) established from pediatric patients. CalcuSyn analysis of cytotoxicity data showed that combination of tamoxifen and etoposide was synergistic with combination index values ranging from 0.243 to 0.369 at IC₅₀ level among different pediatric brain tumor cell lines. Tamoxifen as a single agent is also cytotoxic at higher concentrations (>20 mM) in brain tumor cells. To understand the mechanism underlying the tamoxifen modulation of etoposide cytotoxicity, we analyzed expression of P-glycoprotein, Insulin-like Growth Factor I receptor (IGF-1R), IGF-1, IGF-II, and estrogen receptor (ER) as well as protein kinase C (PKC) activity. While P-glycoprotein, IGF-IR, and IGF-1 were not affected, enhanced inhibition of PKC, and IGF-II were observed in brain tumor cells treated with tamoxifen and etoposide combination as compared to cells treated with single agents alone. Tamoxifen at 10 µM when combined with etoposide at 0 - 100 µM reduced PKC activity 77% compared to only 58% with etoposide alone. IGF-II expression decreased to 48.6% of the untreated control in the combination treatment as compared to 31.2% for etoposide alone and 26.2% for tamoxifen alone treatments. These results suggest that inhibitory effect of tamoxifen on brain tumor cells manifest through different mechanisms involving inhibition of targets such as PKC and IGF-II (Supported by seed grant funds from Miami Children's Hospital Foundation).

BIO 31: FAVORABLE BIOLOGY NEUROBLASTOMA (NB): LEPTOMENINGEAL METASTASES REMAINING STABLE IN A CHILD WITHOUT CHEMORADIOTHERAPY

Kim Kramer, MD^1, Brian H. Kushner, MD¹, Jeffrey C. Allen, MD², George Krol, MD³, Nai-Kong V. Cheung, MD, PhD¹

Department of Pediatrics¹, and Neuroradiology³, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY; Department of Neurology², Beth Israel Medical Center, New York, NY

Background: Intrinsic tumor biological features are critical prognosticators of survival of NB. Patients with localized neuroblastoma and favorable biological parameters may be observed without treatment. Conversely, leptomeningeal metastases in patients with primary extracranial neuroblastoma is highly unusual and despite aggressive multi-modality therapies, invariably fatal.

Methods: We present the case of a 7 month-old infant who presented with acute paraplegia; MRI revealed an extensive, multi-level epidural mass (T12-L4) causing compression of the conus and cauda equina with intradural, leptomeningeal extension.

Results: The patient underwent emergent spinal cord decompression and subsequent surgical resection of the adrenal tumor. The primary tumor revealed favorable biology (favorable Shimada classification. single copy MYCN, low serum lactate dehydrogenase, and favorable VMA/HVA ratio). Metastatic work-up was unrevealing except for MRI evidence of abnormal nodular foci of intradural enhancement coating the conus medullaris and cauda equina suspicious for subarachnoid disease . An 18-fluoro-deoxyglucose (FDG) positron emission tomography scan was compatible with NB in the abdomen; longitudinal linear tracer uptake was also noted in the lower thoracic and

Conclusion: Some infants with favorable biology neuroblastoma may be observed without treatment despite the advanced INSS stage.

BIO 32: MONOCLONAL ANTIBODIES (MABS) AGAINST SOMATOSTATIN RECEPTOR 2A (SSTR2A) AS POTENTIAL MEDULLOBLASTOMA THERAPEUTIC REAGENTS

Chien-Tsun Kuan, Carol J. Wikstrand, T. Shelley Davis, Michael R. Zalutsky, and Darell D. Bigner

Department of Pathology, Duke University Medical Center, Durham NC, USA

SSTR2A, a G protein-coupled molecule, is overexpressed in virtually all medulloblastomas (MED) as investigated by RT-PCR and localization to the cell surface (CS) by FACS analysis. As only low levels of synthetic ligand octreotide uptake via internalization are observed, the use of immune reagents to SSTR2A for therapy has been proposed. Characterized by 7 transmembrane domains, SSTR2A presents an initial extracellular domain (ECD) of 43 amino acids, a 12-mer of which has been shown to be specifically immunogenic in rabbits. We isolated MAbs to the 12-mer ₃₃QTEPYYDLTSNA₄₃-KLH as well as the SSTR2A amino terminal (amino acids 1-47) (ECD)-GST fusion protein, screened by FMAT analysis on D341 and D283 MED cells, and demonstrated specific binding by Western blot and homogeneous CS binding, indicating 100% of the cells express CS epitope. Following iodination, four MAbs (all IgG2a) were tested for immunoreactive fraction (IRF), K_A determination (BIAcore analysis vs immunizing peptide; Scatchard analysis vs D341 MED cells), and internalization by MED cells. Four MAbs [IRF values 50-94%], exhibited a range of K_As vs intact cells (0.32-1.0 x 10⁸ M^-1) and similar K_As vs peptide (1.7-2.4 x 10⁷ M^-1). Following binding of ¹²⁵I-MAbs to D341 MED at 4^oC, measurement of intracellular, CS, and released ¹²⁵I at time points after warming to 37^oC revealed little or no internalization of the ¹²⁵I-MAb-receptor, consistent with previous studies performed in the absence of ligand. All MAbs exhibited long-term CS binding (50-90% at 2 hrs, 50-62% at 24 hrs) indicating stable CS binding over time at 37^oC. All four MAbs estimate 1-4 x 10⁵/D341 MED cell CS receptors by Scatchard and QFACs analyses, a level exceeding that required for targeting. Affinity maturation of these MAbs, and isolation of higher affinity reagents by MAb generation or phage display are being pursued for development of therapeutic moieties.

BIO 33: NT-3 MEDIATED SIGNALING PATHWAYS IN DAOY MEDULLOBLASTOMA CELLS

Doris Lang, Ratnakar Patti, Johannes Schulte, Alexander Schramm, Tarek Shalaby,

Peter C. Phillips, Angelika Eggert, Michael A. Grotzer

Department of Oncology, University Children‘s Hospital of Zurich, Switzerland

High expression of the neurotrophin-receptor TrkC is associated with a favorable survival outcome in childhood medulloblastoma (MB). However, NT-3/TrkC activated signaling pathways in MB are not well understood.

To determine the gene expression programs resulting in MB cells overexpressing TrkC, we transfected the human MB cell line DAOY with the TrkC cDNA or an empty vector (EV) control. Gene expression profiles were then obtained from DAOY, DAOY-TrkC, and DAOY-EV treated with the TrkC specific ligand NT-3 in a time course of 0 h to 24 h using Affymetrix HGU95Av2 oligonucleotide microarrays screening a total of about 12600 genes.

Upon NT-3 activation of DAOY-TrkC cells, a total of 157 genes were dynamically regulated. These results were validated using quantitative RT-PCR. The majority of the NT-3/TrkC regulated genes is involved in signal transduction, cell proliferation, transcription, and cell cycle mechanisms, but also in cell death/apoptosis. Some of the new target genes of TrkC receptors identified, include bradykinin receptor B2 (BDKRB2), prostaglandin-endoperoxide synthase 2 (PTGS2/Cox2), DNA topoisomerase 2A (TOP2A), hyaluronan-mediated motility receptor (HMMR), EZRIN (Villin 2), tumor-suppressing subchromosomal transferable fragment cDNA3 (TSSC3/IPL), solute carrier family 20 member 1 (SLC20A1), and cysteine-rich angiogenic inducer 61 (CYR61). The expression pattern of these genes was also determined in 55 primary MB samples (grouped as MB with high TrkC and high NT-3 vs. MB with low TrkC and/or low NT-3) and mainly correlated with our microarray data.

These new findings may contribute to a better understanding of the effects of TrkC signaling in medulloblastoma and may point toward potential therapeutic targets for this common childhood brain tumor.

BIO 34: SPECIES SPECIFIC UROKINASE RECEPTOR LIGANDS REDUCE GLIOMA GROWTH AND INCREASE SURVIVAL PRIMARILY BY AN ANTI-ANGIOGENESIS MECHANISM

Walter E. Laug, Xingyao Bu, Vazgen Khankaldyyan, Ignacio Gonzales-Gomez, Susan Groshen, Wei Ye, Shaoqiu Zhuo, Jaume Pons, Jennifer R. Stratton, and Steven Rosenberg

Department of Hematology-Oncology and Department of Pathology, Childrens Hospital Los Angeles, Los Angeles, CA and Chiron Corporation Emeryville, CA

The urokinase type plasminogen activator (uPA) system is highly expressed in brain cancers and implicated in tumor progression. We investigated the effect of pegylated mouse (m) and human (h) specific peptides (PEG-uPA 1-48) of the amino-terminal region of uPA on brain tumor growth. Human glioma cells were injected into the forebrains of nude mice and the peptides were given SC twice weekly after tumor establishment. All control animals died within 70 days. The h peptide showed a slight survival increase (p=0.019).70% of the mice receiving the m peptide alone (p=0.009) and 80% of the mice given both peptides (p=0.002 survived over 140 days. Similar data were obtained with the PNET cell line DAOY. Tumor sizes were significantly smaller in animals treated with either m or combined peptides (p<0.001) or h peptide (p=0.006) than the control animals (5.3mm). The mean vascular density and the Ki-67 index were significantly smaller and the apoptotic index higher in all treatment groups when compared to the control animals and the m and combo treatment groups had large areas of collagen deposition. Vascular tube formation by brain capillary cells in vitro was significantly suppressed in a species-specific manner. While adhesion to vitronectin of tumor and vascular cells was not affected, both peptides inhibited migration of these cells on a vitronectin gradient.

Our data demonstrate that these catalytically inactive uPAR ligands suppress glioma growth and increase survival primarily by anti-angiogenic mechanisms and may prove useful as adjuvant treatment for brain tumors.

BIO 35: POST-TRANSCRIPTIONAL SILENCING OF GENES INVOLVED IN THE DEVELOPMENT OF MEDULLOBLASTOMA

Michael J. Lawman , Selena C. Braccili, Cameron K. Tebbi, Blake D. Singletary, Christina M. Lozano and Joseph Horvath. Division of Pediatric Hematology/Oncology, St Joseph’s Children’s Hospital of Tampa, and Department of Immunology and Medical Microbiology, College of Medicine, University of South Florida, Tampa FL USA

Medulloblastoma is the most common malignancy of the cerebellum in children. Published evidence suggests that this condition is the result of deregulation of gene(s) involved in embryonic development. Genes within the hedgehog (Hh) pathway, namely, sonic hedgehog (shh), smoothen (smo), patched (ptch) and the transcription factor family, gli, have all been implicated. The aim of this study was to evaluate RNA interference (RNAi) technology as a method to determine the significance of hh gene(s) in the development of medulloblastoma and ultimately to utilize this technology as a means to control the disease. The RNAi studies reported here involved both small interfering RNA (siRNA) and short hairpin RNA (shRNA).

In order to investigate the use of RNAi to disrupt the Hh pathway in the cell lines, DAOY and D283, two potential RNAi targets were chosen, shh and gli1. Actin and GADPH were used as positive controls. In vitro transcription was utilized to synthesize probes for all four genes and the probes nonisotopically labeled, post synthesis, with Psoralen-Biotin. Using Northern blot analysis, these labeled probes were used to measure the levels of mRNA in untreated and siRNA or shRNA-treated cells. Based upon the predicted sequence, the siRNA and shRNA were designed to contain a 19-nucleotide sequence with a 3’ UU di-nucleotide overhang. Three RNAi sequences per target gene were designed. For each gene in the study, a RNAi containing a “scrambled” sequence was used as a negative control. To analyze the expression of the targeted genes and other neurogenic markers following RNAi treatment, western blot and/or flow cytometry analysis was used.

The alteration of mRNA levels of the RNAi-targeted genes and in protein expression in these cell lines, in conjunction with changes in the growth characteristics, will be discussed relative to the implications that this technology may have as an innovative treatment protocol.

BIO 36: A NOVEL AND RAPID APPROACH TO PROTEIN EXPRESSION PROFILING OF CEREBROSPINAL FLUID (CSF) FROM MEDULLOBLASTOMA PATIENTS USING FUNCTIONALIZIED MAGNETIC BEADS, ANCHORCHIP^TM TECHNOLOGY, MALDI-TOF AND MALDI-TOF/TOF MASS SPECTROMETRY.

S.-M. Leung*, M. Sheldon^# and C. C. Lau^#. * Bruker Daltonics Inc., Billerica, MA; ^#Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX

Medulloblastoma is the most common malignant brain tumor in children. While the prognosis has improved considerably, leptomeningeal spread of the tumor remains a significantly negative predictor of survival. CSF cytology has been used as part of the routine workup for metastatic disease but the sensitivity and specificity is sub-optimal. Recently we have observed that mitochondrial DNA mutation in cell-free CSF of medulloblastoma patients can be a potential marker for disease monitoring and relapse (1). Here we describe the generation and analysis of protein profiles of CSF from medulloblastoma patients to monitor treatment response and disease progression. Five to ten microliters of CSF taken from patients at various times during their diagnostic workup and treatment was added to magnetic beads with different functionalities (hydrophobic, ionic, metal chelating). After binding and washing, the eluted proteins and peptides were deposited on the AnchorChip^TM targets for Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) analysis. A comparison map showing the changes in the protein profiles between before and after treatment were done using ClinProTools software. The biomarker candidates can then be identified using MALDI-TOF/TOF for MS/MS analysis. Because it only takes 6 to 8 hours from sample preparation to data analysis, this new and rapid approach allows rapid screening and evaluation of protein profiles of CSF, thus aiding in the identification of relevant biomarkers and the subsequent development of diagnostic assays for monitoring disease progression and treatment response.

(1) L-JCWong, M Lueth, X-N Li, CC Lau, and H Vogel. (2003) Cancer Research 63:3866-71.

BIO 37: Anti-Medulloblastoma Effects of Valproic Acid as A Histone Deacetylase Inhibitor

Xiao-Nan Li, Qin Shu, Jack Men-Feng Su, Laszlo Perlaky, Ching C. Lau.

Laboratory of Molecular Neurooncology, Cancer Genomics Program, Texas Children’s Cancer Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX 77030

The anti-tumor effects of VPA, a well-established anti-epilepsy drug with histone deacetylase (HDAC) inhibiting activity, were investigated in two medulloblastoma cell lines (D283-MED, DAOY) as compared with one supratentorial primitive neuroectodermal (sPNET) cell line (PFSK) by various in vitro and in vivo assays. In all three cell lines, VPA (0.6 mM to 5.1 mM) inhibited cell proliferation in a time- and dose-dependent manner, and produced irreversible suppression of cell growth upon extended treatment with clinically achievable doses (0.6 mM for 28 days). The colony forming ability in soft agar was suppressed by 1.0 mM VPA and almost completely eliminated by 2.7 mM VPA in D283-MED and PFSK cells after 7 days of treatment. In D283-MED and DAOY cells, VPA (1.0 mM and 2.7 mM) arrested cells in G₁/G₀ phase, promoted apoptosis, induced expression of glial marker GFAP and neuronal marker synaptophysin (SYN). In PFSK, however, higher dose and/or longer exposure time is required to produce similar effects. The anti-tumor effects of VPA were found to be associated with increased accumulation of hyperacetylated histones H3 and H4, of which the more prominent increase is correlated with the more responsive DAOY and D283-MED cells. Treatment of DAOY cells grown s.c. in SCID mice with VPA (400 mg/kg, daily i.p. for 28 days) resulted in significant growth suppression of the xenografts (P<0.001), increased expression of GFAP and SYN, and elevated apoptotic cells. In conclusion, our data demonstrated that VPA, through inhibiting HDACs, has potent anti-medulloblastoma effects by inhibiting cell proliferation, promoting apoptosis and inducing cell differentiation. These results suggest that further preclinical studies to establish the role of VPA as a novel anti-medulloblastoma agent is indicated.

BIO 38: CORRELATION OF ENDOSCOPIC BIOPSY AND TUMOR MARKERS IN PATIENTS WITH PRIMARY INTRACRANIAL GERM CELL TUMORS

Neal Luther, Erika Mark, Ira J Dunkel, and Mark M Souweidane

Department of Neurological Surgery, Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center, New York, NY USA

Primary intracranial germ cell tumors (GCTs) occur most frequently in children and adolescents. Given the predominant midline, intraventricular location of these tumors and the frequent need for concomitantly treating hydrocephalus with endoscopic third ventriculostomy, patients with intracranial GCTs serve as logical candidates for endoscopic tumor biopsy. A minimally invasive technique for tumor sampling is further supported by the high response rates of GCTs using non-surgical therapy. Numerous studies, including reports from our experience, have established endoscopic biopsy of intraventricular brain tumors as an acceptable method for tumor sampling. However, the small sample size obtained using endoscopic techniques coupled with the frequency of mixed tumor histology within a single GCT raises the possibility of misrepresentative tumor sampling.

A retrospective analysis was performed of ten patients (eight males, mean age 16) treated for intracranial GCTs to determine the correlation between endoscopic biopsy results and biochemistry of serum and cerebrospinal fluid (CSF). The presence of raised a-fetoprotein (AFP) and human chorionic gonadotropin (b-HCG) has served as a reliable method for determining that GCTs have malignant components. Our criteria for tumor marker measurements diagnostic of a malignant, secreting tumor were a serum or CSF AFP value greater than 9 ng/ml or b-HCG measurement greater than 50 U/L. Nine patients had tumor marker values below our threshold for secreting tumors; six of these patients had endoscopic biopsy results of germinoma and one showed only gliosis. One sample disintegrated during processing, while another was misinterpreted as astrocytoma (tissue from open resection revealed teratoma). One patient had increased AFP, and in this case endoscopic biopsy diagnosed a yolk sac tumor. In total, all endoscopic biopsies of GCTs correlated with patient tumor marker status.

In our series, endoscopic biopsy was highly correlated with biochemical tumor markers, thus supporting the reliability of neuroendoscopic tumor sampling for GCTs.

BIO 39: ENDOTHELIAL APOPTOSIS INDUCED BY ALPHA-V-INTEGRIN INHIBITION REQUIRES GENERATION OF CERAMIDE

Melissa Millard, Linda B. Tran, Orla, T. Cox, and Anat Erdreich-Epstein

Angiogenesis, formation of new capillary blood vessels, is critical for cancer growth. The cell surface receptors, integrins avb3 and avb5 are critical in angiogenesis. RGDfV (AKA Cilengitide), a function-blocking peptide that blocks integrins avb3/avb5, has completed phase I trials in adults with recurrent gliomas and is in phase I trial in pediatrics. The molecular mechanism of action of integrin avb3/avb5 inhibition is poorly understood.

We have shown that inhibition of endothelial integrins avb3/avb5 increases ceramide, a pro-apoptotic lipid second messenger (Erdreich-Epstein et al, Cancer Research 2000). To examine whether this ceramide increase is required for endothelial apoptosis we seeded human brain microvascular endothelial cells (HBMEC) on vitronectin and induced apoptosis with RGDfV (25 µg/ml). Treatment of [³H]palmitic-acid labeled HBMEC with RGDfV with/without myriocin (1-50 nM) or fumonisin B1 (25 µM), inhibitors of de novo ceramide synthesis, or desipramine (10 µM), an inhibitor of acid sphingomyelinase (ASMase), suppressed the ceramide increase. De novo inhibitors suppressed both baseline and RGDfV-induced ceramide, but left the difference between ceramide in presence/absence of RGDfV unchanged. Desipramine only suppressed the RGDfV-induced ceramide increase, but not baseline ceramide. This suggests presence of at least two pools of intracellular ceramide, only one of which is regulated by integrin binding. Furthermore, sphingomyelin was decreased by RGDfV in [³H]palmitic-acid pre-treated HBMEC, supporting the role of SMase(s) in the ceramide increase. And most importantly, desipramine and imipramine (5-20 µM), and another ASMase inhibitor, SR33557 (1-10 µM), but not myriocin or fumonisin, effectively suppressed RGDfV-induced apoptosis (25-90%), suggesting that ASMase activation by RGDfV and the resultant ceramide increase are necessary for endothelial apoptosis.

These data suggest for the first time a causal role for ASMase-generated ceramide in mediating endothelial apoptosis induced by inhibition of integrins avb3/avb5. This will allow rational design of combination therapy using RGDfV in anti-angiogenic treatment of brain tumors.

BIO 40: Detection of JC virus DNA sequences in pediatric brain

tumors

Toshihiro Mineta, Hiroaki Okamoto, Shigeo Ueda, Yukiko Nakahara, Tetsuya Shiraishi, Takashi Tamiya, and Kazuo Tabuchi

Department of Neurosurgery, Faculty of Medicine, Saga University, Saga, Japan

Object: JC virus (JCV) is a human neurotropic polyomavirus that causes progressive multifocal leukoencephalopathy and is closely related to simian virus 40. Several recent reports have indicated a possible association between JCV and the development of various human brain tumors. We examined the presence of JCV deoxyribonucleic acid (DNA) sequences in primary pediatric brain tumors to elucidate the possibility that certain pediatric brain tumors can arise through infections with JCV.

Methods: Genomic DNA sequences were isolated from 62 pediatric brain tumors (32 medulloblastomas, 18 ependymomas, 5 choroid plexus papillomas, and 7 pilocytic astrocytomas) and analyzed for the presence of JCV DNA by Southern blot hybridization and direct sequencing. JCV DNA was detected in 5 ependymomas and one choroid plexus papilloma. Immunohistochemical studies revealed nuclear expression of the large T-antigen in a choroid plexus papilloma. However, none of the medulloblastomas or pilocytic astrocytomas contained JCV DNA.

Conclusions: These results provide molecular evidence of the association between JCV and the development of certain ependymomas and choroid plexus papillomas in contrast to medulloblastomas and pilocytic astrocytomas.

BIO 41

GLIA MATURATION FACTOR BETA (GMFb), A MEMBER OF THE COFILIN-ACTIN DEPOLYMERIZING FACTOR SUPERFAMILY, PROMOTES PROCESS OUTGROWTH AND IS DIFFERENTIALLY PHOSPHORYLATED DURING CNS DEVELOPMENT AND GLIOMA PROGRESSION

Christy Murdock, Priti Baijal, Shunzhen Zhang, Tao Fan and David D Eisenstat

Manitoba Institute of Cell Biology and Departments of Pediatrics & Child Health, Human Anatomy & Cell Science and Physiology, University of Manitoba, Winnipeg, MB Canada

Malignant gliomas respond poorly to combined modality therapy, including surgery, radiation and chemotherapy. Response to therapy fails, in part, due to infiltration of tumor cells into peritumoral white matter. There is considerable interest in unraveling the intrinsic and extrinsic factors affecting glioma invasion, including regulation of the peripheral actin cytoskeleton. Glia maturation factor beta (GMFb) was initially described as a factor promoting process extension and reduced cell proliferation in primary glioblastoma multiforme (GBM) cultures. GMFb is a 141 amino acid 17 kD protein with significant homology to actin depolymerizing factors (ADFs) that affect cell shape through direct regulation of actin filament polymerization. ADFs exist in active (dephosphorylated) and inactive (phosphorylated) forms. Adult (differentiated) brain and low grade gliomas (7/9) express both forms of GMFb, whereas the active dephosphorylated form is the predominant band found in embryonic brain and high grade gliomas (16/22 AA and GBM). The upper, phosphorylated band of GMFb is extinguished following treatment with phosphatases. In primary culture, GMFb is expressed in astrocytic processes and the axonal compartment of neurons. The cytoplasmic localization of GMFb is confirmed by the transfection of green fluorescent protein (GFP) constructs. Deletion of the putative C-terminal actin-binding domain of GMFb does not affect subcellular localization but abrogates process extension in glial and neuronal tumor cell lines. After treatment of U87 malignant glioma cells with cytochalasin D to disrupt the actin cytoskeleton, GMFb and actin co-localize within the cytoplasmic compartment. Co-immunoprecipitation experiments confirm the GMFb and actin form protein-protein complexes in vivo. Our results show that GMFb is an actin-binding protein that promotes process outgrowth through its interaction with the peripheral actin cytoskeleton. Determining the role of GMFb during CNS development may contribute to our understanding of its role during astrocytoma progression and invasion and towards novel biological approaches directed against these malignancies.

BIO 42: Analysis of genome-wide copy number profiles of medulloblastomas using array-based comparative genomic hybridization

Yukiko Nakahara, Tetsuya Shiraishi, Toshihiro Mineta, Hiroaki Okamoto, and Kazuo Tabuchi

Department of Neurosurgery, Faculty of Medicine, Saga University, Saga, JAPAN

Introduction: Array-based comparative genomic hybridization (array-CGH) is a novel genomic analysis technology that allows high throughput screening for abnormal gene amplifications and deletions with the sensitivity to detect single gene copy change in a tumor genome. We studied genomic changes in medulloblastomas using array-CGH technology and analyzed possible association of genomic changes with the sensitivity of adjuvant therapies.

Materials& Methods: DNA was extracted from each frozen medulloblastoma tissues from 8 patients. DNAs of tumor and reference control were labeled with Cy3 and Cy5. We performed hybridization using commercially available genomic DNA microarray slide, GenoSensor Array^TM 300 (Vysis Inc). It contained 287 clones including telomeres, oncogenes, and tumor suppressor genes. After hybridization, arrays were analyzed by the GenoSensor Reader System (Vysis Inc).

Result: The copy number gains were observed on MSH2, CDK6, TIF1, THRA, and TK1 (3/8 cases). The copy number losses were detected on CTSB, FGFR1, MYC, HRAS, HIC1, FLI, and PDGFB (3/8 cases). Our results show that the sensitive group has a tendency to include chromosomal gains or losses. The copy number gains were detected at chromosome 7, 17q and copy number losses were observed on chromosome 6,8,10q,11,17p in the sensitive group.

Conclusion: Array-CGH is a useful tool for the identification of genetic changes of brain tumors and potential genetic markers to correlate with the sensitivity of adjuvant therapy for patients with medulloblastoma.

BIO 43: VARIABILITY OF CONSTITUTIVE HETEROCHROMATIN OF CHROMOSOMES 1, 9 AND 16 IN 57 CHILDREN WITH SOLID TUMORS AND 64 CONTROLS

Melita Nakić, Iskra Petković, Mladen Čepulić, Ante Čižmić

University Children’s Hospital Zagreb, Zagreb, Croatia

Several studies point to an association of variable heterochromatic segments of chromosomes 1, 9 and 16 and malignant diseases. Investigations carried out so far have mostly dealt with populations of adult patients, whereas studies in children with solid tumours are rare.

In this study, variability of constitutive heterochromatin of chromosome 1, 9 and 16 was analysed in 57 children with solid tumors (of those 20 with neuroblastoma and 19 with nephroblastoma) and in 64 healthy controls. The aim of this investigation was to determine the correlations between the quantitative changes and the changes in the distribution of constitutive heterochromatin and the malignant process in children.

The analysis was carried out on slides obtained by routine method of peripheral blood culture and stained for C-banding. The quantitative variability of constitutive heterochromatin of chromosome 1, 9 and 16 was determined by objective method of linear measuring. C-segment length was expressed in relative units as a relation of the C-band length and the length of the short arm of chromosome 16. Significant differences between two groups were found in C-band size of chromosome 1, 9 and 16, but no difference in inversion frequency was, however, observed. This data might indicate a possible relationship between the amount of constitutive heterochromatin on chromosome 1, 9 and 16 and malignant transformation in children with solid tumors. This investigation established quantitative differences and difference in frequency of localization variants between specific histologic types of tumor. The analysis revealed significantly higher amount of C-heterochromatin on chromosome 9, and increased frequency of C-segment inversions in the group of 20 children with neuroblastoma as compared to 19 children with nefroblastoma. Additional study are, however necessary in order to substantiate these preliminary results.

BIO 44: NEUROFIBROMATOSIS 3: A REPORT OF PATIENTS MEETING DIAGNOSTIC CRITERIA FOR BOTH NEUROFIBROMATOSIS 1 AND 2

Bryan C. Oh, Mark D. Krieger, David I. Sandberg, Jonathan Finlay, and J. Gordon McComb

Divisions of Neurosurgery and Neuro-Oncology, Children’s Hospital Los Angeles, Keck School of Medicine/University of Southern California, Los Angeles, CA USA

Neurofibromatosis (NF) has been classified into two distinct types: NF1 and NF2. Both NF1 and NF2 have well-established diagnostic criteria. However, there have been no reports in the literature of patients meeting diagnostic criteria for both NF1 and NF2. We conducted a retrospective review of patients diagnosed with either NF1 or NF2 from 1993 to 2003. In all, 17 patients were studied. Of these patients, three met diagnostic criteria for both NF1 and NF2.

There were 17 patients (9 males and 8 females) in our study population. Eleven patients met diagnostic criteria for NF1 only. Three of these patients had a positive family history of NF1. Three patients met the criteria for a diagnosis of NF2 only. One of these patients had a positive family history for NF2. Of note, 2 of these 3 patients also had peripheral neurofibromas. While they failed to meet strict NF1 diagnostic criteria, they nonetheless had physical findings consistent with NF1. One female and two males met diagnostic criteria for both NF1 and NF2. The female patient and one of the male patients had bilateral vestibular schwannomas, numerous café au lait spots and peripheral neurofibromas. Her sister also had NF2. The other male patients had bilateral vestibular schwannomas, multiple peripheral neurofibromas, and marked scoliosis.

NF is an extremely variable disorder. As it has been well described, NF ranges from extremely mild cases that present in adulthood to severe cases in which serious complications may develop. In our study, we present evidence that the traditional classification scheme of NF1 and NF2 may miss a group of patients who meet diagnostic criteria for both disease processes. We propose that these patients be given the diagnosis of NF3. Performing direct gene testing on these patients may be helpful in better understanding the neurofibromatoses.

BIO 45: GLUTATHIONE S-TRANSFERASE (GST) POLYMORPHISMS AND OUTCOMES IN CENTRAL NERVOUS SYSTEM (CNS) TUMORS IN CHILDHOOD AND ADOLESCENTS

M. Fatih Okcu, Qin Shu, Xiao-Nan Li, Bogdan Dinu, Laszlo Perlaky, Melissa Bondy, Dawna L. Armstrong, Adesina Adekunle, Meenakshi B. Bhattacharjee, Robert C. Dauser, Murali Chintagumpala, Susan Blaney and Ching Lau.

Texas Children’s Cancer Center, Departments of Pediatrics, Hematology-Oncology, Neurosurgery and Pathology, Baylor College of Medicine, Houston, TX USA.

Background: CNS tumors are the most common solid tumors of childhood. With current therapy approximately 50 % of children and adolescents with brain tumors are cured. At the time of diagnosis it is impossible to predict in advance which patients will benefit from therapy and which patients will experience significant toxicity. The GST family of enzymes is responsible for clearance of chemotherapy agents including alkylating agents and platinum compounds used in brain tumor treatment. We hypothesized that GST polymorphisms may be responsible, in part, for individual differences in response to cancer treatment hence determining the outcome for survival and toxicity.

Methods: We conducted polymerase chain reaction (PCR) for GSTM1 and GSTT1 polymorphisms to investigate the relationship between GST genetic variations and survival in 45 patients with medulloblastoma. Results were analyzed for overall survival (OS) by Kaplan-Meier and Cox-proportional hazard analyses adjusting for age and risk group at diagnosis, and ethnicity.

Results: Patients with the GSTM1 null genotype had significantly shorter survival compared to patients with GSTM1 non-null (3-year OS 52% vs. 87%; p<0.05). We observed a much higher rate of GSTT1 null genotype (31%) compared with the published frequency of this genotype in normal populations values (15-25%), possibly due to the high frequency of GSTT1 null genotype in the African American patients (83% n=5/6).

Conclusions: These results indicate that GSTM1 polymorphisms may be a potential prognostic factor in children and adolescents with medulloblastoma. The GSTT1 genotype may be risk factor for development of medulloblastoma in the AA children.

GSTM1, GSTT1 and GSTP1 genotyping is still ongoing in the remaining 30 medulloblastoma and 60 glioma patients. Updated results will be presented for survival and toxicity outcome.

BIO 46: CLINICAL AND GENETIC STUDIES OF MEDULLOBLASTOMA WITH EXTENSIVE NODULARITY (NMB) IN PATIENTS WITH AND WITHOUT GORLIN’S SYNDROME

Torsten Pietsch*, Alessandro Raso^^, Valeria Capra^^, Hendrik Brune*, Arent Koch*, Ulrik Milde*, Armando Cama^, Claudio Gambini#, Massimo Brisigotti°, Paolo Nozza#, Felice Giangaspero@, Riccardo Occella§ Claudia Milanaccio&, Andra Rossi**, Gianluca Piatelli^, Marcello Ravegnani^, Giorgio Perilongo” and Maria Luisa Garrè&

Dept. Neuropathology, University of Bonn, Germany *; Dept. Pathology, Brescia Hospital°; Neuropathology, Regina Elena, Rome@; Pediatric Oncology, University of Padua”; Dept. Neuroradiology **, Dept. Neurosurgery^, Neuro-Oncology &, Pathology#, Giannina Gaslini Children’s Hospital, Genoa, Italy.

Correspondence address: Maria Luisa Garrè Neuro-Oncology Giannina Gaslini Hospital L.go G. Gaslini 5 16147 Genova Italy.

Genetic characterization of medulloblastomas (MB) has provided evidence of different genetic variants of MB overlapping with well characterized pathological MB entities (NMB, desmoplastic MB (DMB), classic MB (CMB)). NMB has been recently more extensively defined in its clinico-pathological features. In this study we wanted to focus on the involvement of alterations of the Sonic hedgehog / patched pathway in NMB as compared to DMB and CMB. A total of 10 cases of NMB were diagnosed at G. Gaslini Children’s Hospital in the period 1992-2002 (7M, 3F, median age 18 months). All cases are long-term survivors. Two presented with characteristics of Gorlin’s syndrome (GS). All cases reviewed by a pathologists’ panel displayed a similar morphological and immunohistochemical pattern consistent with NMB.

In 7/10 cases frozen tissue was available. Genetic study included SSCP analysis of the coding exons of PTCH1 (exons 2– 23). Quantitative RT-PCR for the specific hedgehog target genes GLI-1 and SOX18 was performed both in 7 NMB and in cases of DMB and CMB. SSCP analysis of PTCH1 gene in 7 cases of NMB showed altered migration pattern in 6/7 NMB cases. Sequencing of the fragments uncovered the presence of DNA polymorphisms in exons 2, 22 and 23. In addition, one case of a GS patient with NMB presented a SNP at exon 23 and a 4 bp deletion in exon 2, which was present in the peripheral blood and in the tumor. The second (wt) allele was obviously lost in the NMB indicating that there was no functional PTCH1 copy present in the tumor (LOH). Quantitative RT-PCR studies showed high GLI-1 mRNA levels as a sign of an activated sonic hedgehog/patched pathway in both, NMB and DMB but not in CMB. We compared the levels of NMB (6 cases) with CMB (12 cases) and DMB (10 cases). Using t-test, 2 sided, p-values were CMB vs. NMB, p= 0.007; DMB vs. NMB p= 0.251 (n.s.). Similarly, the novel SOX18 target gene of SHH was found overexpressed in NMB.

This indicates that NMB and DMB are similar in their patched signalling and they differ from classic MB in their activation status. However, genetic and clinico-pathological features suggest that NMB and DMB could carry different additional genetic alterations and that the patched pathway may be activated at an earlier stage in NMB. This information would be consistent with the high frequency of NMB in early infancy and its peculiar biological features (better prognosis). Our data did not show obvious clinical or genetic differences between NMB associated with GS and sporadic NMB. More data are needed to confirm a possible higher frequency of NMB in Gorlin’s syndrome.

BIO 47: Identification of genes from chromosome 1q THAT ARE up-regulated in ependymoma progression

Emma Prebble¹, Gregory Riggins², Brian Fee², Richard Grundy¹. ¹Institute of Cancer Studies, University of Birmingham, B15 2TT, U.K. ²Duke University Medical Center, Durham, NC27710 U.S.A.

The outcome of paediatric ependymoma is difficult to predict based on clinical and histological parameters. To address this issue we previously performed a CGH screen of paediatric ependymomas. Using hierarchical cluster analysis, we described three genetic groups of primary ependymoma, structural, balanced and numerical. The structural group showed few mainly partial imbalances and multivariate survival analysis showed that this group had a significantly worse outcome when compared with the numerical and balanced tumours. Gains of chromosome 1q were frequently observed in the structural group and in a group of recurrent tumours we analysed. In addition, gain of 1q was observed in three recurrent tumours from patients whose primary tumours showed a balanced profile. Taken together this data suggests that gain of 1q is involved in the progression of paediatric ependymoma. In order to identify specific genes on chromosome 1q that may be important, we have generated Serial Analysis of Gene Expression (SAGE) libraries from two matched pairs of primary and recurrent paediatric ependymomas. The primary and recurrent tumours from matched pair A had a balanced CGH profile, whereas in matched pair B the primary tumour was balanced and the recurrent tumour had a gain of chromosome 1q. We used a pairwise test based on a Monte Carlo simulation of tag counts from the SAGE 2000 software and Fisher’s exact test to identify 27 SAGE tags from chromosome 1q that were significantly up-regulated in the recurrence of matched pair B, but not matched pair A (p<0.1). Tag to gene mapping was performed using SAGEGenie (http://cgap.nci.nih.gov/SAGE). Genes identified will be discussed and include the human cartilage glycoprotein CHI3L1, which has been shown to be highly expressed in the serum of patients with malignant, but not low grade glioma and the brain-enriched hyaluronan-binding protein Brevican, which has been shown to be up-regulated in a rat model of invasive glioma.

BIO 48: Genomic Imbalances in Supratentorial Primitive Neuroectodermal Tumours (stPNETs)

Emma Prebble¹, Sara Dyer^1,2, Marie-Anne Brundler³, David Ellison⁴, Val Davison² & Richard Grundy^{1 1}Institute of Cancer Studies, University of Birmingham, B15 2TT, U.K. ²Regional Genetics Lab. BWH, Birmingham, B15 2TG, U.K. ³Department of Pathology, Birmingham Children’s Hospital, UK ⁴Department of Neuropathology, Newcastle General Hospital, UK

The current World Health Organisation’s classification of Primitive Neuroectodermal Tumours (PNETs) groups together infratentorial and supratentorial tumours, this implies a common origin. However, a recent gene expression study strongly suggests that stPNETS are molecularly distinct from their infratentorial counterparts [Pomeroy et al., Nature 2002: 415, 436-442]. While the outcome for children with medulloblastoma has improved in recent years, the management of PNETs occurring outside the cerebellum remains more difficult and patients generally have a poor prognosis. Achieving significant advances in the diagnosis, prognosis and therapy and of stPNETs relies on extensive molecular characterisation of these tumours. To this end, we have analysed 17 primary and 1 recurrent formalin-fixed, Paraffin-embedded stPNET for genomic imbalances by high-resolution Comparative Genomic Hybridisation (hr-CGH). Thirteen out of 18 (72%) of the tumours had genomic imbalances and the median number of imbalances per tumour was 4 (range 0 – 18). Out of 68 abnormalities 43 (63%) were whole chromosome imbalances while 25 (37%) were partial chromosome imbalances involving a chromosomal region or chromosome arm. Four high level gains were detected at 12q21, 20q, 1q and 6p. The most common chromosomal gains were of chromosome 2 or 2q in 7 out of 18 (39%) cases and gains of chromosome 17 or 17q in 5 out of 18 cases (28%). The most common losses were losses involving chromosome 9 in 4 out of 18 (22%) cases and loss of 13 or 13q in 3 out of 18 (17%) of cases. Correlation with clinical factors will be discussed. This study represents the largest series of genetic aberrations analysed in stPNETs by CGH to date and reveals characteristic chromosomal imbalances that differ from those observed in medulloblastoma.

BIO 49: GENE EXPRESSION PROFILING OF PEDIATRIC MEDULLOBLASTOMAS BY MICROARRAY HYBRIDIZATION

Cheppail Ramachandran¹, Ziad Khatib², Enrique Escalon², Sonia Rodriguez¹, P.K. Raveendran Nair¹, Perseus Jhabvala¹ and Steven J. Melnick³

¹Research Institute, ²Division of Hematology/Oncology, ³Department of Pathology, Miami Children's Hospital, Miami, FL 33155

Medulloblastoma is the most common posterior fossa tumor, representing about 20% of all intracranial tumors in the United States. Microarray hybridization of Clonetech cancer 1.2 arrays was used for expression profiling of pediatric medulloblastomas for identifying upregulated and downregulated genes that could be used for identifying both diagnostic and prognostic markers. All patients except MCH-BT-38 were under 15 years of age. Medulloblastoma patients with progressive disease (MCH-BT-16, 39 and 76) also had fourth ventricle foraminal extension and larger tumors. Of the 10 patients, three patients showed progressive disease following treatment. One patient died of sepsis/meningitis in the post-operative period and had metastatic lesions. Patients with aneuploid tumors responded better to treatment than patients with diploid tumors. Patients with hyperdiploid and tetraploid tumors had a complete response to therapeutic intervention. Gene expression profiles analyzed by microarray hybridization showed a few upregulated and a large number of downregulated genes among medulloblastoma specimens, as compared to an adult and fetal normal brain RNA samples. Membrane arrays were hybridized with PCR amplified cDNAs of tumor RNAs and the data was analyzed using EpClust and GeneCluster software. Of the 1176 cancer-associated genes in the array, matrix metalloproteinase-11 and 12, desmoplakin I, moesin-ezrin-radixin-like protein, placental plasminogen activator inhibitor 1 and 2, ephrin -A5 precursor, wnt-5A, A-raf proto-oncogene serine/threonine-protein kinase, and fibroblast growth factor 6 precursor were upregulated in medulloblastomas as compared to normal brain cells. However, several genes including neurotrophin -3 receptor (TRKC) were downregulated in medulloblastomas. The highly downregulated genes include BRAC-1-associated ring domain protein (BARD1), Calmegin , DNA-binding protein RFX5, placental calcium-binding protein, autoimmunogenic cancer/testis antigen NY-ESO-1, platelet basic protein precursor, STAT-induced STAT inhibitor 3, and bone morphogenetic protein 8. The altered gene expression profiles may provide new targets for diagnosis and treatment of pediatric medulloblastomas (Supported by funds from Miami Children’s Hospital Foundation).

BIO 50: ZD6474, A VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 (VEGFR2) INHIBITOR, INHIBITS GROWTH OF MULTIPLE PRIMARY CENTRAL NERVOUS SYSTEM TUMOR TYPES

Jeremy N. Rich, Dragutin Loncar, Sith Sathornsumetee, Stephen Keir, Catherine Wheeler, Isaiah Dimery, Darell D. Bigner, Henry S. Friedman

Division of Neurology and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710.

Pediatric primary central nervous system (CNS) tumors remain poorly responsive to most therapies. Although CNS tumors display marked heterogeneity in associated molecular alterations, many tumor types display increased activity of the vascular endothelial growth factor (VEGF) receptor and ErbB family member pathways. Novel therapies targeted at these pathways have been linked to blockade of tumor neoangiogenesis and growth. ZD6474 is a novel orally active inhibitor of the VEGF receptor 2 (KDR) tyrosine kinase and also has activity at higher doses against the tyrosine kinase associated with epidermal growth factor receptor (EGFR/HER1). Orally administered ZD6474 was well tolerated by athymic nude mice with an LD₁₀ of greater than 200 mg/kg/day when administered for 10 days of a 12-day period. Oral administration of ZD6474 (200 mg/kg/day on 10/12 days) to athymic mice bearing several established, histologically distinct (glioblastoma, medulloblastoma, and ependymoma) pediatric human CNS tumor xenografts produced significant inhibition of tumor growth in all cases (range 10.4-25.4 days of growth delay relative to controls). Most xenografts displayed a high rate of partial regressions with treatment (range 10-100%). Additionally, a glioma cell line selected for resistance to procarbazine (PR), a chemotherapeutic agent frequently used in glioma therapy, displayed near identical sensitivity to the parental cell line. Toxicity was low with rare deaths and only mild weight loss in the treatment group. Examination of tumors revealed decreased vascularity of treated tumors suggesting an anti-angiogenic effect.
Conclusion: ZD6474 is a novel agent with significant activity against a broad range of pediatric CNS tumor xenografts, including chemotherapy-resistant tumors. Further consideration of clinical development is warranted.

BIO 51: CONSEQUENCES OF DELETIONAL AND EPIGENETIC EVENTS AT 17p13.3: TARGETS OF HIC1 MEDIATED TRANSCRIPTIONAL REPRESSION

Brian R. Rood, Sébastien Pinte, Huizhen Zhang, Keri Ramsey, Dietrich Stephan and Dominique LePrince

Dept of Hematology/Oncology, Children’s National Medical Center, The George Washington University School of Medicine, Washington, DC

The short arm of chromosome 17 is the genomic region most frequently affected by deletional events in human neural tumors. The area of minimal deletion has been isolated to 17p13.3 in medulloblastoma, the most common malignant brain tumor of childhood. This region contains a gene cloned from a CpG island that demonstrates frequent hypermethylation in tumors, HIC1 (hypermethylated in cancer 1). HIC1 is a tumor suppressor gene whose transfection into tumor cell lines has been shown to decrease clonogenic survival. In a murine heterozygous knockout model, deletion and epigenetic silencing marked by DNA methylation have been shown to cooperate to inactivate HIC1 and promote tumorigenesis. The protein encoded by HIC1 is a transcriptional repressor with five Krüppel-like C₂H₂ zinc finger motifs and an N-terminal BTB/POZ domain. We have identified potential repression targets of HIC1 through the use of microarray expression profiling of U2OS cells infected with an ad-HIC1 construct. In silico analysis has been used to confirm the presence of HIC1 binding motifs proximate to target gene regulatory elements. Electromobility shift assays have been performed to demonstrate binding of HIC1 to target sequence specific DNA probes. Finally, reduction of target gene translation has been assayed using western blot of ad-HIC1 infected cells. The identification of HIC-1 target genes establishes the first links between the most common chromosomal event in human neural neoplasms and molecular biological alterations.

BIO 52: INTEGRATIVE PROFILING OF CHROMOSOMAL COPY NUMBER ABNORMALITIES AND GENE EXPRESSION IN PEDIATRIC EPENDYMOMAS

Michael Sheldon, Rebecca H Johnson, Yaojuan Lu, Pulivarthi Rao, Adekunle M Adesina, Dawna Armstrong, Meenakshi B Bhattacharjee, Robert C Dauser, Peter Burger, Jackie Biegel, Charles G Eberhart, Kenneth D Aldape, Ching C Lau
Section of Hematology-Oncology, Department of Pediatrics, Baylor College of Medicine, Houston TX USA

Ependymomas are gliomas that arise in the ependymal layer of the brain and spinal cord. They account for 3-9% of all neuroepithelial tumors and 6-12% of all intracranial tumors in children. Ependymomas are slow growing tumors with marked variations in histology, with anaplastic features sometimes seen in focal areas. Due to the paucity of histological markers, we endeavor to generate genetic profiles that can be used at the time of diagnosis to facilitate outcome prediction and therapeutic decision-making. The biology research arm of the Children’s Oncology Group (COG) ependymoma trial ACNS-0121 comprises a portion of this study. This trial is the largest investigation ever undertaken of the genetic bases, treatment alternatives, and clinical outcomes for ependymoma.

We are using microarray comparative genomic hybridization (aCGH) and gene expression profiling on Affymetrix Human U133 Plus 2.0 arrays to study genetic alterations in childhood ependymoma. Using tumor samples from a variety of institutions, we will identify characteristic patterns of chromosomal copy number aberration (CNA) and classify tumors into clinically relevant subtypes. We will then look for correlations between these CNAs and gene expression profiles from the same tumors. In this way, we will identify new markers as well as generate testable hypotheses that address the etiology and progression of ependymoma.

To expand the number of cases that can be included in this and other types of genomic profiling studies, we are refining new techniques for the analysis of cells harvested by laser capture microdissection of tumor samples embedded in paraffin, and also whole genome amplification of nanogram amounts of DNA. Molecular classification by genomic profiling, in conjunction with a prospective clinical trial such as ACNS-0121, will improve risk assessment and outcome prediction for patients with ependymoma, and identify new prognostic markers for objective patient stratification in future clinical risk-based therapeutic trials.

BIO 53: SPARC/OSTEONECTIN EXPRESSION INDUCES GLIOMA INVASION AND SURVIVAL UNDER SERUM-FREE CONDITIONS

Qing Shi, Shideng Bao, Mark Hjelmeland, Elisabeth Reese, Xiao-Fan Wang, Jeremy N. Rich

Division of Neurology and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710.

Tumor invasion into normal brain is a major contributor to the failure of pediatric glioma treatment. Osteonectin, also known as secreted protein acidic and rich in cysteine (SPARC), is overexpressed by gliomas at the tumor-brain interface, reactive astrocytes, and neoangiogenic vasculature implying a role in invasion. We have shown that the expression of osteonectin in human malignant glioma cell lines induces an invasive phenotype through extracellular matrix and into normal brain with expression of specific matrix metalloproteinases (MMPs) [Rich JN, et al., J. Biol. Chem. 278: 15951]. Despite gaps in the understanding of invasion process, specific cell signaling pathways have been linked to regulation of MMP expression and invasion. We now show that treatment of malignant glioma cultures with exogenous osteonectin acutely induces phosphorylation of FAK, JNK, and Akt/PKB and increases activity of the small G-protein, Rac1. As invading cells may experience relative restriction of nutrients relative to the primary tumor, we examined the survival of polyclonal glioma cultures expressing either vector control or osteonectin under prolonged serum starvation. Cells expressing osteonectin did not differ from vector control in cell cycle fraction under normal serum conditions, but osteonectin expression dramatically lowered the level of apoptosis (4.91 ± 0.01% vs. 0.18 ± 0.02%) and increased G₂ cell cycle fraction (4.16 ± 1.70% vs. 51.85 ± 0.36%) with serum starvation. Combination treatment of serum starved glioma cultures serum and osteonectin induced a moderate increase in the phosphorylation of both FAK and Akt suggesting cooperation between the mechanisms by which osteonectin and serum activate these pathways. Thus, glioma expression of osteonectin may promote tumor invasion through induction of cell motility and survival under stress through the activation of specific intracellular pathways involved in cell movement, protease expression, and cell survival.

BIO 54: C-MYC OVEREXPRESSION RECAPITULATES THE LARGE CELL/ANAPLASTIC MEDULLOBLASTOMA PHENOTYPE IN XENOGRAFTS

Duncan Stearns, Aneeka Chaudhry, Peter C. Burger, Charles G. Eberhart

Departments of Oncology and Pathology, Johns Hopkins Hospital, Baltimore, MD.

Risk assessment in patients with medulloblastoma (MB) has historically rested entirely upon clinical staging criteria. However, histopathologic and molecular features are providing additional insights into progression risk and tumor biology. Tumor anaplasia, defined by increased nuclear size, pleomorphism, increased mitotic and apoptotic rates, has been identified in approximately 25% of MB and has a negative impact on outcome. Amplification of myc oncogenes has been strongly associated with the large cell/anaplastic phenotype (LC/A), but occurs relatively infrequently. More common is elevation of c-myc mRNA levels, which has been shown in several retrospective studies to be a negative prognostic indicator. We show here that c-myc overexpression results in more aggressive MBs that mimic the LC/A phenotype.

DAOY medulloblastoma cells, which have low endogenous c-myc expression, were stably transfected with c-myc cDNA. In vitro, c-myc mRNA levels were elevated 10-fold and protein levels 2-3 fold in several subcloned lines. Luciferase reporter assays revealed a 1.5-2 fold increase in Myc dependant transcriptional activity. Cell cycle analysis showed an increase in the S and G2/M fractions in the c-myc expressing subclones. When injected subcutaneously into NOD/SCID mice, c-myc expressing DAOY xenografts grew on average 75% larger over 8 weeks than control cell lines (P < 0.01, two-tailed T test). More remarkably, the histopathology of the c-myc expressing xenografts recapitulated that of LC/A medulloblastoma, with increased nuclear size, prominent macro-nucleoli, increased mitotic activity and widespread apoptotic bodies including confluent “apoptotic lakes”. Immunostaining with cleaved caspase 3 and Ki67 confirmed the increases in apoptotic and proliferative activity. We are using this model to identify c-myc targets by examining gene expression profiles in vitro and comparing them to those in primary tumors with low or high c-myc levels.

BIO 55: Gene Expression Profiles of Cerebellar Tumors, External Granular Layer (EGL) Neurons, and hyperplastic EGL Nodules in patched-deficient mice

Jack M Su, Charlotte R Nicholson, Cindy C Rho, Chris T Man, Laszlo Perlaky, Xiao-Nan Li, John YH Kim, Barbara A Antalffy, Dawna A Armstrong, Ching C Lau.

Department Pediatric Hematology-Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, 6621 Fannin Street, MC 3-3320, Houston, Texas, USA

We have studied the cellular origin of medulloblastoma in Patched-deficient mice that have spontaneously developed cerebellar tumors. Gene expression profiles of 13 cerebellar tumors were analyzed in comparison with external granular layer (EGL) neurons, internal granular layer (IGL) neurons, and purkinje cells harvested by density gradient separation or laser capture microdissection at various embryonic and post-natal ages.

Hierarchical clustering and Multi-Dimensional Scaling analysis of the expression profiles showed that the majority (9/13) of cerebellar tumors (group 1) most closely resemble the profiles of P0-2 EGL neurons. These data suggest that critical events of tumorigenesis occur in early post-natal periods, coinciding with the period of maximal proliferation of EGL neurons. A second group (4/13) of tumors (group 2), however, resemble the profiles of purkinje cells, and two of these tumors appeared to have originated from the cerebellar vermis, in contrast to the nine tumors in group 1 that were hemispheric in location. EGL-specific genes such as Cxcr4 and purkinje-cell specific genes such as Grid2 (glutamate receptor, inotropic, delta 2) were differentially expressed in tumor group 1 and 2, respectively. Our data suggest that, while the majority of cerebellar tumors in Patched-deficient mice arise from EGL neurons, a subgroup of tumors may originate from purkinje cell precursors in the subventricular zone.

We also observed inappropriately proliferating EGL layers and nodules in otherwise asymptomatic mice. We hypothesized that these hyperplastic EGL nodules represent pre-malignant lesions and compared their expression profiles to those of the cerebellar tumors and Patched-deficient EGL neurons. Preliminary analysis showed that dysregulated cell cycle regulation (Cyclin D1, Cdk4) and transcriptional control (Hmga2) may represent early events in tumorigenesis, and impaired DNA-repair and global genomic instability (Rad51, Chk1) appear to be prominent events in subsequent tumor transformation. Further functional characterizations of these candidate genes should elucidate the mechanisms of tumorigenesis.

BIO 56: Epidermal growth factor receptor gene amplification in disseminated pediatric low grade gliomas

Uri Tabori^1,2, Shlomit Rienstein¹, Yaara Dromi¹, Ayala Aviram¹ , Rina Dvir², Yoav Burstein², Eleonora Trejo², Shlomi Constantini², Lianna Beni-Adani², Amos Toren¹, Shai Izraeli¹ and Gideon Rechavi¹.

¹Sheba Medical Center, Tel-Hashomer, Israel. ²Dana Children Hospital, Tel-Aviv Medical Center, Tel-Aviv, Israel.

Background: Disseminated low grade gliomas (LGGs) represent 5-10% of pediatric LGGs. The genetic and biological nature of these tumors is poorly understood.

Epidermal growth factor receptor (EGFR) gene is located on the short arm of chromosome 7. EGFR amplification is characteristic of primary glioblastomas proliferative and invasive behaviour. We looked for certain molecular abnormalities which may differentiate disseminated from the other LGGs

Methods: Comparative genomic hybridization (CGH) was applied to 18 pediatric patients with LGG. 6 cases with disseminated LGGs were compared to 12 non metastatic LGG controls. FISH analysis and immunohistochemistry were used to further highlight specific genetic targets.

Results: CGH revealed multiple chromosomal abnormalities in 5 of 6 disseminated cases and in 5 of 12 controls. There was no correlation between the amount of chromosomal abnormalities and clinical course. Amplification of chromosome 7 was noted in 4/6 cases as opposed to 2/12 controls (P=0.078). FISH analysis revealed EGFR gene amplification in one case negative for +7 at CGH. Immunohistochemistry for EGFR was positive in 6/6 cases and in 2/9 controls (P=0.08).

At a mean follow-up of 7.2 years all patients are alive with variable but slow disease progression.

Conclusions: The high rate of EGFR gene amplification and protein expression in disseminated pediatric LGGs may have implication on our understanding of its role in gliomagenesis. Targeted therapies may be possible for these children. Larger scale studies are needed to further establish these findings.

BIO 57: Neurotrophic factors expression in childhood low-grade astrocytomas and ependymomas

Gianpiero Tamburrini, *Antonio Chiaretti, *Marco Piastra, **Alessia Antonelli,***Antonio Ruggiero, ***Riccardo Riccardi, *Giancarlo Polidori, Concezio Di Rocco.

Paediatric Neurosurgery, Catholic University of Sacred Heart, Rome, Italy. *Paediatric Intensive Care Unit, Catholic University of Sacred Heart, Rome, Italy. **Department of Neurobiology, CNR, Rome, Italy ***Paediatric Oncology, Catholic University of Sacred Heart, Rome, Italy.

Background: Neurotrophic factors [Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF)] are growth factors implicated in growth and differentiation of brain nerve cells. An involvement of these factors in biology and progression of some specific tumors has been suggested. According to the role of neurotrophic factors in tumor behaviour the aim of the present study was to investigate their expression in two childhood brain neoplasm, namely low-grade astrocytomas and ependymomas.

Materials and Methods: We investigated the NGF, BDNF, GDNF and NGF-receptors (TrkA and p75) expression in tumor tissues, cerebrospinal fluid (CSF) and plasma of 10 children affected by low-grade astrocytomas and ependymomas. Control tissue samples (together with CSF and plasma samples) were obtained from patients who underwent surgery for cerebral vascular or epileptogenic lesions.

Results: The expression of NGF decreases both in tumor samples and in CSF of affected children, as compared to controls. The expression of GDNF remains unchanged both in tissues and in CSF, whereas BDNF increases in CSF. No differencies were found in neurotrophic factors plasma levels in patients and in controls. Gene expression of NGF and its high-affinity receptor (TrkA) are reduced in tumor tissues, whereas the number of cells immunopositive to the low affinity NGF-receptor (p75) is increased.

Conclusion: A reduced expression of NGF and TrkA has been shown in low-grade astrocytomas and ependymomas. These findings might be related to differentiative and apoptotic roles of this neurotrophin. The different expression of NGF, BDNF and GDNF in low-grade astrocytomas and ependymomas suggests that a different redundancy exists among members of the neurotrophic factors family and that their expression may be correlated with biology and behaviour of these tumors.

BIO 58: GENETIC POLYMORPHISMS IN FOLATE METABOLIZING GENES AS RISK FACTOR FOR CHILDHOOD PRIMITIVE NEUROECTODERMAL -, AND ASTROCYTIC TUMORS

Jan M. Van Tornout, Yue-Xian Tu, Stefanie Gilinsky, Jane Kim, Ignacio Gonzalez, Shahab Asgharzadeh

Children’s Hospital Los Angeles and University of Southern California, Los Angeles, CA.

Despite increasing insights into the somatic genetic changes associated with childhood malignant brain tumors, their etiology remains unknown. Various risk factors, including nitrosamine/micronutrient intake, pesticide/fertilizer exposure, and parental occupation in chemical industries have been inconsistently associated with childhood brain tumors. We have previously shown that a polymorphism in methylene-tetrahydrofolate reductase (MTHFR^C677T), a gene involved in folate metabolism and neural tube development, was associated with stage and risk of developing neuroblastoma, a childhood cancer derived from neuroepithelial tissue. We hypothesized that polymorphisms among MTHFR and other genes involved in the folate pathway could influence the risk for developing other tumors of neuroepithelial origin, i.e., PNET family of tumors (medulloblastoma, supratentorial PNET) and astrocytic tumors. We evaluated the genotype distribution of various polymorphic genes involved in the folate pathway, i.e., methionine, MTHFR, methionine synthase (MS), methionine synthase reductase (MTRR), cystathione beta-synthetase (CBS), and reduced folate carrier (RFC1) among 101 pediatric PNETs and 63 astrocytomas. DNA isolated from patients oral swab specimens or whole blood was used for PCR-based genotyping. Genotype distributions of cases at loci of interest were compared with the distributions published in the six largest historical control populations to date. The prevalence of genotypes MTHFR^677T was significantly higher (p-value range <0.001 - 0.026) in the PNET cases compared to 3 out of 5 published control populations (n=598,403,510,554,1147 respectively), while the CBS^699T genotype was more prevalent (p-value=0.0386) compared to published control data (n=364). Similarly, we identified a significant increase (p<0.0001, control n=598) in the MTHFR^677T genotype among the astrocytoma cases vs. the historic control data, and we found a suggestion of trend (p=0.058, control n=371) for increase in CBS^1080T genotype among the cases. These data show that polymorphisms among the genes involved in the folate pathway may significantly affect the risk for developing a childhood PNET and/or astrocytic tumors.

BIO 59: IMMUNOTHERAPEUTIC TARGET ANTIGEN PHENOTYPES OF PEDIATRIC VS ADULT CENTRAL NERVOUS SYSTEM (CNS) TUMOR BIOPSIES, XENOGRAFTS, AND CULTURED CELLS

Carol J. Wikstrand, Chien-Tsun Kuan, Kenji Wakiya, R. Ian Cumming, Katrin Lamszus, Manfred Westphal, and Darell D. Bigner.

Department of Pathology, Duke University Medical Center, Durham, NC, USA and University Hospital Eppendorf, Hamburg, Germany

We and others have reported the feasibility of therapeutically targeting the adult glioma-associated, cell surface-expressed epitopes EGFRwt, EGFRvIII, gpnmb, MRP3, and IL-13Ra2. Their relevance for therapy of pediatric CNS malignancies is unknown. We initiated a prospective analysis of the antigenic phenotype of pediatric astrocytic tumor (A; astrocytomas and GBM, glioblastomas multiforme) and ependymoma (EP) biopsies and xenografts and cultured cell lines derived therefrom by Quantitative Fluorescent Cell Sorter Analysis using directly fluoresceinated monoclonal antibodies specific for these target molecules. Adult astrocytic tumor biopsies and xenografts (AA and GBM) exhibit high frequency of EGFRwt expression (21/33, 71%), and moderate frequencies of EGFRvIII (13/33, 39%), gpnmb (12/30, 40%), and MRP3 (11/19, 58%). Only 9/15 (60%) adult GBM biopsies expressed cell surface IL-13Ra2, while 13/15 (87%) exhibited higher IL-13Ra2 densities in the cell cytoplasm; 0/2 adult AA expressed IL-13Ra2. Pediatric A and GBM mimic the adult phenotype for EGFRwt (3/5, 60%) and MRP3 (3/5, 60%) expression, but exhibit decreased gpnmb expression (1/5, 20%), and a lack of EGFRvIII (0/5). 2/4 pediatric A and GBM biopsies expressed cell membrane IL-13Ra2 and ¾, cytosolic receptor. Cultured cell line analysis supports these trends; 5/5 adult and 5/5 pediatric astrocytic tumor biopsy-derived cell lines express EGFRwt; modest diminution of MRP3 expression (adult cases 4/6, 67%, pediatric cases 2/5, 40%) and a decrease in gpnmb expression (3/6 in adult cases; 1/5 in pediatric cases). We have studied 3 EP xenografts; 1/3 expressed EGFRwt and gpnmb; all lacked EGFRvIII, 0/3 expressed cell surface IL-13Ra2; 3/3 expressed MRP3. Continuing accrual of cases will allow discrimination of diagnostic subclass antigen phenotypes, and determine which therapeutic models defined for adult disease may be applicable to pediatric CNS tumor therapy. As pediatric tumors exhibit lower frequencies of these antigens, it is imperative that new pediatric CNS tumor-associated target epitopes be identified.

BIO 60: MOLECULAR GENOMICS OF MEDULLOBLASTOMA

Hai Yan, Chunhui Di, Daniel Broderick, Roger McLendon, Sridharan Gururangan, Henry Friedman and Darell Bigner

Department of Pathology, Duke University Medical Center, Durham, NC, USA

Primary brain tumors are the leading cause of cancer deaths in children. The most frequent malignant CNS neoplasm in this age group is the medulloblastoma. Despite advances in therapy, 50% of the children afflicted with medulloblastoma die of the disease, and aggressive treatment of children with radiation and chemotherapy results in impaired neural development, growth deficits and endocrine dysfunction in the survivors. Research to demonstrate the pathogenic basis of medulloblastoma, and thereby facilitate the development of new approaches to managing this disease, is sorely needed.

Genome-wide evaluations of genetic alterations in medulloblastoma will provide unprecedented improvements in molecular classification and novel therapeutic targets. We are applying a recently developed state-of-art genetic approach called digital karyotypying to look at genomic alterations in primary medulloblastoma samples. Digital karyotyping is a genome-wide approach to detect copy number alterations at high resolution. This approach appears uniquely powerful in detecting chromosomal aberrations, homozygous deletions, and amplifications, alterations known to target genes involved in tumorigenesis. We have generated six digital karyotyping libraries from medulloblastoma cell lines and primary tumors. The data have not only confirmed the most common genomic abnormalities which have previously been identified by conventional technologies, but also mapped to several genes that have tumorgenesis potential in medulloblastoma. The results will elucidate medulloblastoma pathogenesis and set the stage for a wealth of future basic scientific and translational research.

BIO 61: Neuroprotection with riluzole in the immature rat cerebellum after radiation exposure

Kaleb Yohay, Sunny Kim, Janice Lee, Grace Kim and Jeffrey Rothstein

Department of Neurology and Division of Child Neurology, Johns Hopkins University, Baltimore, MD USA

Ionizing radiation is an essential part of the treatment of many types of central nervous system (CNS) tumors in children. However, severe long-term cognitive effects, especially in younger patients, frequently limit its utility. Modulation of glutamate excitotoxicity has been shown to attenuate CNS injury in neurodegenerative disorders, hypoxia/ischemia and traumatic injury. Excitotoxicity may also play a role in neuronal injury and death after exposure to ionizing radiation. The aim of our current study is to evaluate the glutamate release inhibitor riluzole in inhibition of neuronal apoptosis in the cerebellum of rat pups exposed to ionizing radiation.

Six-day-old rat pups were injected with 8mg/kg of riluzole or vehicle IP 1hour prior to irradiation. They were subsequently exposed to a single dose of 0 Gy, 3Gy or 5 Gy, limited to the head. Six hours after irradiation, the pups were sacrificed and perfused. After brain sectioning, terminal dUTP nick-end labeling (TUNEL) was used to determine the extent and location of apoptosis. Results: The cerebellar granule cell layer showed high levels of radiation dose-dependent apoptosis. Preliminary data shows pups receiving pretreatment with riluzole had a 66% reduction of cells undergoing apoptosis in the granule cell layer when exposed to 3 Gy (n=4) and a 40% reduction when exposed to 5 Gy (n=4).

Our preliminary data suggests that pre-treatment with the glutamate release inhibitor riluzole can significantly decrease neuronal apoptosis in the cerebellum of the immature rat brain exposed to ionizing radiation. Further study needs to be done to determine if riluzole could be an effective means of limiting some of the long-term side effects of therapeutic radiation on the developing brain.

BIO 62: SOMATOSTATIN TARGETED RADIOTHERAPEUITCS FOR MEDULLOBLASTOMA

Ganesan Vaidyanathan, Abraham Boskovitz, Henry S. Friedman, Donna J. Affleck and Michael R. Zalutsky

Departments of Radiology, Pathology and Pediatrics, Duke University Medical Center, Durham, NC USA

Medulloblastoma are relatively radiosensitive malignancies; however, curative doses of radiation often can not be delivered because of dose limiting normal tissue toxicity. This is of particular concern in patients with neoplastic meningitis where radiation induced side effects can be severe. In this disease, targeted radiotherapy is a promising alternative to external beam therapy because of the potential for achieving selective destruction of tumor cells while sparing normal CNS tissues. We have previously shown that radioiodine could be targeted to somatostatin receptors (SSTR) on human medulloblastoma xenografts using the peptide glucosyl-Phe¹-[I-Tyr³]octreotate (GluTOCA). The goal of the current study was to develop an SSTR-avid peptide that could be used to treat medulloblastomas with ¹³¹I or ²¹¹At, a highly cytotoxic a-particle emitting radionuclide with a range in tissue (50-80 mm) well matched to neoplastic meningitis geometry.

Octreotate with a Dde-protected lysine was reacted with N-succinimidyl 4-(bis-Boc)guanidinomethyl-3-(trimethylstannyl)benzoate, the Dde group removed, and the resultant stannylated molecule reacted with ¹³¹I or ²¹¹At and tert-butylhydroperoxide at 70^oC to produce 4-guanidino-3-[¹³¹I]iodobenoyl-Phe¹-octreotate (GMIBO) or 4-guanidino-3-[²¹¹At]astatobenoyl-Phe¹-octreotate (AGMBO), respectively. The specific internalization by SSTR-expressing D341 Med medulloblastoma cells of [¹²⁵I]GMIBO increased from 2.5 ±0.2% at 30 min to 16.9 ±0.3% at 4 h, and was significantly higher than that for [¹³¹I]GluTOCA; a second study showed that the internalization of AGMBO was not significantly different than that of GMIBO. In addition, retention of radioactivity in D341 Med cells for GMIBO was better than that of GluTOCA. The tissue distribution of [¹³¹I]GMIBO and [¹²⁵I]GluTOCA were directly compared in athymic mice with subcutaneous D341 Med xenografts. The tumor uptake of both radioiodine labels was comparable through 8 h, but at 24 h, there was an about twofold higher tumor retention for [¹³¹I]GMIBO.

In summary, our results suggest that GMIBO and its ²¹¹At-labeled analogue warrant further investigation as targeted radiotherapeutics for medulloblastoma.

EPI 1: CNS TUMOR EPIDEMIOLOGY & OUTCOME IN ADOLESCENTS AND YOUNG ADULTS IN THE UNITED STATES, 1975-1998

Anne Bendel, Lynn Ries, Orren Beaty, Krystal Bottom, Archie Bleyer

Children’s Hospitals and Clinics, Minneapolis, MN and Children’s Oncology Group, Arcadia, CA. USA

Purpose: Data from the NCI Surveillance, Epidemiology and End-Results (SEER) were analyzed to determine the incidence and outcome of brain tumors in adolescents and young adults in the United States.

Methods: The SEER incidence, U.S. mortality, and SEER 5-year survival of CNS tumors was determined for each 5-year age group from 0 to 44 years of age from 1975 to 1998. Average annual percent change was derived from linear regression of each reported annual rate. Adolescents and young adults were defined as individuals 15-29 years of age.

Results:

Incidence, 1975-1998

• CNS tumors accounted for 6% of all neoplasms.
• The nadir in incidence of CNS neoplasms occurred in the 15-29 year age group, at a rate of 22.3 per million.
• The incidence of CNS tumors increased over time, mainly due to an increased incidence of “other glioma”.
• Males and White, non-Hispanics had a higher incidence of CNS tumors compared to females and other ethnic groups, respectively.
• Astrocytoma accounted for 64% of CNS neoplasms, “other gliomas” 19%, PNET 8%, and ependymoma 6%.

Outcome, 1975-1998

· Mortality rates for individuals 15-29 years of age were similar to the pediatric age group, and showed modest improvement over time.

· Mortality rates were higher for males than females.

· Survival rates for the 15-29 year age group improved over time, but lagged behind survival rates seen in all other age groups.

· Among astrocytoma, no improvement in 5-year survival rate was observed in 15-24 year-olds, in contrast to an average annual improvement of 0.5-3% per year in the other age groups.

· 20-year survival for astrocytoma was 65% for age 15-19 years, 40% for 20-24 years and 25% for 25-29 years.

· 20-year survival for individuals 15-29 years of age with “other gliomas” was 50% and ependymoma 65%.

EPI 2: OLDER ADOLESCENTS AND YOUNG ADULTS WITH BRAIN TUMORS IN THE UNITED STATES: LACK OF CLINICAL TRIAL PARTICIPATION AND OF SURVIVAL PROLONGATION AND MORTALITY REDUCTION

Archie Bleyer, Maha Hag-Alshiekh, Michael Montello, Troy Budd, Anne Bendel, Orren Beaty and Maura O’Leary; University of Texas MD Anderson Cancer Center, Houston TX and Children’s Oncology Group, Arcadia CA USA

Background: Young adults with cancer have been found to have had less survival improvement than either younger or older patients (Proc. ASCO 21: 389a, 2002). We questioned if this observation applied to brain tumors and if so, why.

Method: Annual rates in SEER incidence and 5-year survival from 1975 to 1998 were analyzed for each 5-year age <40 years. Trends were derived from linear regressions of the reported annual rates for 1975-80, 1981-6, 1987-92, and 1993-8 cohorts. National brain tumor trial data were obtained on 2,213 <40 year-olds entered on cooperative group and consortia (PBTC, NABTT and NABTC) treatment trials during 1997-2001.

Results: Above age 10-15, the clinical trial participation and survival rates co-declined precipitously for all malignant brain tumors, and also for gliomas, ependymoma and primitive neuroectodermal tumors (PNET). In patients with the most common malignant CNS tumors, astrocytomas (including glioblastoma multiforme), the correlation of clinical trial participation and survival rate improvement was statistically significant, with the 15-25 year-olds having the least improvement and the lowest clinical trial participation rate, whether considered as a function of the absolute number of entries (Table) or as a function of the proportion of entries relative to the number of patients diagnosed with the type of tumor.

Correlation of National Clinical Trial Participation and Survival Prolongation in Anaplastic Astrocytoma and Glioblastoma Multiforme (p < .05)
Age (Years)	0-4	5-9	10-14	15-19	20-24	25-29	30-34	35-39
Clinical Trial Accruals, 1997-2001	127	129	54	27	54	97	169	222
Average Annual Change in 5-Year Survival, 1975-1998	1.3%	0.6%	1.1%	0%	0%	1.6%	3.0%	1.9%

Conclusion: The failure in 15-25 year-olds in the U.S. to improve brain tumor survival over the last quarter century appears to be due to their under-representation on clinical trials. Reversing the deficit will require increased clinical trial availability, access, and participation.

EPI 3: INCIDENCE AND PATTERNS OF NEURAXIS METASTASIS (NM) IN PATIENTS (PTS) WITH DIFFUSE PONTINE GLIOMA (DPG)

James Brashears, Sridharan Gururangan, Colleen McLaughlin, Marcello Morgan, James Provenzale, Edward C. Halperin, and Henry S. Friedman

The Brain Tumor Center at Duke, Duke University Medical Center (DUMC), Durham, NC USA

Diffuse pontine gliomas comprise 8% of all intracranial tumors in children. Treatment for this tumor is focal radiotherapy but almost all patients suffer local recurrence and ultimately succumb to the disease.

Neuraxis spread of DPG has only rarely been reported in the literature. We therefore estimated the incidence and characterized the patterns of NM in pts with DPG. The radiation oncology database at DUMC was used to identify pts treated for DPG and a chart review conducted of those diagnosed with NM (defined as leptomeningeal (LM), parenchymal (PM), and/or subependymal (SE) based on neuroimaging studies). Findings from flurodeoxy glucose positron emission tomography (FDG-PET), magnetic resonance spectroscopy (MRS), and histology were also used to assess NM when available.

Between 1986-2000, 86 pts were treated for DPG at our institution. Sixteen pts (18%) [median ages, 9 years, range, 4-17] had NM and were identified at a mean time of 15 months (range, 3-96) from diagnosis. Metastatic patterns included WM in 9 pts, PM in 7 and SE in 6. Five pts showed ≥ 2 metastatic patterns. The FDG uptake on PET was increased in 5 pts in the sites of NM. Five pts had MRS and showed increase in the choline and decrease in the N-acetyl aspartate peaks characteristic of tumor. Three pts had histologic confirmation of high-grade glioma on biopsy. All pts have died of disease despite salvage therapy.

A variety of metastatic patterns occur in a significant proportion of patients with DPG at the time of recurrence Although the outcome is uniformly fatal, identifying NM by non-invasive neuroimaging modalities may help guide palliative treatment and improve quality of life.

EPI 4: Sacrococcygeal Myxopapillary Ependymoma in a Patient with Spinal Dysraphism and Dwarfism

Karen W. Braune, Richard S. Tubbs, Keith E. Georgeson, W. Jerry Oakes, David R. Kelly, and Alyssa T. Reddy.

Departments of Pediatrics, Neurosurgery, Cell Biology, Pediatric Surgery, and Pathology, The University of Alabama at Birmingham and The Children’s Hospital of Alabama, Birmingham, AL USA.

Myxopapillary ependymoma typically occurs in the distal spinal cord and cauda equina and rarely is diagnosed in pediatric patients. Rare reports also describe subcutaneous sacrococcygeal myxopapillary ependymoma that occurs as a primary lesion. The origin of this tumor outside of the nervous system is unclear but it has been theorized to arise from an ependyma-lined cavity that is a remnant of the caudal portion of the neural tube.

A 20 month-old female presented to our institution with a chief complaint of inability to walk. Past medical history was significant for mesomelic dwarfism. She had myelopathic neurologic findings with leg weakness and increased lower extremity reflexes. There were no stigmata indicative of occult spinal dysraphism. Her initial evaluation included cervical spine radiographs and MRI of the brain and spine. Findings of these studies included C1-2 instability, osteodysplasia, syringomyelia from T10-T12, tethered cord and the presence of a 1 centimeter, round cystic lesion in the presacral region. Following recovery from occipitocervical fusion, the patient underwent resection of the presacral mass. Histopathology revealed an epidermoid cyst with a myxopapillary ependymoma present within the adjacent soft tissues without invasion of the coccyx. Although, the follow up interval is short, the patient is clinically well and will undergo spinal cord de-tethering soon.

This is the first case, to our knowledge, in the literature of a subcutaneous myxopapillary ependymoma associated with an epidermoid cyst and also the first in a patient with dwarfism. Rare case reports have noted the coincidence between spinal ependymomas and both split cord malformation and dermal sinus tracts. We theorize that derangement of secondary neurulation may lead to concomitant occult spinal dysraphism in the presence of extraaxial vestiges of ependymal cell rests. This would embryologically unify why our patient had the constellation of an extraaxial epidermoid cyst, myxopapillary ependymoma, and tethered cord.

EPI 5: SYMPTOMS AND SUFFERING AT THE END OF LIFE IN CHILDREN WITH BRAIN TUMORS

Bridget M. Brown and David N. Korones, MD

University of Rochester Medical Center, Rochester, NY USA

Background: Cancer is the second leading cause of death in children, and brain tumors account for 25 percent of these deaths. However, little is known about the experience of children in the terminal phase of this disease. We hypothesized that children with brain tumors have different signs, symptoms, and needs during the terminal phase of their illness than children with other types of cancer.

Methods: We performed a retrospective review of records from all children with cancer treated at Golisano Children’s Hospital who died between the years 1993 to 2003. Records of the last three months of life were examined for demographics, signs and symptoms, hospitalization and treatments, end of life care, and location of death. Information collected about children with brain tumors was compared to that of children with other malignancies.

Results: Of 103 records reviewed, 29 patients (28%) had brain tumors and 74 (72%) had other cancers. The most common symptoms were pain and fatigue (>80% in each group). Children with brain tumors were more likely to suffer from a functional loss such as focal motor deficit (p<0.0001), vision loss (p=0.005), wheelchair use (p<0.0001), dysphagia (p<0.0001), and difficulty speaking (p=0.0001). They were less likely to suffer from dyspnea (p=0.006), appetite loss (p=0.02), and fever (p=0.0002). Patients who had brain tumors were more likely to die at home (p<0.0001), had do-not-resuscitate (DNR) orders documented earlier (p=0.004), and were less likely to receive radiation therapy (p=0.02), surgery (p=0.04), or other invasive procedures (p=0.003) during the last three months.

Conclusions: Children with brain tumors have a different experience at the end of life than children with other types of cancer. Healthcare providers should be aware of these differences to better anticipate needs of children with brain tumors. Discussion of end-of-life issues must begin earlier to assure the child’s ability to participate.

EPI 6: MEDULLOBLASTOMA IN CHILDREN. MONOINSTITUCIONAL EXPERIENCE FROM A DEVELOPING COUNTRY

D. Calle.

Deparment of Paediatric Haematology and Oncology, Instituto Oncológico Nacional "Dr Juan Tanca Marengo" (SOLCA), Guayaquil, Ecuador.

Brain tumors are the second cancer in our center.

Objective: To study retrospectively the clinical features therapeutic reponse and survival of medulloblastoma in children below 15 years.

Methods: Between 1996 and 2003 twenty-one children were diagnosed and treated with surgery, chemotherapy and craniospinal irradiation.

Reponse to treatment was assessed clinically and radiologically. Survival was calculated using percentages values.

Results: Of 21 patients with medulloblastoma, median age at diagnosis was 5 yrs (1 to 15 years). Male: Female radio was 1.6:1. More than 80% of children had headache and vomiting. 12 children had unsteadiness of gait. Majority has the symptoms of de 2-3 months duration. 16 had midline vermian lesion, three had cerebellar hemisphere lesion and 2 had CP angle tumor. 7 had obstructive hydrocephalus. One had spinal metastasis. 21 children were evaluated for treatment outcome. All children had surgery (subtotal excision in 20). 9 children received chemotherapy (8 Vp-Cb and 1 M-SFOP) after surgery and before irradiation (55GysFCP-35gy spinal axis). 3 children received only chemotherapy (2 Vp-CB and 1 BB-SFOP) after surgery. 4 children received chemotherapy (Vp- CB) after surgery, before and after craniospinal irradiation. 5 children had only surgery. Of 21 children 10 (48 %)are alive free of disease. Estimate response in 15 pts: CR in 10 pts, PR in 5 pts. Median follow up 12 months (1 –39). 11 pts are dead, 6 (progression) 4 (recurrence) and 1 (second cancer). 19% were out of follow-up.

Conclusions: 1.-The OS is the highest in the group tried with Surgery+chemotherapy and radiotherapy. 2.-Rate response was 71%. 3.- A better optimization in the quality of resection is necessary to offer a better future to these patients.

EPI 7: OUTCOME OF TREATMENT INDUCED HIGH-GRADE GLIOMA (HGG) IN CHILDREN: A STUDY OF THE CANADIAN PEDIATRIC BRAIN TUMOUR CONSORTIUM

Anne-Sophie Carret, Uri Tabori, Bruce Crooks, Juliette Hukin, Isaac Odame, Donna L. Johnston, Daniel L. Keene, Carolyn Freeman, Eric Bouffet.

On behalf of the Canadian Pediatric Brain Tumour Consortium (CPBTC)

Pediatric Hematology-Oncology, The Montreal Children’s Hospital/McGill University Health Center, Montreal, Quebec, Canada, H3H 1P3.

As cure rates increase, more children treated for cancer are at risk for long-term toxicities including the development of a second malignancy. Reports of treatment related-HGG in survivors of childhood cancers are scarce and data on their evolution and response to therapy are limited. We report treatment related-HGG in 16 children who were treated with irradiation +/- chemotherapy for either acute lymphoblastic leukemia (n=7) or solid tumour (n=9). Among the solid tumour patient group, four had an initial diagnosis which can predispose to a second cancer (3 NF1 with optic glioma, 1 bilateral retinoblastoma).

Median age at cranial radiation was 4.5 yrs (range, 0.5-9). Median age at diagnosis of HGG was 14.5 yrs (range, 7-19) with a median interval between radiation therapy and diagnosis of HGG of 8 yrs (range, 6-14). All gliomas occurred within the previous radiation fields (unifocal:15; multifocal:1). Fifteen patients had a diagnostic biopsy with histology consistent with glioblastoma multiforme (n=13), anaplastic astrocytoma (n=1), gliomatosis cerebri (n=1), gliosarcoma (n=1). For one patient the diagnosis was based on imaging only. Twelve patients received chemotherapy and 2 patients palliative treatment only. Surgical resection was part of the treatment for 9 patients including 2 gross complete resections. Six patients were re-irradiated. The overall median survival for all the patients was 8 months (range, 0.1-82 months) with no difference between leukemia and solid tumour patients. Re-irradiation was associated with a better median overall survival (8.5 versus 13 months). Three patients are still alive with disease.

High-grade glioma may occur in children as a consequence of therapy for a prior malignancy. Cranial radiation therapy may affect the multi-step course of tumorigenesis and should be consider as a risk factor. The optimal therapy and particularly the role of surgery and re-irradiation is still not clear and would require a larger epidemiologic study.

EPI 8: Incidence of Brain Tumor in Chinese Children: A 4 Year Prospective Review

GCF Chan,¹ MMK Shing,² ACW Lee,³ CW Luk,⁴ CK Li,⁵ HL Yuen,⁴ CK Li², SY Ha¹

Departments of Paediatrics & Adolescent Medicine of The University of Hong Kong,¹ The Chinese University of Hong Kong,² Tuen Mun Hospital,³ Queen Elizabeth Hospital, ⁴, Princess Margaret Hospital, ⁵ Hong Kong Pediatric Hematology Oncology Study Group (HKPHOSG), Hong Kong SAR, China.

Objective: We have little information about the incidence and histology types of brain tumors in Chinese children. We analyzed the data from the HKPHOSG surveillance study and compared it with the SEER data.

Methods: New cases of childhood (≤15yrs) with brain tumors diagnosed in the five major regional hospitals (included nearly all cases of local children with cancer) were registered with pre-designed data form at diagnosis from Jan 1999 to Dec 2003. The data were double checked with the Hong Kong Cancer Registry database annually. Classification of brain tumors were based on the WHO classification. Data were compared with the published SEER data for children ≤15yrs (1975-95).

Results: There were 131 new cases of childhood brain tumors diagnosed within this 4 years period but only 122 were ≤15yrs. For these 122 children, median age was 7.81 yrs (range 0.2-15.6 yrs) and M:F=1.4:1. The distribution of tumor histological types ranking from most to least common were: Glioma n=42/122 (34% vs. 65% in SEER) [astrocytoma n=30, brainstem glioma n=9, other glioma n=3], PNET n= 39/122 (31.9% vs. 22.9% in SEER) (medulloblastoma n=34, cerebral PNET n=5); Germ cell tumor n=19/122 (15.6% vs <3% in SEER), ependymoma n=8 (6.5% vs. 9.3%in SEER), craniopharyngioma n=3, choroids plexus tumor n=3 and other miscellaneous tumors. Compared to the SEER data, we have a much higher incidence of germ cell tumors and relative high incidence of PNET. Our analyses based on the short term follow-up data relating to their respective treatment outcomes were comparable with the published results.

Conclusions: Our preliminary data supported previous impression that CNS germ cell tumors are more common among oriental. We also found that medulloblastoma is relatively more common in our locality. However, our outcome data did not suggest that any genetic difference in terms of treatment response.

EPI 9: MALE PREDOMINANCE IN CNS GERM CELL TUMORS: ANALYSIS OF SEER DATA

Paul J Chuba, Richard K Severson, Kanta Bhambani, DR Sharadashree, Ron Thomas, and Merlin R Hamre.

Department of Radiation Oncology, St. John Macomb Hospital, Webber Cancer Center, Departments of Radiation Oncology, Pediatrics, and Family Medicine, Wayne State University, Detroit, MI.

The origin of intracranial germ cell tumors is not known with certainty. Most likely, aberrant migration of germ cells from the wall of the yolk sac (allantois) to the genital ridge takes place during embryogenesis prior to neoplastic transformation. It is also possible that CNS germ cell tumors arise from early gonadal lesions which then migrate to extra-gonadal sites.

Male predominance among cases of CNS germ cell tumor was investigated using data from the National Cancer Institute’s Surveillance Epidemiology and End Results (SEER) Program. Four-hundred-eleven cases (M=312, F=99) of central nervous system germ cell tumors (ICCC code) were identified in SEER registry data from 1973 to 1999. Cases were grouped by histology as Germinoma (ICD-0-2 code 9064, n=257), Dysgerminoma (9060, n=35), Mixed Germ Cell Tumor (9085,9100-2, n=41) and Malignant Teratoma (9080-4, n=58). Cases were also grouped as localized and non-localized according to the SEER staging system. Contingency table analysis was performed to determine the differences of sex by age, race, localized disease and histology.

Results indicated that both the incidence of CNS germ cell tumors and the male predominance increased about the time of puberty. The peak in incidence (3.8 per million population) occurred in the 15-19 year old age group and the greatest male to female ratio (19.5:1) occurred in the 20-24 year old age group. This ratio was markedly reduced after In females, the peak incidence occurred earlier (10-14 year old age group). CNS germ cell tumors were rarely detected after age 20 in females, or after age 35 in males.

Two possible explanations for sex-ratio imbalance for CNS germ cell tumors seem most likely. Differences in numbers of available male or female germ cells could account for this if transformed germ cells migrate from the gonads to the brain. Alternatively, it is possible that pre-existing ectopic germ cells in the CNS are influenced by accelerating endocrine activity at the time of puberty. Gender specific hormonal signals may lead to maturation delay or differentiation arrest, somehow leading to neoplastic transformation. It is possible that germ cells in pineal and/or suprasellar locations (gonadotropin regulatory centers) are particularly susceptible to such influences.

EPI 10: DESMOPLASTIC MEDULOBLASTOMA IN YOUNG CHILDREN: A FEATURE OF GORLIN’S SYNDROME

Cruz O, Mora J, Vila J*, Guillen A**, Costa JM**

Departments of Pediatric Oncology, Pathology* and Neurosurgery** Hospital St Joan de Déu. Esplugues de Llobregat. Barcelona. Spain

Introduction: Gorlin syndrome is an inherited condition that confers a high predisposition to cancer, mainly basal cell carcinomas in young adults. We describe an infant with no familial antecedents who presented with desmoplastic medulloblastoma.

Case Report: A 12 months old boy, born to nonconsanguineus parents, consulted for developmental delay and vomiting. On physical examination megacephaly, frontal bossing and hypertelorism were evident. He had no skeletal or skin abnormalities. MRI revealed a left hemispheric cerebelar tumor with a lamellar pattern. He underwent suboccipital craniectomy with total removal of the tumor. Pathology showed a typical pattern of desmoplastic medulloblastoma. Molecular genetic analysis of his blood showed an heterozygous novel frameshift mutation of the PTCH gene, 385ins, changing the codon 129 from TGG to TTGG resulting in protein truncation. This mutation is a novel disease-causing mutation. The mutation was not detected in the DNA from the parents suggesting de novo mutation or germline mosaicism. Following surgery, the patient was enrolled in the Spanish Pediatric Oncology Society Baby Protocol for medulloblastoma with the aim of avoiding radiation therapy.

Commentary: Nevoid basal cell carcinoma syndrome (NBCCS or Gorlin’s syndrome) is a fully penetrant, autosomal dominantly inherited syndrome with variable expressivity. The disorder is caused by mutations in PTCH. The syndrome comprises multiple basal cell carcinomas, keratocysts of the jaw, spine and rib anomalies and calcification of the falx cerebri. NBCCS-associated medulloblastomas present earlier than medulloblastomas not associated with the syndrome, and with “desmoplastic” phenotype.

Conclusion: This and other similar cases in the literature show the importance of considering Gorlin’s syndrome in the differential diagnosis of any young child who present with desmoplastic medulloblastoma, especially before the age of 5. Chemotherapy only based protocols should be considered in these patients because radiotherapy has a marked effect on the early development of skin and other cerebral tumors.

EPI 11: PAEDIATRIC SPINAL CORD EPENDYMOMA (SCE) IN THE MRI ERA:

A CANADIAN PAEDIATRIC BRAIN TUMOUR CONSORTIUM STUDY

Ugonwa Dag-Ellams, Beverly Wilson, Paul Steinbok, Mariana Silva, Keith

Aronyk, Normand Laperriere, Eric Bouffet,

The Canadian Paediatric Brain Tumour Consortium (CPBTC), Paediatric Brain Tumour Program, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8

Intramedullary tumours are rare in the paediatric population. It is recognized that the MRI has considerably influenced the management of these tumours. However, the impact of this change has never been evaluated, particularly in terms of postoperative management.

A retrospective review of 18 patients, (11 males, 7 females) paediatric cases of SCE during the MRI scan era was conducted in the CPBTC.

Median age at diagnosis was 11.6 years (1.5-17.4). Back pain and gait disturbances were the most common presenting symptoms. Median duration of presenting symptoms was 11.5 months (0.75-48).

In addition to preoperative MRI, 1 patient had a Myelogram, 2 a CT. 16 MRI were with Gadolinium enhancement. MRI revealed spinal dissemination in 2 patients. Most tumours were located in the lumbar region (12/18). The median number of segments involved was 4 (2-12).

All patients underwent surgery. Resection was total in 10 cases, subtotal in 6 and partial in 1, (unknown in one). Postoperatively, 17 MRI were performed. 9 showed no residual disease, 4 residual tumour and 4 equivocal residue. 2 tumours were anaplastic, 3 grade 2 and 12 grade 1/myxopapillary, (no grade in one). Four patients, (including two with anaplastic ependymoma) received postoperative radiation for residual tumour. One of these patients received additional chemotherapy.

Five patients relapsed, all locally. Four had repeat surgery, followed by radiation in 3. One patient had radiation only. Two patients had a second relapse. At the last follow-up, all patients were alive. 12 of the patients were alive and in remission, 4 patients alive with residual disease and one alive receiving treatment. Median follow-up was 2.6 years (0.16-10.16). One patient was lost to follow-up.

Conclusions: Myxopapillary ependymomas account for the majority of SCE in the paediatric age. The introduction of MRI in follow-up has enabled decreased use of postoperative radiation.

EPI 12: SECOND NEOPLASMS (SN) OF THE CENTRAL NERVOUS SYSTEM (CNS) IN CHILDREN TREATED AT A SINGLE INSTITUTION

Bożenna Dembowska- Bagińska¹, Iwona Filipek¹, Wiesława Grajkowska³, Marta Perek- Polnik¹, Marcin Roszkowski², Danuta Perek¹

¹Oncology Department, ²Neurosurgery Department, ³Patomorphology Department, Children’s Memorial Health Institute, Warsaw

The aim of our study was to analyze SN treated in our center.

Between 1997 and 2004 9 pts: 7 boys and 2 girls, aged 5 4/12 to 21 6/12 were treated for second neoplasms in the CNS. Six patients had a primary diagnosis of CNS tumor (PNET, MB, LGG- 3 pts, Meningioma ), 1 pt- parameningeal RMS, 1 pt- HD and 1 pt ALL. Median time from diagnosis of primary disease to second neoplasm was 6 8/12. 7 out of 9 pts underwent radiotherapy for primary tumor. In 4 pts SN occured in irradiated field , in 1- at margins, in 2 – outside of irradiated area. SN were diagnosed within 2 yrs to 14 yrs 11/12 from radiotherapy. The 2 pts who didn’t undergo radiotherapy developed second tumor 3 10/12 and 5 7/12 from diagnosis. Onset of disease was symptomatic in 6 pts. 3 others were diagnosed with SN at routine CNS radiological check up. Second tumors were localised in the brain in 8 pts, 1 pt had tumor in the spinal cord. In 3 pts with shortest time of observation from primary diagnosis relapse was suspected but pathology confirmed otherwise. 8 pts underwent surgery of their SN. The pt with intraspinal tumor did not qualify for any kind of surgical intervention . 7 pts had a radical resection of the tumor, 1- subtotal. 3 pts were diagnosed with meningioma, 1pt with AI, 2 pts with anaplastic Astrocytoma, 1 pt PNET and 1 pt Glioblastoma multiforme.In 1 pt diagnosis of intraspinal glioma was based on MRI scan. Surgery was the only treatment in 4 pts ( Meningioma and LGG ). 5 pts had adjuvant chemotherapy, 3 additional radiotherapy. Six pts are alive, 1 pt with intraspinal glioma died of treatment complications. 2 pts (PNET, GM) were lost from observation.

Supported by grant nr C 028/P05/2002

EPI 13: Childhood Cancer Registry in Argentina (Registro Oncopediatrico Hospitalario Argentino: ROHA). Primary CNS tumour cases

Blanca Diez., Enrique Schwartman, Marcelo Scopinaro, Mercedes Garcia Lombardi, Maria Davila, Dora Loria, Florencia Moreno.

Roha Buenos Aires, Argentina

ROHA is a not-for-profit institution committed since 2000 to provide a resource for gathering and disseminating epidemiologic data on childhood cancer, describing their incidence and survival patterns, evaluating diagnosis and treatment and establishing awareness of the disease. It gathers data from 69 sources, 7 registries and 2 cooperative groups. This report includes 3368 cancers diagnosed during 2000-02 in children younger than 15 years. Total Argentine population is 36.260.130 (Census 2001) and 28 % is younger than 15 years. Data are grouped by histologic type and primary site based on the International Classification of Childhood Cancer 1996 (ICCC) and International Classification of Diseases for Oncology, 3er Edition. In ROHA 95% of cancers are histologically confirmed. The percentage does vary by ICCC category from 86% for CNS (ICCC III + Xa) to 100% for leukaemia (ICCC I). For epidemiologic purposes we group CNS tumours into broad histologic categories. For the 3 year period of 2000-02, there were 591 CNS tumours (18% of all malignancies). Embryonal tumours 29.2%, pylocitic astrocitoma 10.6%, low grade glioma 11.1%, high grade glioma 8.7%, ependymoma 11.3%, germinal cell 4%, others 24.7%. Incidence of medulloblastoma (F/M 53/84), ependymomas (F/M 24/43) and germ cell tumors (F/M 9/15) in males is higher than in females. For other types there is little difference (F/M 175/188). In younger than 10 years localization was brain stem 19.8%, cerebrum 17.8% and cerebellum 62.3%. In older than 10 years localization was brain stem 21.3% , cerebrum 23.3% and cerebellum 55.3%. Although survival differs by histology behaviour, size and location, the survival for all cases was 64%. No differences in survival by sex (F 63% vs. M 64%); age was an important factor (older than 3 years 66% vs. younger than 3 years 57%). This data while not yet rip are very similar to other published registries.

EPI 14: CEREBRAL TUMORS IN CHILDREN LESS THAN ONE YEAR OLD

Concezio Di Rocco, Luca Massimi, Massimo Caldarelli, Giampiero Tamburrini, Aldo Iannelli

Pediatric Neurosurgery Unit, Catholic University Medical School, Rome, Italy

Intracranial tumors in infants less than one year old account for about 10% of all intracranial tumors occurring in the pediatric population. However, they constitute a still obscure clinical entity in terms of management and late outcome. Indeed, most of the series reported in literature concern either relatively small series or large series, which, however, group patients from various Institutions. Furthermore, the follow up period is limited to a few years.

The present paper concerns a population of 105 infants, treated at a single Institution in the period 1980-2002, and subdivided in two cohorts, the first (1980-2002) consisting of 51 patients and the second (1993-2002) 54 patients. Surgical treatment was performed in 98 cases with 42.8% of total, 11.2% of subtotal and 46% of partial removal of the tumor. The surgical excision was often carried out in two or more steps to minimize mortality and morbidity. Thirty-seven children received adjuvant chemotherapy but radiotherapy was administered to 12 children when at least 3-4 years old. Mortality during the immediate post-operative period (within one month from the operation) was 2%; surgical complications occur in 15 patients; late mortality (until 5 years from the operation) was 39.8%.

The comparison between the two cohorts shows almost a similar outcome. The long-term survivors (mean follow up = 16.2 years) represent the 43.1% of the infants of the first cohort and most of them are in excellent/good clinical conditions (63.5%). The second cohort presents the 64.8% of survivors after a mean follow up period of 5.8 years with the 74.3% of them in the excellent/good result class. The higher overall mortality was found among the PNETs (66.6%) and astrocytomas (47.6%), especially if located in the optic-hypothalamic region, which also represented the two most common oncotypes of the entire series. Lower mortality was observed among papillomas (37.5%), teratomas (28.5%), ependimomas (16.6%), meningiomas (0%), gangliogliomas (0%).

EPI 15: THE PRE-DIAGNOSTIC PROBLEMS IN CHILDREN WITH PRIMARY BRAIN TUMORS

Monika Drogosiewicz, Marta Perek-Polnik, Bożenna Dembowska-Bagińska, Danuta Perek, Iwona Filipek

Department of Oncology, the Children’s Memorial Health Institute, Warsaw, Poland

Aim of the study was to assess the pre diagnostic period and diagnostic difficulties in patients with primary brain tumors treated in one institution.

Material and Method: pre-diagnostic period of 172 pts (101 boys, 71 girls, aged 3 months –17 years 3 months, median 8 years 3 months) treated in oncology department from 01.1997-12.2000 was analyzed based on patients carts. Analysis of tumor location and related symptoms and symptoms interval was performed in correlation with mean diagnostic period and performed diagnostic procedures and involved specialists.

Results: In the whole group 50% of pts was diagnosed within 1 month, 25 % up to 3 months, 16% - 4 to 12 months, 9% more than 12 months. According to tumor location the shortest pre-diagnostic period was obviously in the brain stem tumors group (mean 2,08 months) while the longest in the midline tumors (mean 9 months) and hemispheral tumors (mean 7 months). The cause of diagnostic delay in this group was epilepsy treatment without MR check-up. In 47% of cases patient was diagnosed by first specialist (mainly neurologists and he was refered to. Only 28% of general pediatritians who saw the patients ordered CT/MR and diagnosed the tumor. The most common misdiagnosis was gastric problems and was most heavily diagnosed including invasive procedures.

Comments: The pre-diagnostic period in children with primary brain tumors is too long, so more education is needed on the basic level of health care about epidemiology of brain tumors and sign and symptoms of the disease.

Supported by grant nr C 028/P05/2002

EPI 16: OPTIMIZING CARE FOR BRAIN TUMOR PATIENTS

Claudia Epelman, Sidnei Epelman, Alejandro Arancibia.

TUCCA – Associação para crianças e adolescentes com tumor cerebral and Santa Marcelina Hospital. Av. Nove de Julho, 4275 - São Paulo - Brasil

Malignant brain cancer is a devastating illness. Because of the brain injury caused by the tumor itself and subsequent treatment (surgery, radiation and chemotherapy), most brain tumor patients develop neurological, emotional and intellectual difficulties that compromise their ability to live independently, to study and to work.

In Brazil, 1400 new brain tumor patients are diagnosed per year, mainly between 4 and 9 years. Unfortunately, a high incidence of late diagnosis and lack of facilities for adequate approach in public hospitals are still the reality of the country.

In order to improve cure rates and decrease sequelae of those patients, the Brain Tumor Association for Children and Adolescents - TUCCA, a charitable organization was founded in 1998. The association is dedicated to improving the treatment, quality of life and the long-term outlook for young patients with brain and spinal cord tumors through research, multidisciplinary support, education and advocacy for families and survivors.

TUCCA has joined in a collaborative effort with different institutions in Brazil to fund support programs and quality of life improvements for children and their families that go beyond the budgetary scope of the institutions. The association has already supported 120 patients, distributes a free Guide for parents of children with brain tumor all over the country, co-sponsors educational seminars, conferences and provides online information through www.tucca.org.br .

Last year, TUCCA also developed strategies to decrease late diagnosis and improve prognosis in patients with retinoblastoma. A international campaign to call attention of leucokoria to pediatricians, ophthalmologists and population in general was developed together with International Network for Cancer and Treatment Research.

The Brain Tumor Association raises funds through donations and different events like annual gala dinner and various recitals and other performances.

EPI 17: PRESENTING FEATURES OF BRAIN TUMOURS

Rebecca Ferris, Colin Kennedy, Ashok Nathwani

Department of Paediatric Neurology, University of Southampton, U.K.

Address for correspondence: Dr R Ferris, Consultant Paediatrician, Royal Hampshire County Hospital, Romsey Road, Winchester, Hants, SO22 5DG, U.K.

Objectives: Analysis of patterns of presenting symptoms and signs in children with brain tumour.

Methods: Retrospective review of case notes of 200 consecutive children presenting over 14 years at a tertiary referral centre in the U.K.

Results: The most frequent symptoms were headache (56%), vomiting (51%), behavioural and/or educational problems (44 %), unsteadiness (40%), and visual problems (38%). 74 of the 112 patients with headache had it as their first and 14 as their only symptom. Headache was more than 4 months in duration in 36 % including 57 % of those in whom it was the only symptom. A diurnal pattern of headache suggestive of raised intra-cranial pressure was present in two thirds. Headache was commonly associated with vomiting (77%), visual difficulties (57%), unsteadiness (45%), behaviour change (32%) and disturbed sleep (26%). The median duration of symptoms was 3 weeks for visual problems and unsteadiness, 5 weeks for vomiting, and 2 months for headaches, educational and behavioural problems.

On examination, cranial nerve abnormalities (49%), cerebellar signs (48%), papilloedema (38%), motor abnormalities (27%) were the commonest findings. 12 % had no neurological signs at presentation including 8.5% with new onset seizures (1 simple partial, 13 complex partial and 3 generalised).

Conclusions: In children with a brain tumour, headache has a recognisable pattern. New onset behavioural/educational problems or unsteadiness are the next commonest presenting symptom after headache and vomiting. Cranial nerve signs, cerebellar signs and papilloedema are commoner than upper motor neurone signs. Seizures will usually not be associated with abnormal signs. Recognition of this could expedite the diagnosis of brain tumours in childhood.

EPI 18: LENNOX GASTAUT SYNDROME CAUSED BY A PECULIAR CYST: A CASE REPORT OF AN UNEXPECTED TUMOUR

Johanna M. Fock, Eelco W. Hoving, Annet Kingma, Jan A. Leeuw

Pediatric Neuro-oncology Workgroup, Departments of Neurology, Neurosurgery and Pediatric Oncology. University Hospital Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands

Lennox Gastaut syndrome is one of the malignant child epilepsy syndromes with tonic, atonic, myoclonic seizures and atypical absences. The pathology of this epilepsy syndrome is not yet solved, although many cases show diffuse brain lesions. Cases resulting from brain tumours are rare.

A right-handed boy, aged two, was referred to our hospital with atonic seizures in clusters. He used valproicacid(VPA) resulting in a good control of the attacks. After a flu-like period the attacks increased and changed into atonic, tonic and generalized tonic clonic seizures, and atypical absences of 30 seconds. Electroencephalogram confirmed the diagnosis Lennox Gastaut syndrome. Vigabatrin was started with good effect. MRI of the cerebrum was performed, showing an arachnoidal cyst in the left fissura Sylvii. Because of the unsure effect of surgery on amelioration of the epilepsy treatment, a wait and see policy was chosen. 18 Months after the initial seizures another period of severe attacks evolved. Craniotomy for marsupialisation of the cyst was the treatment of choice now. A solid tumour appeared peroperatively and a gross total resection was accomplished. There were no postoperative complications. Histologically the tumour was a low grade glioma. No adjuvant therapy with radiation or chemotherapy was given. The boy recovered well and didn’t suffer any more from epileptic seizures. His postoperatively neuropsychological assesment showed a developmental delay for language and visualmotor integration apart from a severe attention-deficit disorder. The parents had never noticed delay or change in mental development; it was therefore assumed that both tumour, Lennox Gastaut epilepsy and a genetic factor contributed to the cognitive dysfunctioning. 4 Years postoperatively the boy is doiing well with significant mental improvement (IQ rise from 50-94) but he still needs special education. Neither the epilepsy syndrome nor the tumour recurred.

EPI 19: NEUROCUTANEOUS MELANOSIS (NCM) – A CHALLENGE FOR

THE PEDIATRIC NEUROONCOLOGIST

Michael C. Frühwald, Kathy Keyvani, Hans-Jörg Schulze, Otfried Debus, Heribert Jürgens, Ronald Sträter

University Children's Hospital Muenster, Department of Pediatric Hematology and Oncology; Albert-Schweitzer-Straße 33; 48149 Münster / Germany fruhwald@uni-muenster.de

Tumors of the spinal cord represent between 1 and 6% of all CNS

malignancies. Clinical symptoms comprise radiating or localized nerve root pain accompanied by sensory and motor dysfunction. As these neoplasms are rarely amenable to neurosurgical resection or even biopsy, they represent a special diagnostic and therapeutic challenge for the multidisciplinary team.

Case Report: Our index case is a 11 month old boy, who presented at birth with multiple nevus cell nevi on the neck, face and extremities. At 5 months the boy developed signs of raised intracranial pressure with increased head circumference and irritability. After diagnosis of an internal hydrocephalus by ultrasound and CT a VP-shunt was placed. At 11 months the patient presented symptoms of incomplete paraplegia. MR-imaging revealed an enhancing mass extending from the medulla oblongata to the cervical spine. Analysis of CSF obtained from the VP-shunt was positive for nests of tumor cells stained by the antimelanosomal antibody HMB-45. The diagnosis of a spinal leptomeningeal melanoma was made. The combination of nevus cell nevi (> 3) associated with melanoma of the spine is by definition diagnostic of neurocutaneous melanoma (NCM). Therapy with dexamethasone, temozolomide and thalidomide brought initial stabilization, but could not stop the disease process. The patient succumbed to the neoplasia at 15 months of age.

Neurocutaneous melanosis is a rare congenital phacomatosis. Melanomas of the leptomeninges and spinal cord occuring in the course of this syndrome pose a great therapeutic challenge. The outcome is almost inevitably fatal. Herein we discuss current knowledge of the tumor biology and potential therapeutic or at least palliative approaches.

Supported by the Karl Bröcker Stiftung, Geseke/ Germany

EPI 20: PREDICTORS OF TUMOR PROGRESSION IN PEDIATRIC GANGLOGLIOMAS

Lynn Gargan, Bradley E. Weprin, Linda R. Margraf, Daniel C. Bowers

The Neuro-Oncology Program, Children’s Medical Center of Dallas and the University of Texas Southwestern Medical Center, Dallas, TX USA.

Few reports describe the outcome and prognostic factors for children with gangliogliomas. The purpose of this study is to identify prognostic factors for tumor progression for children with newly diagnosed ganglioglioma. The medical records of consecutive children < 18 years old with a low-grade ganglioglioma were examined.

25 children with ganglioglioma were identified (mean ± SD age, 7.7 years ± 4.9 years; range: 1.3 to 17.6 years). There were 17 (68%) males. The mean duration of follow-up was 5.7 years ± 4 years (median: 6.4 years; range: 0.3 – 13.9 years). Tumor locations included the cerebral cortex (n = 16), brainstem (n = 4), cerebellum (n = 3), thalamus (n = 1) and suprasellar region (n = 1). 22 children were treated with surgery alone (complete resection = 12, subtotal resection = 10) and 3 were treated with subtotal surgical resection and radiation therapy. Kaplan-Meier 5 year PFS was 91 %. Three tumors located in the brainstem progressed at 0.25, 1.2, and 10 years following diagnosis (Brain Stem vs. All other sites, p-value = 0.011). One of these tumors underwent malignant transformation to an anaplastic ganglioglioma and the child died 10 years following initial diagnosis.

The progression-free survival and overall survival of children with ganglioglioma are very good. Tumors located in the brainstem are more likely to progress than in other locations. Malignant transformation is an uncommon event for children with ganglioglioma.

EPI 21: DISSEMINATED JUVENILE PILOCYTIC ASTROCYTOMA IN CHILDHOOD

J. Russell Geyer, Ji Hye Kim, Richard G. Ellenbogen and Dennis W.W. Shaw

Children’s Hospital and Regional Medical Center, Seattle, WA USA

Purpose: To evaluate the outcome of the patients with disseminated pilocytic astrocytoma compared to non-disseminated disease.

Methods: We identified 12 patients with disseminated pilocytic astrocytoma from our Tumor Registry over a 21 years period and reviewed medical records and neuroimaging to determine tumor location, pattern of dissemination, clinical characteristics, treatment, and outcome. 24 controls without dissemination, matched for age, histology, and primary location were use for case/control comparison using Kaplan-Meier survival analysis.

Results: 12 (12%) out of 99 cases with pilocytic astrocytoma were identified with CSF dissemination. Primary tumor sites: hypothalamus/chiasm [8], cerebellum [3], medulla/vermis [1], and pineal region [1] (2 sites in one patient). Two cerebellar tumors were gross totally resected, subtotal/partial resection (8) or biopsy (3) in the remaining. 10 patients received postop chemotherapy and/or irradiation. Dissemination was present at initial diagnosis in two and from 2 to 99 months after diagnosis in 10. Diagnosis was by MRI [12] and histology [7]. 9 patients had tumor or treatment-related symptoms and 3 were asymptomatic at time of metastatic detection. 8 patients were treated with single or combinations of resection, irradiation, and chemotherapy for dissemination. On follow-up, 11 of 18 cranial and spinal metastatic lesions improved or remained stable, minimal progression noted in 7. 4 of 12 patients died over a mean follow up of 78 months since dissemination detection, compared to 2 of 24 controls over a similar period. Mean survival time; disseminated = 161 +/-15 months [CI 130-191], control = 183 +/-11 months [CI 161-205] (p= 0.33).

Conclusion: CSF dissemination of pilocytic astrocytoma occurs most commonly in tumors of the chiasm/hypothalamic region. Though the mortality with disseminated tumors appeared greater, the difference didn’t reach statistical significance in this series. Treatment options should be considered carefully appreciating the potential indolent nature of these tumors even with dissemination.

EPI 22: CYSTIC CENTRAL NEUROCYTOMA OF THE FOURTH VENTRICLE: A CASE REPORT

Hasan Ghaffar, Shan Li, Richard J. Hicks, Luis A. Moral

Departments of Pathology and Radiology, Baystate Medical Center and Tufts University School of Medicine, Springfield, Massachusetts, U.S.A.

Central neurocytomas are neuroepithelial neoplasms occurring in young adults that usually arise in the region of the lateral ventricles and/or the third ventricle. They have also been described in atypical locations without any association with the ventricles, and rarely in the posterior fossa. We describe the radiologic, histologic and immunohistochemical features of a central neurocytoma of the fourth ventricle in a 13-year-old female. Magnetic resonance imaging showed a 3.5x2.0x1.8 cm cystic mass arising in the fourth ventricle and extending superiorly to the tectum. The mass filled the fourth ventricle completely and obstructed the aqueduct of Sylvius. Biopsy revealed a low-grade neoplasm comprised of nests and cords of isomorphous small round cells with perinuclear halos in a gliofibrillary background. Anaplastic features were absent. The neoplastic cells were immunoreactive with synaptophysin and negative with neurofilament, glial fibrillary acidic protein, epithelial membrane antigen and cytokeratin. The Ki-67 labeling index was less than 1%. Since central neurocytomas occur rarely in the fourth ventricle and can display pronounced cystic changes, they should be considered in the differential diagnosis of cystic lesions within the posterior fossa.

EPI 23: INTRACRANIAL CHILDHOOD EPENDYNOMAS: AN ANALYSIS OF PROGNOSTIC PATTERNS OF FAILURE IN THE GROUP OF 154 CONSECUTIVE PATIENTS.

Wieslawa Grajkowska¹, Slawomir Barszcz², Marcin Roszkowski², Marta Perek³, Monika Drogosiewicz³, Danuta Perek³, Elzbieta Jurkiewicz⁴, Pawel Daszkiewicz².
¹ Pathology Departament, Children’s Memorial Health Institute, Warsaw, Poland, ² Neurosurgery Departament, Children’s Memorial Health Institute, Warsaw, Poland, ³ Oncology Department, Children’s Memorial Health Institute, Warsaw, Poland, ⁴ Radiology Department, Children’s Memorial Health Institute, Warsaw, Poland
Pathology Department The Children’s Memorial Health Institute , Aleja Dzieci Polskich 20, 04-730 Warsaw, Poland

From 1981 to 2003, 154 children were treated for intracranial ependynoma at Children’s Memorial Health Institute, Warsaw, Poland.

In this study, all cases of ependynoma, were evaluated according to the influence of

age, of the patients, primary tumor location, histopathological type, mitotic activity,

extent of surgical resection, type of radiation therapy and contemporary chemotherapy

(from 1997) on the survival probability.

All the recurrences were evaluated according to secondary tumor location and a spinal relapse.

EPI 24: Registry for Central Nervous System (CNS) Atypical Teratoid Tumor / Rhabdoid Tumor (AT/RT): Treatment and Outcome in 42 Cases

Joanne M. Hilden, Sharon Meerbaum, Peter Burger, Jonathan Finlay, Anna Janss, Bernd W. Scheithauer, Andrew W. Walter, Lucy B. Rorke, Jaclyn A Biegel

Department of Pediatric Hematology/Oncology, The Children’s Hospital, The Cleveland Clinic, Cleveland, OH USA

The registry was established to collect treatment and outcome information for children with CNS AT/RT, a very rare and aggressive childhood tumor. We report data for 42 registry patients. There were 28 males and 14 females, ranging in age from 1.5 to 118 months (median 24 months) at the time of diagnosis. Twenty (48%) children underwent gross total resection (GTR); twenty-two had a subtotal resection or biopsies. All children received chemotherapy at diagnosis. Thirteen (31%) children received primary radiation therapy (XRT). Sixteen (38%) children received intrathecal (IT) chemotherapy. High dose chemotherapy and peripheral blood stem cell or marrow rescue (SCR) was given to 13 (31%) children. One child died of toxicity (fungal pneumonia).

Twenty-six children (62%) had progression (15) or relapse (11); median time to progression was 12 months (range 3-62 months). Among the 20 children with primary GTR, 10 (50%) are free of disease 9.5-76 months post diagnosis; 10 are dead of disease with a median survival of 15 months (3-76 months). Thirteen children received XRT as initial therapy with a median survival of 48 months (7-96 months post diagnosis), five died of disease 7-62 months, and 8 show no evidence of disease 19-96 months. Children treated with IT had a recurrence rate of 9/13 (69%) with a median survival of 23 months (10-90 months). Of the 13 children treated initially with SCR rescue, 6 (46%) recurred at 10-22 months from diagnosis, 1 died of toxicity, and 6 remain disease-free 9.5 to 90+ months from diagnosis. Fourteen patients (33%) show no evidence of disease (NED) (median survival 42 months; range 9.5-96 months). Median age of the NED children is 30 months (range 5-78 months). Ten (72%) underwent a GTR, 7 (50%) received XRT, 6 (43%) received IT, and 4 (28%) received SCR. Table shows NED patients’ treatment:

Patient #	Age (mon)	GTR	XRT	IT	SCR	NED Survival (mon)
6	29	v	v	v		53
9	28	v				76
13	30	v		v		44
16	40		v	v	v	90
22	78	v	v			72
23	19	v	v	v	v	48
28	48		v			96
29	15	v	v	v		60
31	6	v			v	24
32	22					38
44	44				v	21.5
50	47	v				11
57	49	v				9.5
58	5	v	v	v		21

Aggressive resection, radiation therapy, and intensive chemotherapy for this rare, often fatal CNS tumor is recommended; stem cell rescue and intrathecal chemotherapy should also be considered. Physicians should continue patient enrollment onto the CNS AT/RT registry. Prospective multi-institutional clinical trials should be designed specifically for CNS AT/RT.

EPI 25: Reducing the risks and increasing the benefits; an integrated clinical pathway for children with CNS tumours

Monica Hopkins, Conor Malluci, Nicki Thorp, Barry Pizer, Heather McDowell

Royal Liverpool Children’s NHS Trust, Eaton Rd Liverpool.L12 2AP UK.

Complex interdisciplinary collaboration is becoming the central tenet of effective high quality care of children with CNS tumours, often across multiple institutions. Meeting the varied needs of these children throughout their cancer journey, is challenging. These teams must be co-ordinated, effectively communicate and be pro-active in their planning of care, always seeking to ensure a sound evidence base.

The neuro-oncology team in Liverpool set about constructing a robust framework to facilitate these goals, whilst developing a parallel audit facility to generate data for effective evidenced based care. An integrated care pathway (oncology, surgery and

radiotherapy) for children with CNS tumours was finally launched in September 2004 after extensive consultative work and a pilot strategy. The pathway consists of three sections for the three modalities of therapy plus outpatient documentation for long term follow up and surveillance. It commences at referral, through treatment and follow up thereafter, with detailed guidance on the strategy for care and space for the documentation of variance as well as audit questions which generate evidence for and against currently held standards of care.

Ten patients have been commenced on this pathway. All of the children have so far completed the surgical section, diagnosis and initial care. Preliminary analysis has been undertaken to evaluate the pathway and standard surgical care thus far. Themes for analysis were; referral process and timings, presentation status and action, diagnostic imaging efficacy in relation to surgical findings, operative morbidity and rehabilitation progression. The surgical pathway can now be modified to remove redundant data, re phrase misinterpreted questions and adapt care for the variances stated.

EPI 26: UNCOMMON PRESENTATION OF CRANIOPHARYNGIOMA WITH ANEMIA AND SCHOOL FAILURE IN AN ADOLESCENT

Khalid Kamal, Bulent Ozgonenel, Sureyya Savasan, Kanta Bhambhani 
Children's Hospital of Michigan, Division of Hematology/Oncology, Wayne State University, Detroit, MI, USA
 

A thirteen-year-old girl with a history of decline in academic performance in the last six months was referred for the workup of mild normocytic anemia. Physical examination and review of systems were remarkable for delayed puberty, constipation and weight gain despite decreased appetite. Initial investigations led to the diagnosis of panhypopituitarism and a suprasellar mass, 2.5 cm in diameter. The patient underwent total resection of the tumor and histopathologic examination revealed craniopharyngioma. At presentation, the patient did not have any headache, focal neurologic findings, visual field defects, behavioral symptoms or any clinical or laboratory evidence of diabetes insipidus. Craniopharyngioma can present in children with various symptoms related to endocrine, hypothalamic, visual, and neurologic dysfunction. Although readily explained with an endocrinopathic basis, school failure and anemia in an adolescent are uncommon forms of presentation of craniopharyngioma and the diagnosis may be elusive, requiring careful evaluation of the patient.

EPI 27: Creation of a Website for Families of Children with Brain Tumours (BT)

Phyllis McGee, Eric Bouffet, Darren Hargrave, and Ross Hetherington

Pediatric Brain Tumour Program (PBTP) and Family Health Media Group (FHMG), The Hospital for Sick Children and University of Toronto, Toronto, ON Canada

Consumers of online health information are concerned with quality and reliability. In the case of families with a child diagnosed with a paediatric brain tumour (PBT), these concerns are justified. Although over 80% of these parents turn to the Internet as source of information, a review of >4000 websites pertaining to PBT demonstrated that many sites are of poor quality, inconsistent, contain errors, are of poor readability, and not well designed from the user’s perspective. Thus, there is a significant need for high quality websites with comprehensive, reliable, and accessible information to serve families of children with BT.

To meet this need, FHMG and the PBTP have collaborated to create a website to serve families of children with BT. The FHMG includes pediatric specialists, medical journalists, a medical illustrator, a Flash animator, a creative director, a user experience consultant, and a team of web developers. Interdisciplinary members of the PBTP provided expert content. Families were consulted both formally and informally throughout the development process.

The website will launch in early 2004. There are >200 pages of material on PBT. The Information Architecture follows the natural history of the disease, providing an overview of PBT and specific information on the most common tumour types (including medulloblastoma, ependymoma, brainstem glioma, craniopharyngioma, low-grade glioma), treatment, care at home, and late effects. The material is comprehensive, providing medical information, but also addressing safety, nutrition, psychosocial, educational, and quality of life issues. The website presents current research in an accessible manner. Interactive animations provide parents and children knowledge regarding neuroanatomy, radiation, and MRI. The content scores high using the DISCERN tool. Readability scores using the Flesh-reading system are >50 throughout the document.

In the future, secure e-mail and discussion fora will be implemented to facilitate communication between patients/families, community professionals, and members of the PBTP.

EPI 28: END-OF-LIFE CARE OF CHILDREN WITH CENTRAL NERVOUS SYSTEM (CNS) TUMOURS

Phyllis McGee, Eric Bouffet, Ruchi Sinah, Maggie Breen and Darren Hargrave.

Neuro-Oncology Programs, The Hospital for Sick Children (HSC), Toronto, Canada and The Royal Marsden Hospital (RMH), London, UK.

The provision of end-of-life care is a crucial element of any paediatric neuro-oncology teams practice but the clinical course and management interventions have not been well reported.

Purpose: The purpose of this study was to retrospectively examine the end-of-life care provided to children dying of a brain tumour in two different oncology programs in two different countries to discern similarities and differences.

Methodology: A retrospective analysis was performed to describe each child’s clinical course, and the care provided by members of the oncology team in terms of health care professionals involved, and symptom assessment and management.

Results: There were 102 (RMH 52, HSC 50) cases between 01-01-97(RMH)/01-01-01 (HSC) and 31-12-03. The diagnoses were diffuse pontine glioma 28.4%, PNET/ medulloblastoma 23.6%, high-grade glioma 19.6%, ependymoma 10.8% and other tumours 17.6%. The mean age at diagnosis 6.9 (range 0.1 to17) years, mean age at death 8.1 (0.2 to 18.9) years. The mean times from diagnosis and tumour progression to death; were 15.1 (0.1 to 100) and 4.1 (0.1 to 19) months respectively. Overall 53% of children died at home, whilst 44% died in hospital and 3% in a hospice. The commonest symptoms managed included; pain 60.5%, immobility 57%, seizures 46%, swallow difficulties 36% and impaired speech 32%. The commonest interventions were; dexamethasone 70%, opiate analgesia 55%, palliative chemotherapy 54%, anticonvulsants 48% and nasogastric tube 28%. The main differences between the two centres included: place of death (60% RMH & 46% HSC), dexamethasone usage (55% RMH & 75% HSC) and types of health care professionals involved.

Discussion: The end-of-life care of children with a CNS tumour requires close collaboration between the family, the hospital and community based interdisciplinary teams. This study elucidates the care issues related to end-of-life care for children with CNS tumours, and provides direction for future palliative care strategies.

EPI 29: FIRST POLISH CENTRAL NERVOUS SYSTEM (CNS) TUMOR GROUP (PCNST G)

INTEGRATED CARE FOR CHILDREN WITH CNS NEOPLASMS IN POLAND

D.Perek¹, B.Dembowska¹, A Balcerska², W Balwierz³, A.Chybicka⁴,J.Kowalczyk⁵, M.Krawczuk – Rybak⁶, W.Madziara⁷, J.Peregud – Pogorzelski⁸,J.Wachowiak⁹, M. Wysocki¹⁰, E.Zielińska¹¹

¹Department of Oncology Children’s Memorial Health Institute Warsaw, Department of Hematology and Oncology , Medical Academy ²Gdańsk, ³Kraków, ⁴Wrocław,⁵Lublin,⁶Białystok,⁷Katowice,⁸Szczecin, ⁹Poznań,¹⁰Bydgoszcz, ¹¹Łódź

Background: In Poland children with CNS tumors have in the recent past been treated mainly with surgery and radiotherapy. They have been operated of their tumors in neither adults or children neurosurgery departments followed by radiotherapy and there was no unified multidisciplinary approach to their diseases and there was no common chemotherapy protocols.

There was also lack of cooperation and integration among specialists in the field of neuroimaging, pathology, surgery, radiotherapy, chemotherapy.

Aim: To change this status and improve diagnosis and treatment results of CNS tumors in children the PCNSTG was created.

PCNSSG focused on organization of child service across the country involving several specialties in 11 centers in Poland. In each region pediatric oncologist is a coordinator of the care team. Each specialty has a national coordinator and the whole program is coordinated by the team from The Childrens Memorial Health Institute.

Based on our own experience and utilizing data from literature analysis a project on diagnosis and combined treatment of CNS tumors in children was launched.

The project includes sequence of multidisciplinary interventions: diagnostic and therapeutic for each tumor type. For MB/PNET, HGG and children under 3 years of age own protocol was introduced and for LGG,GCT - SIOP protocol.

A computer database was created and each new patient is registered in the study. Information on treatment methods applied, follow up, late effects are collected. in the data base.

By introducing this program we want to target important issues as: interdisciplinary communication and cooperation, introduction of evidence based medicine. This will result in ruling out anecdotal practices and improvement of treatment results.

A pathway for children with CNS tumors should be developed in terms of diagnostic, therapeutic procedures as well as psychosocial support and monitoring of late effects.

Organization schema and preliminary results will be presented.

Supported by grant nr C 028/P05/2002

EPI 30: incidence, TIMING, and OUTCOME of Central nervous system metastasis in pleuropulmonary blastoma

John Priest, Gretchen Williams

The Pleuropulmonary Blastoma Registry, Children’s Hospitals and Clinics, 345 N. Smith Ave., St. Paul, MN 55102

PPB is a rare dysembryonic childhood tumor of lung and pleura. Three pathologic Types are: I (cystic), II (cystic & solid), III (solid). The median age at diagnosis in Type I disease is 10 months; II : 34 months; III: 44 months.

Incidence: cerebral parenchymal metastases (CM) occur in Types II and III (II-III) PPB: in most complete Registry series, CM occurred in 11/43 cases (26%). In larger Registry series with less follow-up, CM occurred in 17/64 cases (27%). Literature cases with variable follow-up: CM in 10/122 cases (8%).

Interval: in Registry and literature cases combined, the interval from PPB diagnosis to CM is 1-60 months, median 11 (n = 24 [literature data incomplete]). In 22 of these, CM developed within 32 months. Registry and literature patients with CM range in age at PPB diagnosis from 24-141 months, median 36 (n = 26), consistent with overall II-III cases. Metastases were not observed in Type I disease.

Outcome: 18/24 CM patients died. Of 6 alive: 3 are 12, 24, and 80 months after CM (therapy: surgery, radiation, +/- chemotherapy); 3 are early in therapy after CM. Brain was only site of recurrent PPB in 8/17 Registry patients with CM; the 6 surviving patients were among these 8.

Cord compression was noted in 3 cases; mechanism is epidural compression from disease in epidural space or vertebrae. CSF cytology was not positive in any Registry or literature case. Autopsy found leptomeningeal disease in 2 Registry patients with very advanced disease.

These data suggest: 1. CM not rare in II-III PPB; 2. surveillance brain MRIs every 3 months in II-III PPB from PPB diagnosis to 36 months after diag