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Abstracts from the Thirteenth International Symposium on Pediatric Neuro-Oncology

	ATRT

Top ATRT BIOLOGY BRAINSTEM GLIOMA CHOROID PLEXUS TUMORS CASE REPORTS CRANIOPHARYNGIOMA EPENDYMOMA EPIDEMIOLOGY GERM CELL TUMOR HIGH-DOSE CHEMOTHERAPY HIGH-GRADE GLIOMA NEURO-IMAGING INFANT TUMORS LATE EFFECTS LEPTOMENINGEAL AND DISSEMINATED... LOW-GRADE GLIOMA NURSING AND MULTIDISCIPLINARY... NEUROFIBROMATOSIS AND ASSOCIATED... NEUROPATHOLOGY NEUROSURGERY OUTCOME STUDIES PNET/MEDULLOBLASTOMA QUALITY OF LIFE RADIATION ONCOLOGY RARE TUMORS THERAPEUTIC AND NEW DRUG...

Diane Birks,¹ Andrew Donson,¹ Sean Mcnatt,² Nicholas Foreman,² and Michael Handler²; ¹University of Colorado Health Sciences Center, Aurora, CO, USA; ²The Children's Hospital, Aurora, CO, USA.

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive malignant CNS tumors of early childhood. Prognosis is extremely poor with median survival times ranging from 6 to 17 months. It has been recognized since the late 1990s that both AT/RTs and malignant rhabdoid tumors of the kidneys and other soft tissues are characterized by a loss of INI1 (aka SMARCB1, BAF47, hSNF5), a component of the SWI/SNF protein complex that regulates transcriptional activity through chromatin remodeling. In fact, loss of INI1 protein, as determined through immunohistochemistry, has become the de facto standard for diagnosis of AT/RTs. Any pediatric brain tumor showing loss of INI1 will generally be diagnosed as an AT/RT, regardless of histological characteristics. However, loss of INI1 has been found in other tumor types, including schwannomas and epithelioid sarcomas. Also, INI1 is not absent in approximately 15% of pediatric brain tumors that show diagnostic histological features for AT/RTs. Thus, INI1 may not be wholly specific to malignant rhabdoid tumors or AT/RTs. To identify potential diagnostic markers specific to AT/RT, 110 pediatric and adult brain tumor samples and 66 normal brain samples were analyzed for gene expression using Affymetrix U133Plus2 GeneChip microarrays. These arrays measure the expression of >54,000 probe sets, including all known human genes. The tumor samples analyzed included eight AT/RTs, as well as glioblastoma, medulloblastoma, large-cell medulloblastoma, ependymoma, pilocytic astrocytoma, rhabdomyosarcoma, meningioma, and radiation-induced tumors. AT/RT expression was compared individually to each other tumor type as well as to normal samples; p-values were adjusted using a false discovery rate of 0.05 which took into account all tests both within and across all groups. Fifty-nine unique genes were found to be overexpressed in the AT/RT samples when compared to all other groups (including normal); five genes were underexpressed. While generally INI1 was expressed at lower levels in AT/RTs than in all other samples, this did not achieve statistical significance for two groups (normal and meningioma). This result is not surprising, because microarrays measure mRNA levels rather than protein levels. Of the 59 genes statistically overexpressed in AT/RTs, 58 showed overlap in their range of expression with other tumor types. However, one gene was found that was consistently expressed in AT/RTs but showed almost no overlap in its range of expression compared to the tumor or normal samples: claudin 6 (CLDN6), a key component of tight junctions. There was an average difference of >50-fold between CLDN6 levels in AT/RTs versus all other tumors and similarly versus normal samples. Compared to medulloblastomas, the most difficult tumor type to discriminate from AT/RTs, CLDN6 was expressed >53-fold higher in AT/RTs with no overlap at all in expression levels. Analysis of protein abundance using Western blotting confirmed the microarray results. Thus the combination of INI1 as a negative marker and CLDN6 as a positive marker may be useful in defining AT/RTs for diagnostic purposes.

ATRT 2. CLINICAL FEATURES OF ATYPICAL TERATOID/RHABDOID TUMOR IN JAPAN

Hidehiro Oka¹ and Kiyotaka Fujii¹; ¹Neurosurgery, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan.

Introduction: The clinical features of atypical teratoid/rhabdoid tumors (AT/RTs) in Japan and the importance of the differential diagnosis from PNET/medulloblastoma and germ cell tumor are described.

Methods: Forty-one patients with AT/RTs in Japan were analyzed for clinicopathological features.

Results: The 41 patients with AT/RTs (23 male and 18 female) had ages ranging from 1 month to 37 years (mean, 2.9 years). They had tendency to show increased intracranial pressure by obstructive hydrocephalus and/or tumor volume. Tumors were located in the posterior fossa (63%), brain hemispheres (22%), spinal (12%), and pineal region (3%). Interestingly, AT/RTs of younger patients (<2 years) tended to be located in the posterior fossa or spinal region. Leptomeningeal dissemination was present in >50% of AT/RT cases. Histologically, AT/RT is defined as a polymorphous neoplasm often featuring rhabdoid, PNET, epithelial, and mesenchymal components. AT/RTs usually include PNET components and occur mainly in the posterior fossa, mimicking medulloblastoma. AT/RT is characterized by the cytogenetic finding of monosomy 22 rather than i(17q). The tumor is similarly mistaken for PNET because of supratentorial immunophenotypic diversity, particularly features indicative of epithelial and mesenchymal differentiation. Nonetheless, the remarkable spectrum of tissues typical for teratoma are absent in AT/RT. The prognosis of this tumor is far less favorable than that of PNET/medulloblastoma or germ cell tumor.

Conclusions: This study describes the clinicopathological features of 41 AT/RTs in Japan, and we emphasize the necessity for distinguishing this unique tumor from other pediatric CNS neoplasms.

ATRT 3. CNS ATYPICAL TERATOID RHABDOID TUMOR (ATRT) IN CHILDREN LESS THAN 36 MONTHS: A CANADIAN PEDIATRIC BRAIN TUMOR CONSORTIUM (CPBTC) EXPERIENCE

Lucie Lafay-Cousin,¹ Daniel Keene,² Anne-Sophie Carret,³ Bruce Crooks,⁴ David Eisenstat,⁵ Chris Fryer,⁶ Donna Johnston,⁷ Valerie Larouche,⁸ Albert Mograbi,³ Beverly Wilson,⁹ Anthony Whitton,¹⁰ Shayna Zelcer,¹¹ and Eric Bouffet¹²; ¹University of Calgary, Calgary, AB, Canada; ²Children's Hospital of Eastern Ontario, Ottawa, ON, Canada; ³Montreal, QC, Canada; ⁴Halifax, NS, Canada; ⁵Winnipeg, MB, Canada; ⁶Vancouver, BC, Canada; ⁷Ottawa, AB, Canada; ⁸Quebec City, QC, Canada; ⁹Edmonton, AB, Canada; ¹⁰Hamilton, ON, Canada; ¹¹London, ON, Canada; ¹²Toronto, ON, Canada.

Background: CNS ATRTs are extremely rare and agressive tumors occuring primiraly in very young children. Data on this relatively recent entity remain limited to institutional experience or single registry report.

Rationale/aim of the study: To provide a population-based review on this entity to better define incidence, demographic, outcome data, and potential prognostic factors.

Methods: Subgroup analysis of patients with pathology confirmed diagnosis of ATRT found as part of a larger retrospective review of CNS tumors in children <36 months of age diagnosed between 1990 and 2005 in Canada.

Results: Data were obtained from 14 out of 16 Canadian centers. ATRT was listed as histological diagnosis in 24 of the 531 reported cases of CNS tumors (4.5%). Seventeen patients (71%) were male. Mean age at diagnosis was 12.6 months (±9.44). Tumors were infratentorial in 58.4%, supratentorial in 33.4%, and spinal in 8.3%. Ten patients (41.7%) had evidence of metastatic disease on imaging at diagnosis. Gross total resection was achieved in five patients (21%). One third of the patients did not receive postoperative therapy. Sixteen patients (66.7%) received adjuvant chemotherapy, combined with radiation in 37.5%. High-dose chemotherapy (HDC) with stem cell rescue was administered in five children (31.5%). Median time to progression was 9.1 months (0–35) and median survival time for the 16 treated patients was 14 months (10.1–12.9). One and 2-year overall survival were 50% (±10.2%) and 18.8% (±8.6%), respectively. The metastatic status, the use of radiation, or high-dose chemotherapy were not found to be significant prognostic factors. Patients who underwent a resection greater than 90% had a better survival (p = 0.006). At completion of the survey, three patients remain alive (20, 21, and 78 months from diagnosis). All underwent resection greater than 90%; one was irradiated, and two received HDC.

Conclusion: Our study confirms the poor prognosis of CNS ATRTs in infants. However, aggressive surgery may favorably alter the outcome of a subset of patients. Cooperative prospective studies are needed to define the respective role of systemic chemotherapy, intrathecal chemotherapy and radiation.

ATRT 4. DE NOVO INI-1 GENE GERMINAL MUTATION CAUSING SYNCHRONIC CONGENITAL PINEAL REGION AND KIDNEY AT/RT: CASE REPORT AND REVIEW OF THE LITERATURE

Ramon Navarro,¹ Astrid Laguna,² Noelia Pérez,³ Carmen De Torres,² Parareda Andreu,² Jaume Mora,² and Ofelia Cruz⁴; ¹Pediatric Neurosurgery, Hospital Sant Joan de Déu, University of Barcelona, Esplugues de Llobregat, Barcelona, Spain; ²Pediatric Oncology, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain; ³Pathology, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain; ⁴Pediatric Oncology, University of Barcelona, Esplugues del Llobregat-Barcelona, Spain.

Introduction: Rhabdoid tumors are rare but highly aggressive CNS malignancies. The management of these tumors remains very challenging, especially as they are usually diagnosed in early childhood. Overall prognosis is dismal, and there is no consensus on their treatment. Molecular genetic studies have led to the identification of a rhabdoid suppressor gene (INI1/hSNF5) at 22q11.2. Germline mutations in this gene predispose children to the development of atypical teratoid rhabdoid tumor (AT/RT), the so-called rhabdoid predisposition syndrome.

Case Report: We present a 6-month-old Chinese female with an initial diagnosis of kidney rhabdoid tumor that had a simultaneous asymptomatic pineal region mass. She was initially managed with complete abdominal surgery and chemotherapy (alternating courses of ifosphamide, VP-16, and carboplatin with vincristine, doxorrubicine, and ciclophosphamide). A partial response of the pineal mass to this regimen was obtained. The simultaneous occurrence in our patient of a renal rhabdoid tumor and a CNS mass was highly suggestive of constitutional mutation in the INI-1 gene which was confirmed by identification of a common C472T in exon 4 mutation in her blood cells. The sequence analysis suggested that there was loss of heterozygosity, so there was only the mutant allele left in the renal tumor. Her parents and siblings did not share the germinal mutation. The patient subsequently underwent cranial surgery with complete resection of the pineal tumor. Histology of the CNS mass confirmed the AT/RT diagnosis. After surgery, maintenance treatment with irinotecan and cisplatin, together with tamoxifen was given. In addition, the CNS tumor was locally consolidated with fractionated stereotactic radiation therapy (4.500 cGy) given to the tumor bed region at the age of 16 months. Currently she is 20 months old, in complete continuous remission and receiving maintenance treatment with tamoxifen.

Commentary: The rhabdoid predisposition syndrome offers an added challenge to the management of these patients. There is still no consensus as to the best approach for AT/RT treatment. The ability to achieve complete surgical resection seems to be a significant prognostic factor in determining overall survival, but it is not enough for cure. Radiation therapy is controversial since most patients are infants. However, the fact that long-term survivors, especially in older patients, were more likely to have had initial radiation favors the use of radiotherapy. As for chemotherapy, these patients have received a variety of regimens, mainly using platinum or alkylating agents, and even high-dose chemotherapy. Temozolomide and intrathecal therapy have been also assayed with some encouraging results. In our patient, we could demonstrate some degree of chemosensitivity to the VAC/ICE alternating regimen. New approaches are needed, and some future avenues might be offered with inmunotherapy or biologically driven therapies. Our patient is on tamoxifen chemopreventive therapy based on the fact that INI1/hSNF5 represses transcription of cyclin D1.

ATRT 5. DELAYED METHOTREXATE EXCRETION IN AN INFANT WITH ATYPICAL TERATOID RHABDOID TUMOR AND A LARGE POSTOPERATIVE INTRACRANIAL PSEUDOCYST: PHARMACOKINETIC ANALYSIS AND IMPLICATIONS FOR BRAIN TUMOR TREATMENT

Robert Sanders,¹ Paula Schaiquevich,² Frederick Boop,³ Deborah Ward,² Zoltan Patay,⁴ Amar Gajjar,¹ and Clinton Stewart²; ¹Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN, USA; ²Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN, USA; ³Department of Neurosurgery, St. Jude Children's Research Hospital, Memphis, TN, USA; ⁴Department of Radiological Sciences, St. Jude Children's Research Hospital, Memphis, TN, USA.

High-dose intravenous methotrexate (HDMTX) has activity against medulloblastoma and is included in several current clinical trials for young children with brain tumors. Experience with other tumor types has shown that sequestration of methotrexate within pathological fluid collections (e.g., pleural effusions) may lead to delayed excretion and an increased risk of systemic toxicity. The similar effect of intracranial fluid collections on methotrexate pharmacokinetic parameters has not been studied. We report the case of an infant with a right frontoparietal atypical teratoid rhabdoid tumor (ATRT) who postoperatively developed a large extracerebral fluid-filled pouch at the tumor resection site. After he experienced delayed methotrexate excretion during his first cycle of HDMTX, an Ommaya reservoir was placed into the pseudocyst, permitting matched plasma and cerebrospinal fluid MTX pharmacokinetic studies during his second course. Pharmacokinetic analysis demonstrated that systemic MTX clearance was within the expected range for his age but that the terminal half-life was prolonged, creating the potential for delayed systemic toxicity. Through careful monitoring, hydration, and leucovorin administration, significant toxicity was avoided. We conclude that patients with intracranial fluid collections are at risk for increased toxicity with HDMTX administration but demonstrate that this drug can be safely administered to such patients with vigilant clinical management.

ATRT 6. DIAGNOSTIC AND TREATMENT PITFALLS OF ATYPICAL TERATOID/RHABDOID TUMOR (AT/RT)

Ai Muroi,¹ Shingo Takano,¹ Takashi Fukushima,² Tetsuya Yamamoto,¹ Atsushi Saito,¹ and Akira Matsumura¹; ¹Department of Neurosurgery, University of Tsukuba, Tsukuba City, Ibaraki, Japan; ²Department of Pediatrics, University of Tsukuba, Tsukuba City, Ibaraki, Japan.

Purposes: The prognosis of the patients with AT/RT younger than 3 years old is very poor. We reviewed six cases of AT/RT in patients younger than 3 years old treated in Tsukuba University in order to investigate the diagnostic and treatment pitfalls.

Materials and Methods: Six cases (two male; four female) were pathologically diagnosed as AT/RT; age ranged from 6 to 36 months. Tumor location was infratentorial in two, intraventricular in two, supratentorial in one, and spinal in one. Initial symptoms were increased intracranial pressure with intracranial AT/RT and paraparesis with spine AT/RT. Dissemination was observed at initial in two cases. Total resection was achieved only in one and subtotal resection in others. After histological confirmation, chemotherapy (ifosfamide, cisplatin and etoposide) radiotherapy was initiated. Stem cells were harvested from bone marrow in four cases and high-dose chemotherapy was done in three cases with stem cell rescue.

Results: Pathologically, typical rhabdoid cells were not observed in two cases, where immunohistochemistry with EMA, SMA, vimentin, and INI-1 was definitely useful to diagnosis. Prognosis of six cases was dead in three cases (3, 9, and 14 months) and alive in three cases (4, 4, and 38 months). Long-term survivors (>38 months alive) have dissemination at initial presentation, but high-dose chemotherapy with stem cell rescue and whole brain and whole spine irradiation (3 months after the diagnosis at the age of 34 months) resulted in complete response for 20 months. In another case of a 4-month-old girl, salvage irradiation was quite useful for intracranial extension.

Conclusion: Diagnostic and therapeutic pitfalls of AT/RT were as follows: (1) Immunohistochemistry for EMA, SMA, and vimentin is important in case of scant rhabdoid cells and prominent PNET features. (2) High-dose chemotherapy could be useful in selected cases; therefore, preparing stem cells should be designed as the initial treatment. (3) cerebrospinal fluid dissemination should be avoided by intrathecal administration of chemotherapy and seamless chemotherapy. (4) Radiotherapy and salvage therapy could be planned as an initial therapy before the age of 3 years.

ATRT 7. GERMAN PEDIATRIC ATYPICAL TERATOID/RHABDOID TUMORS: INTERIM ANALYSIS OF THE ATRT-CNS PILOT STUDY AND RETROSPECTIVE ANALYSIS

Ove Peters,¹ Beatrix Heinzelmann,¹ Jörg Marienhagen,² Monika Friedrich,³ Odo-Winfried Ullrich,³ Torsten Pietsch,⁴ Kortmann Rolf-Dieter,⁵ Monika Warmuth-Metz,⁶ Brigitte Wrede,¹ Petra Turowski,¹ Gerda Demleitner,¹ and Johannes Wolff⁷; ¹Pediatric Oncology, Children's Hospital, Regensburg, Germany; ²Center of Clinical Trials, University Regensburg, Regensburg, Germany; ³Neurosurgery, University Regensburg, Regensburg, Germany; ⁴Neuropathology, University Bonn, Bonn, Germany; ⁵Radiology, University Leipzig, Germany; ⁶Radiology, University Würzburg, Germany; ⁷Houston, TX, USA.

Background: Atypical teratoid/rhabdoid tumors (ATRT) account for <1% of all CNS tumors. Usually newborns and infants are affected. The outcome is dismal (median overall survival, 6–11 months). We present a retrospective analysis of the German historic pediatric ATRT patients (n = 63) from 1988 to 2008, a meta-analysis of international ATRT-outcome data of treated children (406 case reports) from 1985 to 2008, and an interim analysis of the novel ATRT-CNS pilot study (anthracycline-based multimodality therapy).

Methods: The retrospective analysis of the German historic pediatric ATRT-patients (n = 58) from 1988 to 2002 and the meta-analysis of international ATRT-outcome data of treated children (401 case reports) from 1985 to 2002 served as the backbone for the development of the ATRT-CNS pilot study. Children were enrolled in this study after the diagnosis of ATRT was confirmed by the German Neuropathology Reference Center. After two induction chemotherapy cycles (each 3 weeks: doxorubicin 25 mg/m² 12-h i.v., d1–3; dactinomycin 45 µg/kg i.v. push, d1; cisplatin 70 mg/m² 6-h i.v., d4; vincristine [VCR] 1.5 mg/m² i.v. push, d8, 15; MTX 2 mg iventr., d1–4) conventional local 3D radiation therapy (54 Gy) with simultaneous chemotherapy (carboplatin 80 mg/m² 6 h i.v., d1–4) was given. Thereafter, reinduction chemotherapy (same as first/second cycle) was implemented, followed by consolidation chemotherapy (6 cycles/9 months: CCNU 75 mg/m², d1; cisplatin 70 mg/m² 6 h i.v., d1; VCR 1.5 mg/m² i.v. push, d1, 8, 15; MTX 2 mg iventr., d1–4).

Results: Eleven out of 30 children were excluded from the analysis (eight foreign patients, and three patients due to therapy violation). Nineteen German patients (15 male, 4 female; median age, 22 months) have been treated according to the study criteria. Tumor site: 10 hemispheric (one bifocal), 6 cerebellum, 1 pineal, 1 cerebellopontine angle, and 1 spinal. Additionally, in 32% of the children leptomeningeal dissemination/malignant pleocytosis was present. Primary surgery: one complete, five subtotal, nine partial resections, and four biopsy only. Response to the two primary doxorubicin-based cycles (induction) could be evaluated in 19 children: 1 continued complete response, 2 complete response, 13 partial response, and 3 stable disease. The event-free survival and overall survival is 79% (mean survival time, 50 months; CI, 38–63 months) compared to 27% (mean survival time, 12 months; CI, 9–15 months) of the German historic ATRT group and 28% (mean survival time, 24 months; CI, 19–29 months) of the international ATRT-patient group (meta-analysis). Main toxicity (NCIVers.2) in 19 children after the induction/reinduction chemotherapy: grade III–IV mucositis and infection in 42% and 37%, respectively. Reported neurotoxicity: one transient transverse myelits, one cerebral edema with seizures 2 weeks after local radiochemotherapy, and one postoperative death after ventriculoperitoneal shunt implantation. No encephalomyelopathy has been reported so far.

Conclusion: This anthracycline-based induction regimen shows that ATRTs are chemosensitive but has significant toxicity. The survival data are significantly improved compared to the German historic ATRT outcome data of treated children.

ATRT 8. INI1 PROTEIN EXPRESSION IN PRIMARY MALIGNANT EMBRYONAL PEDIATRIC CNS TUMORS FROM EAST DENMARK

Astrid Sehested,¹ Karsten Nysom,¹ Lars Bøgeskov,² John Hauerberg,² Henning Laursen,³ and Helle Broholm³; ¹Paediatric Oncology, Rigshospitalet, Copenhagen, Denmark; ²Neurosurgery, Rigshospitalet, Copenhagen, Denmark; ³Neuropathology, Rigshospitalet, Copenhagen, Denmark.

Atypical teratoid/rhabdoid tumors (AT/RT) of the CNS are rare, highly malignant primary CNS neoplasms seen predominantly in very young children and infants. The definition as a clinical and neuropathological entity is relatively recent, and the differentiation from PNET may be difficult. Analysis of nuclear expression of INI1 protein in tumor cells by immunohistochemistry shows negative staining (lack of expression) in nearly all AT/RT tumors. Negative staining may also be seen in a small fraction of primitive neuroectodermal tumors without rhabdoid phenotype, and when seen in such cases is associated with a poorer prognosis. There is growing consensus that such tumors should be classified and treated as AT/RT tumors and that INI1 staining should be carried out in all pediatric malignant embryonal tumors. Ninety tumor specimens from 87 children with primary malignant embryonal CNS neoplasms treated at our institution from 1971 to 2007 inclusive were examined for expression of nuclear INI1 protein by immunohistochemistry. Four tumor specimens from four patients could not be stained due to poor specimen quality (all were medulloblastoma patients, three are long-term survivors). Positive INI1 staining in tumor cells was seen in 82 specimens from 79 patients, whereas four tumor specimens from four patients demonstrated lack of nuclear INI1 protein in tumor cells. Concerning these last four patients, the first was a girl diagnosed in 1991 13 months of age with a central supratentorial PNET and treated with radical tumor resection, chemotherapy, and radiotherapy; she died of disease 7 months postdiagnosis. The second was a girl diagnosed in 1992 18 months of age with a supratentorial central tumor and histology described as malignant anaplastic tumor, possibly sarcoma; the patient was treated with tumor resection, chemotherapy, and radiotherapy but succumbed to disease 6.5 months postdiagnosis with widespread systemic metastases. The third was a boy diagnosed in 1992 at age 22 months with a supratentorial central PNET; he died postoperatively due to surgical complications. Pathological review of these three patients now shows AT/RT in all three, although the last patient did not have rhabdoid morphology. The fourth is a girl diagnosed in 2007 at age 6 with a tumor in the cerebello-pontine angle, where histopathology was compatible with AT/RT but without specific rhabdoid features. Here the INI1 analysis was part of her work-up, and she is at present undergoing intensive treatment with chemotherapy and radiotherapy and has stable disease. Of the 79 patients with tumors that showed normal expression of INI1 by immunohistochemistry, the diagnoses were medulloblastoma in 69 patients (29 patients diagnosed between 1971 and 1989, with 8 long-term survivors, and 40 patients diagnosed between 1990 and 2007, with 22 patients surviving today); PNET in six patients (three survivors); and CNS Ewing sarcoma in three patients, all alive with disease, and ependymoblastoma in one patient (DOD). In conclusion, we confirm that a negative INI1 stain is a rare finding (4 of 83 patients), and in three of four cases was associated with young age and poor prognosis despite aggressive treatment in two cases.

ATRT 9. INSIGHTS INTO THE EPIGENETICS OF ATYPICAL TERATOID RHABDOID TUMORS: THE POTENTIAL FOR TREATMENT WITH HISTONE DEACETYLASE INHIBITORS

David Ashley,¹ Andrea Muscat,² Vinod Dagar,³ Christian H. Rickert,⁴ Cw Chow,⁵ J.A. Biegel,⁶ Paul Ekert,⁷ Richard Saffery,² Jeff Craig,² Ricky Johnstone,⁸ and Elizabeth Algar⁷; ¹Royal Children's Hospital, Murdoch Children's Research Institute, Parkville, Victoria, Australia; ²Murdoch Children's Research Institute, Victoria, Australia; ³Murdoch Children's Research Institute, Victoria, Australia; ⁴Paediatric Pathology/Neuropathology, Royal Children's Hospital, Victoria, Australia; ⁵Royal Children's Hospital, Victoria, Australia; ⁶Division of Human Genetics, The Children's Hospital of Philadelphia, PA, USA; ⁷Royal Children's Hospital, Murdoch Children's Research Institute, Victoria, Australia; ⁸Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.

Background: Atypical teratoid rhabdoid tumor (ATRT) is an aggressive but rare tumor of infancy and early childhood resistant to conventional chemotherapies and radiotherapy. The majority of afflicted children succumb to their disease within a year of diagnosis. SMARCB1 (INI1) is part of an ATP dependent SWI/SNF chromatin-remodeling complex and is frequently deleted in rhabdoid tumor. We hypothesized that the oncogenic pathway in ATRT may involve epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodeling function attributable to loss of SMARCB1 and that such effects may be reversed by treatment with histone deacetylase inhibitors (HDACi).

Methods and Results: In this study we demonstrated, using an inducible expression system, that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and that it is transcriptionally activated by increased histone H3 acetylation at its promoter, thereby revealing a novel aspect of SMARCB1 function. The histone deacetylase inhibitor (HDACi) Romidepsin, also known as Depsipeptide, restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC transcription and allelic expression. Significantly, treatment with HDACi induced cell cycle arrest in the rhabdoid tumor cell lines, G401 and STM91-01. Most importantly, CDKN1C expression was absent or negligible in clinical specimens, suggesting that CDKN1C silencing is a common event in ATRT.

Conclusion: We propose that the aberrant epigenetic regulation of the transcription of key cell cycle regulators such as CDKN1C may be fundamental to ATRT biology. Romidepsin and related compounds should be further explored as therapeutic reagents for the treatment of ATRT and that measuring the reversal of epigenetic changes by such compounds may provide unique methods for monitoring disease response.

ATRT 10. MULTIFOCAL NECROTIZING LEUKOENCEPHALOPATHY: A COMPLICATION OF ATYPICAL TERATOID RHABDOID TUMOR THERAPY—PROMPT DETECTION, DIAGNOSIS, AND TREATMENT IS REQUIRED

Michael Palumbo,¹ Sean Quinlan-Davidson,² Jean-Pierre Farmer,² Jose-Luis Montes,² Jeffrey Atkinson,² Carolyn Freeman,² Steffen Albrecht,² Christine Saint-Martin,² and Anne-Sophie Carret³; ¹McGill University, Montreal, QC, Canada; ²Montreal, QC, Canada; ³Montreal Children's Hospital, Montreal, QC, Canada.

We describe a case of multifocal necrotizing leukoencephalopathy (MNL) as a treatment-related complication in a child treated for an atypical teratoid/rhabdoid tumor (ATRT) of the CNS. To the best of our knowledge, MNL has been rarely described in children treated for malignancies. The patient was diagnosed at 20 months of age with an ATRT of the left middle cranial fossa for which she underwent macroscopically complete resection. Urgent radiotherapy (total dose 10.8 Gy) was given 14 days following surgical excision due to rapidly progressive recurrence in the surgical cavity. The patient then underwent two cycles of induction chemotherapy with a combination of high-dose methothrexate, vincristine, etoposide, cyclophosphamide, and cisplatin followed by involved-field radiotherapy (50.4 Gy total dose) resulting in complete tumor regression. This was followed by two cycles of consolidation chemotherapy consisting of high-dose carboplatin and thiotepa with peripheral blood stem cell rescue. Although the protocol calls for three such cycles of consolidation, the child was severely immunosuppressed and experienced severe sepsis along with very subtle signs of neurological impairment (intermittent right eye strabismus and ptosis and bilateral papilledema) following the second cycle. An MRI revealed enlarged ventricles with impressive changes in the pons and mid brain consisting of a "butterfly" diffuse increased signal on flair and T2 images, while the architecture was maintained. A stereotactic biopsy of the lesion, however, revealed white matter necrosis consistent with the diagnosis of MNL. The third cycle of consolidation was omitted, and the patient was started on high-dose steroids and weekly intravenous immunoglobulin (IV-IG) for 4 weeks, which resulted in partial regression of the necrotizing lesion on MRI and marginal improvement in the patient's neurological status. Within a month following the initiation of steroid tapering, the patient presented with worsening neurological symptoms and blindness. A repeat MRI demonstrated necrosis progression. The patient was restarted on high-dose steroids and IV-IG, and although the repeat MRI after the end of steroid therapy demonstrated a more circumscribed necrotic area, there was also evidence of multiple nodular disease in the optic chiasma, left cerebellum, pons, and inferior left temporal lobe. While on palliative care, the patient improved, and the last MRI 1 year following the last cycle of consolidation showed disappearance of both the necrotic area and multiple nodules. With recent use of more aggressive therapies, we document some prolongation of the natural history of this tumor but also significant treatment-related morbidity such as MNL. Combination of severe immunosuppression, sepsis, high-dose chemotherapy, and early radiotherapy can cause MNL probably mediated by an excessive systemic inflammatory response. As early stage can be difficult to detect, prompt investigations and biopsy may help with differential diagnosis. Early initiation of steroids and IVIG may help.

ATRT 11. ORTHOTOPIC ATRT XENOGRAFT PANEL APPROACH FOR THERAPEUTIC TESTING

C. David James,¹ Scott Vandenberg,² Nalin Gupta,¹ Michael Prados,³ Anuradha Banerjee,³ and Mitchel Berger⁴; ¹Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA; ²Pathology, University of California, San Francisco, San Francisco, CA, USA; ³University of California, San Francisco, San Francisco, CA, USA; ⁴Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA.

Atypical teratoid rhabdoid tumors (ATRTs) may well be the most malignant type of primary brain cancer. For children afflicted with ATRT the duration of symptoms is generally brief due to the incredibly high proliferative rate of the tumor cells. This characteristic, however, has not facilitated the identification of effective therapies, cytotoxic or other, for achieving improved treatment of ATRT patients. In addition to the unfortunate biological and clinical behavior of ATRT, the low incidence of this cancer complicates progress in treatment through clinical trial activity: a high-throughput animal model test system could greatly expedite the discovery of more effective therapies for improved treatment of children with ATRT. The same characteristics that confer high-grade malignancy to ATRT also contribute to the ability of these tumors to grow in athymic mice as human tumor xenografts, and in spite of the relative rarity of this cancer, we and others have been successful in establishing ATRT xenografts that can be indefinitely sustained in athymic mice. The development and molecular characterization of an ATRT xenograft panel is therefore feasible, and such a panel would allow preclinical trial assessments of novel therapies in rodents. These studies would more rapidly reach conclusion than corresponding patient clinical trials, and since an ATRT xenograft panel would consist of multiple unique tumors, results from testing several xenografts would allow one to assess whether a particular treatment approach is generally effective or limited to a molecularly-defined subset of ATRT. Here we present results from the histopathologic, immunohistochemical, and orthotopic (intracranial) therapy response characterization of ATRT xenografts that have been modified for bioluminescence imaging. Histopathologically, ATRT xenografts show a uniformly rhabdoid appearance, in spite of being derived from corresponding patient tumors with typical heterogeneous cell populations. Immunohistochemical characterization shows the intracranial xenografts as being BAF47 negative, with immunohistochemistry results in total supporting the ability to compare treated versus untreated tumors for biomarkers of interest. Mice receiving orthotopic injection of ATRT cells experience relatively short periods of symptom-free survival (30–50 days), which is a time frame that facilitates rapid assessment of therapeutic efficacy in therapy-response experiments. Finally, we show through longitudinal bioluminescence monitoring that intracranial ATRT xenografts can be examined for response to therapy in living animals, as well as for acquisition of resistance to therapy. In total, our results support the utility of ATRT xenografts for studying the molecular and cellular biology, as well as therapeutic response, of these tumors.

ATRT 12. RESULTS FROM A SINGLE-ARM, MULTIINSTITUTIONAL PHASE II STUDY OF MULTIAGENT INTRATHECAL AND SYSTEMIC CHEMOTHERAPY WITH AGE- AND RISK-ADAPTED RADIATION THERAPY FOR CHILDREN WITH NEWLY DIAGNOSED CNS ATYPICAL TERATOID/RHABDOID TUMOR (DFCI 02-294)

Susan Chi,¹ William Fletcher,¹ Xiaopan Yao,¹ Kenneth Cohen,² Michael Fisher,³ Jaclyn Biegel,³ Anna Janss,⁴ Stewart Goldman,⁵ Daniel Bowers,⁶ Peter Manley,⁷ Claire Masewski,⁴ Anne Bendel,⁸ Joshua Rubin,⁹ Christopher D. Turner,¹ Charles Roberts,¹ Karen Marcus,¹⁰ Liliana Goumnerova,¹¹ Nicole Ullrich,¹¹ Christine Chordas,¹ Mary Ann Zimmerman,¹ and Mark W. Kieran¹; ¹Dana-Farber Cancer Institute, Boston, MA, USA; ²Johns Hopkins University, Baltimore, MD, USA; ³Children's Hospital of Philadelphia, Philadelphia, PA, USA; ⁴Emory University, Decatur, GA, USA; ⁵Children's Memorial Hospital, Chicago, IL, USA; ⁶University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA; ⁷Hasbro Children's Hospital, Providence, RI, USA; ⁸Children's Hospitals and Clinics, Minneapolis, MN, USA; ⁹Washington University in St. Louis, St. Louis, MO, USA; ¹⁰Joint Center for Radiation Oncology, Dana-Farber Cancer Institute/Children's Hospital, Boston, MA, USA; ¹¹Children's Hospital, Boston, MA, USA.

Background: Atypical teratoid/rhabdoid tumor (ATRT) of the CNS is a highly malignant neoplasm primarily affecting young children, with a median survival ranging from 6.5 to 10 months. Based on a small successful pilot series of patients with newly diagnosed and recurrent disease (Zimmerman et al. J Neurooncol. 2005;72:77), a multiinstitutional trial was conducted using a modified IRS-III regimen for patients with newly diagnosed CNS AT/RT (DFCI 02-294).

Methods: Treatment was divided into five phases: preirradiation induction chemotherapy (weeks 1–6); chemoradiation (weeks 7–12); consolidation (weeks 13–18); maintenance (weeks 19–44); and continuation therapy (weeks 45–51). Intrathecal chemotherapy administration alternated between the intralumbar and intra-Ommaya routes. Patients with M0 stage received conformal radiation therapy at the prescribed time to a total dose of 5,400 cGy. For patients >3 years of age with M+ disease at diagnosis, craniospinal irradiation at 3,600 cGy was prescribed, with boost to primary sites of disease to total dose of 5,400 cGy.

Results: Between February 2004 and February 2007, 25 children were enrolled, of whom 22 were evaluable (2 ineligible, 1 withdrawal). There were nine males and 13 females; median age at diagnosis was 2.5 years (range, 2.4 months to 19.5 years). Primary tumors were located in the supratentorial compartment in 12 patients and in the posterior fossa in 10 patients. Gross total resections (GTR) of the primary tumor were achieved in approximately 50% of patients. Sixteen patients had M0 disease at diagnosis, one had M2 disease, and five had M3 disease. Twelve patients completed prescribed treatment. Ten patients came off study for the following reasons: toxic death (one), progressive disease ranging from weeks 2–33 (five), radiation recall (one), transverse myelitis (one), second malignancy (one, M7 AML in a patient with trisomy 21), and noncompliance (one). All patients received some intrathecal therapy. Seventeen patients received radiation therapy, 12 conformal focal radiation therapy, and five craniospinal radiation therapy. Significant toxicities for this treatment regimen included bone marrow suppression; febrile neutropenia; infection; gastrointestinal, electrolyte, and hepatic function disturbances; neuropathies; transverse myelitis; and high-frequency hearing loss. Also notable were two patients who experienced radiation recall. There was one toxic death from pneumococcal sepsis. The planned 51-week treatment plan required 52–78 weeks to complete. Of the 14 patients evaluable for chemotherapeutic response (pre-RT), the objective response rate (CR + PR) was 62%. The objective response rate from radiation therapy was 38%. The 1-year event-free survival (EFS) and overall survival (OS) are 68 ± 10% and 77 ± 9%, respectively, and the 2-year EFS and OS are 48 ± 13% and 67 ± 10%, respectively. Sites of relapse include local (three patients), distant metastases (two), disseminated (three), and unknown (one). Median OS has not yet been reached. Fifteen tumors have been centrally reviewed as ATRT; four additional tumors demonstrated deletion of INI-1/Baf47 by immunohistochemistry; the remaining three tumors are being collected for review.

Conclusions: For this rapidly fatal disease, significant progress has been made in terms of improving survival. A future study is planned to incorporate growing biologic data.

ATRT 13. SELECTIVE INHIBITORS OF HISTONE DEACETYLASES (HDI)—A NOVEL AVENUE FOR THE TREATMENT OF RHABDOID TUMORS

Kornelius Kerl,¹ Manfred Jung,² Heribert Juergens,¹ and Michael C. Fruehwald¹; ¹University of Muenster, Muenster, Germany; ²Department of Medicinal Chemistry, University of Freiburg, Germany.

Atypical teratoid rhabdoid tumors (AT/RTs) are rare, but highly aggressive, rather chemoresistant neoplasms. The dismal prognosis has not significantly improved since their first description. The epigenetic structure of DNA and its lesions have gained major interest in the discussion on novel therapeutic strategies for AT/RT. The two most important, closely connected mechanisms of epigenetic regulation are DNA methylation and histone deacetylation. The distinct effects of histone deacetylase (HDAC) inhibitors (HDIs) in studies on neuroblastoma and medulloblastoma cells prompt investigation into the effect these substances have on AT/RT. HDAC are divided into four major groups; class III are termed sirtuins (NAD-dependent HDAC). Previously we have presented unselective HDIs as promising compounds in cell culture models for the treatment of rhabdoid-derived cells (GI50: from nM to µM). Several unselective HDIs are currently in phase I and II clinical trials. Strategies are being developed to implement HDI in the treatment of AT/RT. Furthermore, in vitro survival of AT/RT seems critically dependent on the presence of cyclin D1. We used an array of seven selective HDI and sirtuins (ST 13 HDAC 1+6; ST 80 HDAC 6; PE 24/8 HDAC8; PE 11/5 HDAC 8; Sirtinol SIRT 1+2; Hydrazon Sirt 2; PU 46 SIRT 2; RN 128 SIRT 2), which were designed by one of us, for the treatment of rhabdoid cell lines in vitro. Additionally a combination treatment with cyclin D inhibitors (4HPR and 4OHTam) was used. Our results indicate the following: (1) selective HDIs inhibit the growth of rhabdoid tumor cells in culture models. Effective drug concentrations for the different selective HDI vary from 1 µmol/liter to 100 µmol/liter (GI50 values). (2) HDI of class I HDAC (ST13; GI50: 1 µmol/liter) and SIRT II (Hydrazon 1 µmol/liter) seem to be more effective than other selective HDI. (3) The combination of selective (and unselective) HDI with fenretinide (4HPR; cyclin D1 inhibitor) exerts a synergistic inhibitory effect on the growth of rhaboid cells in vitro. These synergisms have not been described in any other tumor cell lines before. Selective HDIs are effective in treating AT/RT cell lines in vitro. The combination of HDI with cyclin D inhibitors acts synergistically. Both HDI alone and in combination are interesting tools to be employed in vivo for the treatment of patients with AT/RT and other rhabdoid tumors. Supported by the Karl Bröcker and the Sonja Wasowicz Stiftung, Germany.

	BIOLOGY

Top ATRT BIOLOGY BRAINSTEM GLIOMA CHOROID PLEXUS TUMORS CASE REPORTS CRANIOPHARYNGIOMA EPENDYMOMA EPIDEMIOLOGY GERM CELL TUMOR HIGH-DOSE CHEMOTHERAPY HIGH-GRADE GLIOMA NEURO-IMAGING INFANT TUMORS LATE EFFECTS LEPTOMENINGEAL AND DISSEMINATED... LOW-GRADE GLIOMA NURSING AND MULTIDISCIPLINARY... NEUROFIBROMATOSIS AND ASSOCIATED... NEUROPATHOLOGY NEUROSURGERY OUTCOME STUDIES PNET/MEDULLOBLASTOMA QUALITY OF LIFE RADIATION ONCOLOGY RARE TUMORS THERAPEUTIC AND NEW DRUG...

 
BIO 1. ACQUIRED RESISTANCE TO THE HSP90 INHIBITOR 17-ALLYLAMINO-17-DEMETHOXYGELDANAMYCIN (17-AAG) IN ADULT AND PEDIATRIC GLIOBLASTOMA CELL LINES

Nathalie Gaspar,¹ Swee Sharp,¹ Simon Pacey,¹ Chris Jones,² Mike Walton,¹ Gilles Vassal,³ Andrew Pearson,⁴ and Paul Workman¹; ¹Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK; ²Paediatric Oncology, Institute of Cancer Research, Sutton, Surrey, UK; ³Institut Gustave Roussy, Villejuif, Paris, France; ⁴Paediatric Oncology, Royal Marsden Hospital, Sutton, Surrey, UK.

Introduction: The molecular chaperone HSP90 has emerged as an exciting anticancer drug target due to its role in maintaining the stability and function of numerous oncogenic client proteins. The inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the intrinsic HSP90 ATPase activity, which is essential for HSP90 function, and is in phase I and II clinical trials in children and adults, respectively. HSP90 inhibitors have been shown to inhibit cell growth in both adult and pediatric glioblastoma (GB) cell lines. The ability for these primary brain tumors to develop drug resistance probably accounts for their high recurrence rate and poor curability. Exploring the mechanism of acquired resistance to HSP90 inhibitors in GB is an important goal for the potential development of these agents as part of the therapeutic arsenal against GB.

Methods and Results: By continuous exposure to increasing 17-AAG concentrations, in vitro acquired resistance to 17-AAG has been successfully generated in four GB cell lines from both adult (SF268, U87MG) and pediatric (SF188 and KNS42) origins. High levels of resistance with resistance indices (RI = IC₅₀ of resistant line/IC₅₀ of parental line, as determined by sulforhodamine B assay) of 20–137 were obtained rapidly (2–8 weeks). RIs were higher in adult (RI 104–137) than in pediatric resistant lines (RI 20–23). After cessation of 17-AAG, RIs were decreased (6–25), except for the pediatric line KNS42, where RI increased from 20 to 33, then RI stabilized in all cell lines for several weeks (7–26 weeks). Cross-resistance was found with other ansamycin benzoquinones (17-DMAG, 17-AG) but not with structurally unrelated HSP90 inhibitors, radicicol, and the inhouse synthetic potent resorcinylic pyrazole/isoxazole amide compounds (VER-49009, VER-50589), ruling out general mechanisms of resistance to HSP90 inhibition. We further demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1/DT-diaphorase) was involved in 17-AAG acquired resistance of all GB lines. NQO1 has been reported to metabolize ansamycin benzoquinones to their more active hydroquinone counterparts and has been implicated in 17-AAG primary resistance. NQO1 protein levels were depleted in all resistant lines (by Western blot analysis) to a greater extent under 17-AAG pressure than after cessation of 17-AAG. A significant inverse correlation between NQO1 enzymatic activity and 17-AAG IC₅₀ was observed with the resistant lines. Finally, the NQO1 inhibitor ES936 abrogated the differential effects of 17-AAG sensitivity between the parental and resistant lines, confirming the major role of NQO1 in 17-AAG resistance. However, after 17-AAG cessation in the pediatric resistant line SF188-RA6, NQO1 protein/activity returned to parental level despite continued insensitivity to 17-AAG, suggesting an additional mechanism of resistance, albeit restricted to ansamycin benzoquinones. The NQO1 mRNA levels (as measured by quantitative RT-PCR) were also inversely correlated with 17-AAG IC₅₀ in the adult resistant lines, but no mRNA level modification was observed in the pediatric resistant lines, suggesting different mechanisms of NQO1 depletion between adult and pediatric cell lines. Interestingly, generation of acquired resistance cell lines was unsuccessful with the resorcinylic pyrazole/isoxazole amide agents in these four GB lines.

Conclusion: Low NQO1 activity is not only a mechanism of primary resistance, but also a likely mechanism of acquired resistance to 17-AAG in both adult and pediatric GB and possibly in other cell types (e.g., melanoma), underlining the problematic metabolism of 17-AAG. Interestingly, mechanisms of NQO1 depletion seemed to be different between adult and pediatric cell lines. New series of HSP90 inhibitors with improved metabolic properties such as the resorcinylic pyrazole/isoxazole amide analogues, which are able to circumvent the NQO1 mechanism of resistant, provide additional support for new clinical development.

BIO 2. BRAF GENE DUPLICATIONS AND ACTIVATING MUTATIONS CAUSE ABERRANT MAPK PATHWAY ACTIVATION IN PEDIATRIC LOW-GRADE ASTROCYTOMAS

Marc Remke,¹ Wibke G. Janzarik,² Aurélie Ernst,¹ Andreas Kulozik,³ Astrid Gnekow,⁴ Guido Reifenberger,⁵ Andrey Korshunov,⁶ Wolfram Scheurlen,⁷ Heymut Omran,⁸ Peter Lichter,¹ and Stefan Pfister¹; ¹Molecular Genetics, German Cancer Research Center, Heidelberg, Germany; ²Department of Neurology, University Hospital Freiburg, Freiburg, Germany; ³Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany; ⁴Children's Hospital, Klinikum Augsburg, Augsburg, Germany; ⁵Neuropathology, Heinrich-Heine University, Düsseldorf, Germany; ⁶Neuropathology, Burdenko Neurosurgical Institute, Moscow, Russia; ⁷Cnopfsche Kinderklinik, Nürnberg, Germany; ⁸Pediatric Neurology and Muscle Disorders, University Hospital Freiburg, Freiburg, Germany.

Astrocytomas (WHO grade I and II) comprise the most common pediatric brain tumors. However, molecular mechanisms of tumor development and recurrence remain poorly understood. In the present study, we performed array-based comparative genomic hybridization (array-CGH) analysis of 66 pediatric astrocytomas of WHO malignancy grades I and II. The most frequent genomic aberration was a circumscribed duplication of the BRAF locus at 7q34 present in 30 of 66 (45%) cases. Activating mutations of the BRAF gene were identified in 4 of 66 (6%) tumors, exclusively affecting cases without BRAF duplication and indicating different mechanisms for BRAF activation. Tumors with BRAF duplication showed significantly increased mRNA and protein levels of BRAF as compared to tumors without duplication. In addition, we observed mRNA abundance of CCND1, a well-established downstream target of BRAF, in tumors with DNA copy-number gains of the BRAF locus. Fluorescence in situ hybridization (FISH) analysis of 36 low-grade astrocytomas from adult patients also revealed a high frequency of duplications restricted to the BRAF locus (24%), as well as larger gains of chromosome arm 7q (35%). Elevated BRAF protein expression in the tumor was associated with favorable prognosis in adult patients. Proliferation of cell lines derived from low-grade gliomas was effectively blocked by stable knockdown of the BRAF gene using lentivirus-mediated transduction of BRAF-specific shRNAs as well as by pharmacological inhibition of MEK1/2, the immediate downstream target of BRAF. Cell cycle analysis revealed a G2/M arrest in cells treated either with shRNAs targeting the BRAF gene or MEK1/2 inhibitors. Overall, our findings implicate aberrant activation of the mitogen-activated protein kinase (MAPK) pathway due to gene duplication or activating mutation of BRAF as a common event in the tumorgenesis of low-grade astrocytomas and provide a promising novel target for future therapeutic strategies.

BIO 3. CHARACTERISATION OF THE 4Q12 AMPLICON IN PEDIATRIC GLIOMAS

Lara Perryman,¹ Dorine Bax,¹ Suzanne Little,¹ Nathalie Gaspar,² Lynley Marshall,¹ Gilles Vassal,³ Paul Workman,² Andrew Pearson,⁴ Michael Taylor,⁵ Richard Grundy,⁶ Suzanne Baker,⁷ Darren Hargrave,⁴ David Ellison,⁸ and Chris Jones¹; ¹Paediatric Oncology, Institute of Cancer Research, Sutton, Surrey, UK; ²Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK; ³Institut Gustave Roussy, Villejuif, Paris, France; ⁴Paediatric Oncology, Royal Marsden Hospital, Sutton, Surrey, UK; ⁵Developmental Biology, The Hospital for Sick Children, ON, Canada; ⁶Children's Brain Tumour Research Centre, Queens Medical Centre, Nottingham, UK; ⁷Developmental Neurobiology, St. Jude Children's Research Hospital, TN, USA; ⁸Neuropathology, St. Jude Children's Research Hospital, TN, USA.

The genomic locus at chromosome 4q12 harbors the known oncogenes PDGFRA, KIT, and KDR (VEGFR2) and has been found to be amplified in human glioma samples. Combinations of these genes are targeted by novel small molecule tyrosine kinase inhibitors such as imatinib and AZD2171, thus providing a strong impetus for understanding the frequency and nature of this genomic event. Estimates of gene dosage and protein expression for these and other genes at this locus are equivocal in adult glioma and lacking in pediatric cases. We have sought to clarify this in the high-grade gliomas (HGGs) of childhood by mapping the amplicon by means of targeted FISH probes and Agilent 44K oligonucleotide array CGH, as well as investigating protein expression in the tumors by immunohistochemistry. In our retrospective study of formalin-fixed, paraffin-embedded cases, we observed three patterns of DNA copy gain at 4q12 in pediatric HGG. First was high-level amplification (>10 copies) of a locus as focal as 985 kb harboring PDGFRA and the proximal genes CHIC2 and LNX1, but not extending further distally. Second was an extended amplicon, as wide as 3.1 Mb, which was also high level and included these genes as well as KIT. Finally, was a low-level copy number gain of between three and six copies of a large (>8 Mb) region spanning up to 36 genes and including PDGFRA, KIT, and KDR/VEGFR2. By mining the publicly available datasets for DNA copy number profiling in adult glioblastoma multiforme, we observed an identical pattern of amplification/gain encompassing the same three discrete events. It is noteworthy that in both primary tumors and cell lines, receptor overexpression was seen in the absence of gene amplification. Of key interest was a pediatric astrocytoma cell line Res259/UW467 that harbored a complex rearrangement at 4q12 involving both focal amplification of PDGFRA and KIT as well as a low-level gain of the larger region encompassing KDR/VEGFR2. These data highlight the different amplification events present at 4q12 in pediatric HGGs and provide evidence for a uniquely useful in vitro model for mechanistic and preclinical investigations of this locus and drugs targeting the products encoded by genes therein.

BIO 4. COMBINATION OF A POLY(ADP-RIBOSE) POLYMERASE (PARP) INHIBITOR AND TEMOZOLOMIDE (TMZ) SUPPRESSES GROWTH OF PEDIATRIC GLIOBLASTOMA MULTIFORME (GBM) AND MEDULLOBLASTOMA (MB) IN ORTHOTOPIC MOUSE XENOGRAFTS

Jack Su,¹ Li-Tiang Yu,¹ Qin Shu,¹ Adekunle Adesina,² Torsten Pietsch,³ Joy Bauch,⁴ Brian Dayton,⁴ Vincent Giranda,⁴ Susan Blaney,¹ Ching Lau,¹ and Xiao-Nan Li¹; ¹Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX, USA; ²Department of Pathology, Baylor College of Medicine, Houston, TX, USA; ³Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany; ⁴Abbott Laboratories, Abbott Park, IL, USA.

Background: PARP is a nuclear enzyme that detects DNA breaks and activates DNA repair proteins in the base-excision repair (BER) and the nonhomologous end-joining (NHEJ) repair pathways. As the majority of TMZ-induced DNA adducts are repaired by the BER mechanism, it has been hypothesized that increased PARP activity may mediate tumor resistance to DNA alkylating agents such as TMZ. We demonstrated PARP overexpression in pediatric GBM and MB specimens, and we confirmed that PARP inhibition enhanced in vivo TMZ efficacy against pediatric GBM and MB xenografts in mice.

Design and Methods: PARP expression in 18 pediatric GBM and 59 MB specimens was evaluated by immunohistochemistry (IHC) using an antibody detecting full-length PARP (Trevigen). Intracranial orthotopic MB and GBM xenografts in SCID mice were established using two MB cell lines (Daoy and MHH-MED1) and two patient-derived pediatric GBM cell lines. Two weeks after tumor injection, TMZ (q.d. x 5 days) and/or ABT-888 (b.i.d. x 5 days), an orally bioavailable PARP inhibitor from Abbott Laboratories, were administered. All treated animals received two courses of treatment (5 days of study drugs, followed by 23 days of rest). Survival durations were compared by log-rank analysis. Plasma and CNS concentrations of ABT-888 were measured by LC-MS. PARP and methylguanine methyltransferase (MGMT) activity was quantitated using published techniques. Ku70, BRCA1, MLH1, and MSH2 protein levels were measured by Western blot analysis.

Results: Intense (3+) nuclear PARP IHC staining was detected in 61% (11 of 18) of GBM and 81% (48 of 59) of MB specimens, and intermediate (2+) PARP staining was seen in 28% (5 of 18) of GBM and 13% (8 of 59) of MB specimens. Normal pediatric cerebrum and cerebellum, in contrast, showed only faint (1+) cytoplasmic staining. TMZ and ABT-888 treatment moderately prolonged the survival of mice with Daoy xenografts (p = 0.054) and significantly prolonged the survival of mice with MHH-MED1 xenografts (p = 0.038), compared to the TMZ-only group. Combining ABT-888 and TMZ led to a similar improvement in survival in the two GBM models (p = 0.179 and p = 0.026). The brain:plasma ratio of ABT-888 concentration ranged from 15% to 69% (mean, 34% ± 17%), indicating good CNS penetration. Pretreatment PARP activity was comparable in the two MB xenografts, and ABT-888 treatment resulted in 20- to 50-fold reductions in PARP activity. Pretreatment MGMT activity and MLH1 and MSH2 protein levels were comparable in all four xenograft models and unaffected by ABT-888. However, Ku-70 and BRCA1 protein levels were reduced in the GBM xenografts after ABT-888 treatment.

Conclusion: We demonstrated differential PARP expression in pediatric MB and GBM tumors versus normal brain tissue. Combining ABT-888 with TMZ dramatically reduced PARP activity and improved survival in mice with MB and GBM xenografts, independent of MGMT activity and mismatch repair proficiency. Reduction of key proteins in the NHEJ and homologous recombination repair pathways was observed in GBM xenografts after ABT-888 treatment. Our data support the addition of ABT-888 to radiation and DNA-damaging drugs to improve the treatment outcome for pediatric CNS tumors.

BIO 5. COMPARISON OF THE WNT PATHWAY STATUS IN SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL TUMORS (SPNET) AND MEDULLOBLASTOMA

Hazel Rogers,¹ Suzanne Miller,¹ James Lowe,² Keith Robson,² Marie-Anne Brundler,³ Beth Coyle,¹ and Richard Grundy¹; ¹University of Nottingham, Nottingham, UK; ²Nottingham, UK; ³Birmingham, UK.

Supratentorial primitive neuroectodermal tumors (sPNETs) are high-grade, predominantly pediatric, brain tumors. Although relatively rare, accounting for 3%–7% of pediatric CNS tumors, sPNETs are aggressive with a poor prognosis. Due to their rarity, relatively little research has been undertaken to understand the molecular genetic mechanisms involved. Previously sPNETs have been grouped together with medulloblastomas, due to their histological similarities. A number of developmental signaling pathways have been shown to be deregulated in medulloblastoma, which may suggest potential candidate genes to investigate in sPNETs. One such pathway is the WNT signaling pathway, which has a key role in cellular proliferation, differentiation, migration and adhesion. Pathway activation through the stabilization and nuclear accumulation of beta-catenin (CTNNB1) has been demonstrated in sporadic medulloblastomas. In the majority of cases this was caused by activating mutations in exon 3 of CTNNB1. Mutations occurred at or adjacent to residues that are phosphorylated to target the protein for degradation. A small study identified a single mutation in CTNNB1 exon 3 in one out of four sPNETs, however, no further research has been undertaken in sPNET. In this study WNT pathway status was investigated in 24 sPNETs and 40 medulloblastomas using immunohistochemistry of CTNNB1, enabling the determination of the cellular location of the protein. This serves as a marker for pathway status, where nuclear staining represents the active state and cytoplasmic inactive. The mutational status of exon 3 of CTNNB1 was also investigated by sequencing of DNA samples extracted from a subset of the tumors and correlated with the IHC results. Nuclear staining and therefore pathway activation was seen in 8 out of 24 (33%) sPNETs. However, mutation of exon 3 of CTNNB1 was seen in only one sample (4%) suggesting pathway activation was caused by an alternative mechanism or factor. The medulloblastoma samples displayed an equivalent percentage of nuclear staining of 35%. The mutation rate of CTNNB1 exon 3 found in sPNETs of 4% was lower than the rate found in medulloblastomas of 19%. The levels of pathway activation and mutation seen in the medulloblastomas agree with previously published results. WNT pathway activation has been demonstrated in 33% of sPNETs, which is equivalent to percentages seen in medulloblastoma. However, only one sPNET contained a mutation of exon 3 of CTNNB1, suggesting the pathway is activated by an alternative means.

BIO 6. CONCURRENT ACTIVATION OF NOTCH CELL SIGNALING AND DELETION OF INK4A/ARF IN RADIAL GLIA CAUSES CEREBRAL EPENDYMOMA

Nicholas G. Gottardo,¹ Helen Poppleton,¹ Robert Johnson,¹ Sarah Sherr,¹ David W Ellison,¹ Karen Wright,¹ Twala L. Hogg,¹ and Richard J. Gilbertson¹; ¹St. Jude Children's Research Hospital, Memphis, TN, USA.

Ependymoma is the third most common pediatric intracranial tumor; yet little is known about the biology of this disease. Up to 40% of children eventually succumb to their disease since total resection is required to ensure cure, and the disease is chemoresistant. Novel treatment strategies are urgently needed for ependymoma, but there has been a lack of validated molecular targets for therapeutic development. We previously reported that ependymomas are likely to arise from neural progenitor cells termed radial glia (RG) that are susceptible to anatomic site-specific genetic alterations (Taylor et al. Cancer Cell. 2005). As an example, we showed that all cerebral ependymomas activate NOTCH signaling and delete INK4a/ARF. To further test the hypothesis that cerebral RG are transformed by concurrent activation of NOTCH and inactivation of INK4a/ARF, we generated a Blbp-NICD transgenic mouse that expresses the intracellular domain of NOTCH1 in RG throughout the CNS. We bred these mice with Ink4a/Arf null mice. Blbp-NICD mice developed classical ependymomas that phenocopy the human disease precisely to include pseudorosettes, glial differentiation, and electron microscopic features of ependymomas. These tumors arise in both Blbp-NICD-Ink4a/Arf+/+ and Blbp-NICD-Ink4a/Arf–/– mice. Tumors develop with much longer latency in the Blbp-NICD-Ink4a/Arf+/+ mice (approx. 16 months) compared to approximately 6 months in Blbp-NICD-Ink4a/Arf–/– mice. Remarkably, tumors in Blbp-NICD-Ink4a/Arf+/+ mice spontaneously delete Ink4a/Arf. No tumors were observed outside the cerebrum despite the expression of the transgene in the hind brain and spine. Our data strongly support the hypothesis that RG are susceptible to transformation in site specific-forms of ependymoma; in the cerebrum this includes aberrant Notch signaling and concurrent Ink4a/Arf loss. This model represents the first spontaneous mouse model of ependymoma and should prove extremely useful for future biological studies and the development of new treatments.

BIO 7. CONTRASTING TELOMERE DYNAMICS BETWEEN NEURONAL AND GLIAL PEDIATRIC BRAIN TUMORS (ON BEHALF OF CCLG BIOLOGICAL STUDIES COMMITTEE)

Ruman Rahman,¹ Lee Ridley,¹ Siobhan Quinn,¹ Beth Coyle,¹ and Richard Grundy¹; ¹Childrens Brain Tumour Research Centre, University of Nottingham, Nottingham, UK.

Telomeres are nucleoprotein structures consisting of highly repetitive DNA that cap chromosomal termini of human cells. Telomere length at birth ranges from 5 to 15 kb and upon successive rounds of cell division, telomeres erode, due in part to the inability of the lagging strand to faithfully replicate its ends. Eventually, telomere attrition triggers replicative senescence and subsequent widespread apoptosis. Thus telomeres are regarded as a cellular "mitotic clock," representing a primitive tumor suppressor mechanism. Eroded telomeres are replenished, however, in highly proliferative cells, germ cells, and tumor cells by a homeostatic mechanism governed by the enzyme telomerase. Telomeric repeats are synthesized at the telomere substrate by telomerase using an intrinsic RNA component (hTERC) as template in a reverse transcription reaction catalyzed by its protein component (hTERT). Telomerase is active in approximately 90% of human tumors, stimulating a plethora of cancer therapeutic strategies targeting telomerase. We and others have previously demonstrated telomerase-mediated telomere maintenance as a key mechanism facilitating tumor progression in childhood Ependymoma. However, telomerase and telomere status of pediatric glioblastoma multiforme (GBM), sPNET, and PNET primary tissue has not been forthcoming, with only limited current data, derived from cell lines. Here we report a comprehensive descriptive characterization of telomerase activity and telomere length from isolated tumor tissue and explore the amenability of targeting telomerase in these tumors. Telomerase activity was evident in 7 of 7 GBM, 8 of 18 sPNET, and 20 of 20 PNET tumors, consistent with high tumor grade. Telomere length for sPNET DNA (mean, 5.2 kb; median, 4.8 kb; n = 15) was significantly shorter than those of patient-matched blood (mean and median, 6.3 kb; n = 5). Telomere dynamics were similar for PNET tumor DNA with telomere length significantly shorter (mean, 4.4 kb; median, 3.8 kb; n = 15) than those of blood (mean, 6.9 kb; median, 6.6 kb; n = 3). This was in contrast to mean GBM telomere length (mean, 8.3 kb; median, 8.6 kb; n = 7), comparable to telomere length from blood (mean, 8.5 kb; median, 8.5 kb; n = 4). Longer telomere length observed for GBM tumors is consistent with our previous findings in ependymoma (mean telomere length, 10.9 kb; median, 10.0 kb; n = 20). Hence we observe telomere length in primary tumors of neuroectodermal origin (4.9 kb ± 0.3) to be significantly shorter than glial tumor telomere length (8.8 kb ± 0.5) (p < 0.001). Furthermore, no significant difference was observed for mean and median age at diagnosis between neuroectodermal (5.8 years; 5.5 years; range, 0.2–12.0 years) and glial tumors (4.7 years; 4.0 years; range, 0.1–8.8 years). This intriguing contrast between embryonal (poorly differentiated) and further differentiated tumors hints at a fundamental difference in early neurogenesis with respect to cellular replicative history. Our results encourage consideration of varying telomere length dynamics when devising therapeutic strategies to achieve telomerase inhibition in pediatric brain tumors. We would predict the onset of replicative senescence and apoptosis to occur more rapidly in sPNET and PNET tumors upon inhibition. Histone deacteylase (HDAC) inhibitors are a category of telomerase inhibitors being studied as a treatment for cancer and neurodegenerative diseases and proposed to be associated with silencing of gene promoters, including that of hTERT. Induction of apoptosis and telomerase inhibition has recently been documented in adult GBM and medulloblastoma using TSA treatment. We are currently investigating this proof of concept using the HDAC inhibitor Trichostatin A on representative tumor lines, assaying for telomerase inhibition, cell viability, cell proliferation, and apoptosis.

BIO 8. CROSS-TALK BETWEEN TUMOR CELLS AND ENDOTHELIUM TRIGGERS A STRONG CHEMOTACTIC SIGNAL RECRUITING T LYMPHOCYTES TO DISTANT MEDULLOBLASTOMA DEPOSITS

Nabil Ahmed,¹ Vita Salsman,² Donald R Shaffer,² Kadikoy Huseyin,³ Xiao-Nan Li,⁴ Laszlo Perlaky,⁴ Meenakshi Bhattacharjee,⁵ Cliona Rooney,⁶ Helen Heslop,⁶ and Stephen Gottschalk²; ¹Texas Children's Cancer Center, Center for Cell and Gene Therapy, Houston, TX, USA; ²TX, USA; ³Baylor College of Medicine, TX, USA; ⁴Baylor College of Medicine, Houston, TX, USA; ⁵Pathology, Baylor College of Medicine, TX, USA; ⁶Houston, TX, USA.

Background: Failure of local control is a poor prognostic factor that heralds incurable disease recurrence in medulloblastoma. In addition, recurrence is multifocal in up to 60% of patients, adding to the dismal prognosis of these patients. We have shown that genetically modified T-cells expressing engineered HER2-specific chimeric antigen receptors (HER2-T cells) induce regression of HER2-positive human medulloblastomas growing in the brains of mice after intratumoral injection. The objective of this project was to study the ability of HER2-T cells to achieve locoregional control in medulloblastoma.

Methods: Mice with established human HER2-positive medulloblastoma xenografts (Daoy) were stereotactically injected into the contralateral hemisphere with HER2-T cells expressing the firefly luciferase (luc) gene to determine T-cell migration and expansion in vivo using bioluminesence imaging. To determine the antitumor activity of T-cells, unmodified HER2-T cells were injected into mice bearing luc-expressing Daoy tumors in the contralateral hemisphere. Chemokine expression and chemokine receptor profiles were determined by standard techniques and transwell assays were used to study T-cell migration ex vivo.

Results: HER2-T cells migrated within the brain to distant primary medulloblastoma and Daoy xenografts as judged by bioluminescence imaging. Within 48 h of injection HER2-T cells clustered at the tumor site and proliferated as judged by imaging as well as immunohistochemistry for the non-G0 marker Ki-67. HER2-T cells had potent antitumor activity after injection into the contralateral hemisphere of tumor bearing mice, inducing tumor regression, which resulted in a survival advantage of treated animals. To investigate the mechanism of in vivo T-cell migration, we analyzed the chemokine expression profile of Daoy cells and primary medulloblastoma cells, revealing only the presence of chemokines like CXCL-8 (IL-8) and MIF for which HER2-T cells did not have chemokine receptors on their cell surface. Consistent with this finding, supernatants from Daoy cells failed to induce T-cell migration in transwell migration assays. To determine if brain tumor endothelial cells trigger T-cell migration we used supernatants from the murine glioma endothelial cells (b.END.3) as a chemoattractant but also did not observe T-cell migration. However, supernatants derived from cocultures of Daoy and b.END.3 cells induced strong T-cell migration. Chemokine expression analysis revealed high levels of human RANTES (CCL5), for which HER2-T cells expressed the corresponding chemokine receptor.

Conclusions: We have shown that HER2-T cells migrate to tumor sites within the CNS, expand at tumor sites, and induce regression of medulloblastoma xenografts in vivo. Our results also suggest that "cross-talk" between tumor and tumor endothelium plays a major role in creating a chemotactic gradient for T-cell migration. Hence, the adoptive transfer of HER2-T cells represents a promising immunotherapeutic approach to control locoregionally advanced, incompletely resected and/or drop-metastatic medulloblastoma.

BIO 9. CYCLIN-DEPENDENT KINASE 6 IS REGULATED BY MICRORNA 124 IN MEDULLOBLASTOMA

Jessica Pierson,¹ Rong Fan,¹ and Rajeev Vibhakar¹; ¹Department of Pediatrics, University of Iowa, Iowa City, IA, USA.

Background: Medulloblastoma comprises approximately 20% of all primary pediatric brain tumors. Despite advances in treatment with surgery, radiation, and chemotherapy, patients with high-risk and infant tumors continue to do poorly. Recently increased expression of cyclin-dependent kinase 6 (CDK6) was identified as an adverse prognostic marker in medulloblastoma. Genomic amplification accounts for some but not all of the CDK6 overexpression. MicroRNAs are a large family of small, noncoding RNAs with gene regulatory functions. They consist of 18–22 nucleotide RNA molecules with posttranscriptional gene silencing activity. In addition to their role in normal development, microRNAs are also associated with carcinogenesis.

Hypothesis: CDK6 expression is regulated by microRNAs in medulloblastoma.

Methods: Medulloblastoma cell lines Daoy, D283 Med, and D341 Med were cultured under standard conditions. Primary cell cultures were derived from biopsy specimens of medulloblastoma patients. Total RNA was isolated from medulloblastoma cell lines or brain tissues using the mirVANA RNA isolation kit (Ambion, Austin, TX, USA). MicroRNA expression was measured by quantitative RT-PCR with MicroRNA primers and probes from Applied Biosystems on an ABI 7700 cycler. Luciferase experiments were done using the the siCHECK dual luciferase system (Promega, Madison, WI, USA). The putative miR 124a target sites from the CDK6 3' UTR were cloned into the 3' UTR of the Renilla luciferase gene. Target sites were amplified by PCR and mutations introduced by alternate primers. Cells were transfected with 0.5 µg siCHECK using LT1(Mirus, Madison, WI, USA), and Renilla luciferase activity was measured 48 h after transfection and normalized to firefly luciferase. Some cells were cotransfected with 50 or 100 nM miR 124a microRNA oligonucleotide (Ambion). Western blotting was performed per standard methods. Cell proliferation was measured at 48 h using Cell Titer AQeous (Promega), and apoptosis was measured by caspase activation using the Caspase-Glo 3/7 assay kit (Promega) according to the manufacturer's instructions.

Results: We identified putative miR sites in the CDK6 UTR by using three well-known bioinformatic algorithms (TargetScan v4.0, PICTAR, and miRBase). Among the potential microRNA (miR) sites was microRNA 124a, a brain-enriched microRNA. Expression of miR 124a was significantly decreased in Daoy, D283 Med, and D341 Med cell lines and in primary cells compared to normal adult cerebellar RNA. To establish a functional association between miR 124a and CDK6 in medulloblastoma we performed analysis of the putative miR 124a binding sites from the CDK6 3' UTR using luciferase assays. Cotransfection of siCHECK-CDK6 with miR 124a oligonucleotides (100 nM) decreased firefly luciferase activity to 34% of vector alone control. Mutation of both target sites restored luciferase activity to 93% of control. Additionally, re-expression of miR 124a in Daoy medulloblastoma cells decreased expression of CDK6 protein levels. Transient transfection of miR124 RNA oligonucleotide significantly decreases D283 medulloblastoma cell growth but does not alter apoptosis. Furthermore, in patient samples expression of miR 124a is significantly decreased.

Conclusions: Our data strongly indicate that CDK6 is regulated by microRNA 124 in medulloblastoma. MiR124 expression is significantly decreased in medulloblastoma, and re-expression of miR 124 in medulloblastoma cells significantly decreases proliferation.

BIO 10. DETECTION OF HUMAN HERPESVIRUS 6 VARIANTS IN PEDIATRIC BRAIN TUMORS: ASSOCIATION OF VIRAL REPLICATION IN LOW-GRADE GLIOMAS

John Crawford,¹ Mariarita Santi,² Hallidora Thorarinsdottir,³ Robert Cornelison,⁴ Elisabeth Rushing,³ Steve Jacobson,⁵ and Tobey Macdonald³; ¹George Washington University, Washington, DC, USA; ²Children's National Medical Center, Washington, DC, USA; ³DC, USA; ⁴Bethesda, MD, USA; ⁵MD, USA.

Human herpesvirus 6 (HHV6) has been implicated in the pathogenesis of encephalitis, epilepsy, and multiple sclerosis. Since HHV6 is a common virus acquired during childhood, we screened a series of pediatric brain tumors with known clinical information for HHV6 DNA by nested PCR and in situ hybridization. HHV6 major capsid protein DNA was detected in 53% (n = 64) of paraffin-embedded and 58% (n = 24) of fresh frozen tumors compared to 22% (n = 32) of age-matched nontumor brain (p = 0.0008). HHV6 large tegument protein DNA was found in 73% of paraffin-embedded, 71% of fresh frozen, and 38% of control brain (p = 0.019); 73% of tumors were HHV6 variant A compared to 33% of control brain. In situ hybridization for HHV6 was found in 54% of brain tumors (n = 137) and 31% control brain (n = 32; p = 0.021), and was not limited to areas exclusively containing tumor on surgical resection. To establish the presence of active HHV6, immunohistochemistry was performed using HHV6 variant nonspecific gp116/54/64 antibody; 40% (n = 124) of tumors were positive compared to 18% (n = 32) of controls (p = 0.013). Interestingly, 58% of low-grade gliomas were immunopositive compared to 19% of high-grade gliomas (p = 0.002) and 25% of nongliomas (n = 36). Immunofluorescence microscopy confirmed the association of HHV6 antigen in cells of glial origin through colocalization with glial fibrillary acidic protein. There was no association between HHV6 immunopositivity and patient age at diagnosis (p = 0.12), gender (p = 0.22), or progression-free survival (p = 0.861). We provide the first reported association between HHV6 and pediatric brain tumors; these findings may provide insight into potential disease mechanisms.

BIO 11. DIFFERENTIAL EXPRESSION OF CELL DIVISION CONTROL PROTEINS IN PEDIATRIC VERSUS ADULT EPENDYMOMAS AND COMPARISON TO OTHER INFILTRATING GLIOMAS: PRELIMINARY RESULTS

Jose Otero,¹ and Tarik Tihan¹; ¹Pathology, UCSF, San Francisco, CA, USA.

Background: Ependymomas are glial tumors thought to be derived from the ependymal cells or their precursors and are among the most common tumors in the pediatric age group. Prognosis in ependymomas is influenced by a number of factors, such as age at diagnosis, extent of resection, and histological grade. Recent studies also suggest a negative correlation between proliferative rate of ependymomas and their survival probability, which raises the possibilities that cell cycle regulation may be differentially regulated in ependymomas with high proliferation rates and differ between pediatric and adult ependymomas.

Methods: In order the determine the activation of a critical cell cycle checkpoint, we examined the activity of cdc2, a cyclin dependent kinase whose activity is required for the G2-M phase transition. We have analyzed 20 ependymomas (nine pediatric cases and 11 adult cases) by immunohistochemical staining for the activated and inhibited forms of cdc2 and its downstream targets in tissue microarrays and compared the results to oligodendrogliomas (20 WHO grade II and 17 WHO grade III tumors, including two patients 15 and 17 months of age), and glioblastomas (45 newly diagnosed and 15 recurrent).

Results: Most ependymomas showed low staining for activated and inhibited cdc2. However, all cases showing elevated staining for activated cdc2 were found within the pediatric population. Most GBMs showed higher staining of both inhibited and activated cdc2 relative to oligodendrogliomas and ependymomas.

Conclusion: In this preliminary study, recognition of increased activation through cdc2 can account for the increase in the proliferative rate of pediatric ependymomas. Mitotic cells positive for inhibited cdc2 were detected in GBM and anaplastic oligodendroglioma, suggesting that these tumors transitioned from G2-M phase despite inhibited cdc2. Since our study did not have sufficient power to correlate cdc2 activation with outcome in a reasonable fashion, a larger study is under way to determine the exact relationship of this pathway with patient outcome.

BIO 12. DIFFERENTIAL MECHANISMS OF TEMOZOLOMIDE RESISTANCE IN PEDIATRIC GLIOMA CELL LINES

Nathalie Gaspar,¹ Lynley Marshall,² Dorine Bax,² Marta Viana-Pereira,³ Suzanne Little,² Lara Perryman,² Gilles Vassal,⁴ Andrew Pearson,⁵ Paul Workman,¹ Rui Reis,³ Darren Hargrave,⁵ and Chris Jones²; ¹Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK; ²Paediatric Oncology, Institute of Cancer Research, Sutton, Surrey, UK; ³Life and Health Science Research Institute (ICVS), University de Minho, Portugal; ⁴Institut Gustave Roussy, Villejuif, Paris, France; ⁵Paediatric Oncology, Royal Marsden Hospital, Sutton, Surrey, UK.

The alkylating agent temozolomide (TMZ) has been demonstrated to have antitumor activity in glioblastoma multiforme (GBM), although sensitivity is restricted to a subset of patients. Resistance to TMZ in adult GBM patients is associated with two major mechanisms: lack of promoter methylation of the gene encoding the DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) and deficiencies in mismatch repair (MMR) enzymes. In the pediatric setting, concurrent with the limited clinical and biological data is a paucity of preclinical information in model systems derived from pediatric glioma patients. We have screened a series of 11 glioma cell lines (six adult high grade, three pediatric high grade, and two pediatric low grade) for TMZ efficacy in vitro and have investigated the differential mechanisms of TMZ resistance involved. The cell line panel comprised adult GBM (LN229, A172, U118MG, U138MG, U87MG, SF268), pediatric GBM (SF188, KNS42), pediatric anaplastic astrocytoma (UW479), pediatric grade II astrocytoma (Res259/UW467), and juvenile pilocytic astrocytoma (Res186/Res199). Response to TMZ was assessed in triplicate by the SRB assay. Three adult GBMs (SF268, LN229, A172) and a pediatric grade II line (Res259/UW467) were sensitive to TMZ, with mean IC₅₀ values ranging from 11.3 to 20.9 µM. The remaining resistant lines had IC₅₀ values of 407.8–949.3 µM. MGMT protein expression was assessed by Western blotting and promoter methylation by methylation specific (MS)-PCR and MS-MLPA. There was a direct relationship between MGMT promoter methylation by both assays and protein expression, and in the majority of cases a lack of methylation, and subsequent overexpression, was linked to TMZ resistance (U118MG, U138MG, SF188, UW479, Res186/Res199). The two exceptions were the adult GBM line U87MG and the pediatric GBM line KNS42. We further screened by Western blotting and MS-MPLA the MMR proteins MLH1, MLH3, MSH2, MSH3, MSH6, and PMS2, however, there were no deficiencies observed in U87MG or KNS42. The U87MG cells are PTEN null, which may explain in part the TMZ resistance due to constitutive Akt activation, however, the KNS42 cells are PTEN wild-type. They do, however, harbor a homozygous TP53 mutation (R342^*), although the link between mutant p53 and TMZ resistance is controversial. In order to investigate alternative mechanisms of TMZ resistance in these cells, we carried out expression profiling by Affymetrix U133 Plus2.0 microarrays. Among 52 differentially expressed genes between sensitive and resistant cell lines was an enrichment of genes associated with phosphodiesterase activity, significantly dysregulated in KNS42, and thus providing a link between TMZ resistance and the base excision repair pathway. These data provide an extensive characterization of the in vitro efficacy of TMZ in pediatric glioma model systems and demonstrate that novel mechanisms of resistance may be active in childhood GBM.

BIO 13. DISABLING THE PROLIFERATIVE AND THE REPLICATIVE ABILITY OF MEDULLOBLASTOMA AND TERATOID/RHABDOID CNS TUMOR CELLS BY A NOVEL TELOMESTATIN DERIVATIVE

Tarek Shalaby,¹ Andre Von Bueren,¹ Marie-Louise Hürlimann,² Giulio Fiaschetti,² Deborah Castelletti,² Kazuo Shin-Ya,³ Alexandre Arcaro,¹ and Michael Grotzer⁴; ¹Department of Oncology, University Children's Hospital of Zurich, Switzerland, Zurich, Switzerland; ²Department of Oncology, University Children's Hospital of Zurich, Switzerland, Switzerland; ³National Institute of Advanced Industrial Science and Technology, Tokyo, Japan; ⁴University Children's Hospital of Zurich, Zurich, Switzerland.

Small molecules that stabilize the G-quadruplex structure in c-myc promoter sequences have shown potential as a new opportunity for cancer therapy. Employing this innovative line of attack to cancer cells, we investigated the effects of S2T1-60TD, a novel telomestatin derivative that specifically targets c-myc promoter, on a representative panel of medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) children's brain cancer cell lines. In remarkable contrast to control cells, treatment (72 h) with S2T1-60TD exhibited a clear antiproliferative effect in all cell lines tested, in a dose- and time-dependent manner and with IC₅₀ at submicromolar levels (0.25–0.39 µM). Importantly, S2T1-60TD demonstrated significantly less cytotoxic effects on normal (not cancer) fibroblast MCR-5 cells (IC₅₀ for MCR-5 was 5x more than the IC₅₀ of MB and AT/RT cells). Seventy-two–hour treatment with S2T1-60TD reduced the mRNA and protein expressions of c-myc and hTERT—that is, transcriptionally regulated by c-myc—and decreased both genes activities. Under conditions where inhibition of both proliferation and c-myc activity were observed, S2T1-60TD treatment decreased the protein expression of the cell cycle activator CDK2 and resulted in cell cycle arrest. Long-term treatment with nontoxic concentrations of S2T1-60TD resulted in time-dependent telomere shortening, which was accompanied by cell growth arrest (starting on day 28) and was followed by cell senescence and induction of apoptosis at day 35 in all of the five cell lines investigated. In this study, we present evidence showing that the use of S2T1-60TD compound to target both c-myc deactivation and telomere maintenance disruption has resulted in the obstruction of proliferation of the MB and AT/RT cancer cells and disabled their ability to replicate. Upon in vivo animal testing, S2T1-60TD may well represent a potential effective and innovative therapeutic strategy for childhood brain tumors.

BIO 14. DISTINCTIVE MOLECULAR PROFILE OF RADIATION-INDUCED GLIOBLASTOMA

Andrew Donson,¹ Nicole Erwin,² B.K. Demasters,³ Diane Birks,⁴ and Nicholas Foreman⁴; ¹University of Colorado Health Sciences Center (UCHSC), Aurora, CO, USA; ²Pediatrics, UCHSC, CO, USA; ³UCHSC, CO, USA; ⁴Aurora, CO, USA.

Radiation-induced glioblastomas (RIGs) represent a significant proportion of glioblastomas (GBMs) seen in children and young adults and manifest poor prognosis. Little is known about their underlying biology, although limited studies have suggested no unique histological or cytogenetic characteristics to distinguish them from de novo GBMs. In this study, we investigated RIGs using gene expression microarray profiling and Western blot analysis. Despite the inability of histological and molecular genetic studies to identify distinguishing features between RIGs and pediatric GBMs, gene microarrays showed significant differences between these two tumor types. Greater overlap was detected in gene expression patterns between RIGs and pilocytic astrocytomas (PAs) than between RIGs and pediatric GBMs, medulloblastomas, ependymomas, atypical teratoid/rhabdoid tumors, or rhabdomyosarcoma, suggesting a common precursor cell for RIG and PA. Examination of the genes that constitute the RIG profile showed that a number of clinically relevant molecules are overexpressed in RIG relative to pediatric GBM. Specifically, we showed that platelet-derived growth factor receptor alpha (PDGFRa) and ErbB3 were strongly expressed at both the RNA and protein levels. These proteins may provide a therapeutic target for novel small molecule inhibitors. As an example of how this information can be applied, we used the unique RIG gene expression profile in order to elucidate the etiology in the case of a GBM arising in the site of a treated medulloblastoma. It was debated whether the new astrocytic tumor represented a progression of the medulloblastoma or a radiation-induced neoplasm. Gene microarray assessment showed that the original tumor clustered with other standard medulloblastomas. However, the secondary tumor manifested a genetic expression profile concordant with other radiation-induced glioblastomas and dissimilar to pediatric GBM or standard medulloblastomas studied in our laboratory. In conclusion, the characterization of the molecular pathology of RIG provides a diagnostic tool as well as insight into the etiology of these neoplasms and rationale for design of novel therapeutic regimens for this highly aggressive tumor.

BIO 15. DO ALL GLIAL TUMORS OF THE CHILD CONTAIN CANCER-INITIATING CELLS?

Barbara Bessette,¹ Pascale Varlet,² Sebastien Dubleumortier,¹ Christelle Dufour,³ Josette Cadusseau,⁴ Catherine Daumas-Duport,² Jacques Grill,³ Christian Sainte-Rose,⁵ Herve Chneiweiss,¹ Stephanie Puget,⁵ and Marie-Pierre Junier¹; ¹Inserm U752, Paris, France; ²Department of Pathology–Neuro-Oncology, Sainte-Anne Hospital, Paris, France; ³Institut Gustave Roussy Villejuif, Villejuif, France; ⁴Inserm U841, Creteil, France; ⁵Necker Sick Children's Hospital, Paris, France.

Background: Physiopathological understanding of malignant high-grade gliomas in children is made difficult by their heterogeneous appearances, and the variability in clinical evolutions of patients bearing tumors of similar histopathological aspects. Recent isolation of tumor-initiating cells (TICs) having some of the properties of neural stem cells in medulloblastomas and adult gliomas opens novel insights into the development and therapeutic resistance of primitive brain tumors. Data being scarce with respect to pediatric gliomas, we aimed at isolating TIC from 18 of these tumors and from four embryonal tumors. Our approach was based on the fact that TICs can be isolated upon their ability to develop in defined medium supplemented with EGF and bFGF under the form of floating cellular spheres (FS), to self-renew and to be clonally derived.

Material and Methods: Tumors corresponded to nine low-grade glial or neuronal-glial tumors (one pilocytic astrocytoma, two ANET, six gangliogliomas), nine high-grade glial tumors (four ependymomas III, one oligoastrocytoma III and its reccurence, one brainstem glioblastoma, two infiltrating brainstem astrocytomas). Six of these high-grade gliomas and one clear-cell ependymoma had the characteristics of malignant glioneuronal tumors according to Hospital Sainte-Anne's classification. Embryonal tumors comprise three medulloblastomas and one ATRT. One cavernoma and one biopsy taken at distance from a pilocytic astrocytoma were used as nontumoral control tissues. Culture ages range from 25 days to >3 months at this time.

Results: Viable cells anchored to the culture dish were observed in all cases. No FS were observed in the ATRT, and nontumoral tissue-derived cultures up to 30 DIV (days in vitro). As expected, all medulloblastoma-derived cultures contained FS within 10 DIV. FS developed in 66% of the low-grade glial and glioneuronal tumors and 100% of high-grade glial tumors within 7 DIV. Medulloblastoma-derived FS self-renewed for at most 2.3 months prior to death, the numbers of FS doubling each 6.4 ± 0.9 days (rate of self-renewal, mean ± SEM). No significant difference was noted with respect of rates of renewal between low- and high-grade tumors (7.9 ± 1.3 days for gangliogliomas over 3 months, vs. 8.1 ± 1.4 rate for ependymomas III over 2–3 months). Brainstem glioma–derived FS are ongoing self-renewal at a rate of 5.5–13.3 days. At this time, 50% of the low-grade tumors, all accounted for by gangliogliomas, and 78% of high-grade glial tumors are still undergoing self-renewal.

Conclusion: Further characterizations will indicate if the pediatric glial tumor derived–FS contain TICs. They will include determination of their karyotype, of their proteomic profile, and, most importantly, of their capability upon grafting in immunodeficient mice brain to generate tumor exhibiting morpho-phenotypes alike to the original tumor.

BIO 16. DUAL-SPECIFIC ANTIBODY, D2C7 (SCDSFV)-PE38KDEL FOR BRAIN TUMOR THERAPY

Vidyalakshmi Chandramohan,¹ Charles N. Pegram,¹ Scott E. Szafranski,¹ Stephen T. Keir,¹ Ira Pastan,² Chien-Tsun Kuan,¹ and Darell D. Bigner¹; ¹Pediatric Brain Tumor Foundation Institute, Duke University Medical Center, Durham, NC, USA; ²National Cancer Institute, Bethesda, MD, USA.

Brain tumors are the second most frequent malignancy among children, comprising about 17% of all pediatric cancer cases. In the United States, approximately 2,200 children are diagnosed annually with invasive brain tumors, of which astrocytomas account for about 52%, primitive neuroectodermal tumors 21%, other gliomas 15%, and ependymomas 9%. The epidermal growth factor receptor (EGFR) is expressed by normal epithelial cells in most tissues but overexpressed in gliomas: 27%–57% in astrocytomas, 71%–94% in anaplastic astrocytomas, and 60%–90% in glioblastoma multiforme (GBMs). In addition, 58%–61% of all GBMs also express the EGFR variant III mutant (EGFRvIII), which is not found in normal tissues. Monoclonal antibodies targeting either the wild-type EGFR (EGFRwt) or EGFRvIII have been developed, and one of them, D2C7, a murine IgG1, recognizes both the EGFRwt and tumor-specific EGFRvIII receptors. In the present study, we have demonstrated cloning of a novel, recombinant single-chain variable-region antibody fragment from the D2C7 hybridoma and have engineered a disulfide-stabilized linkage between variable heavy and light domains to generate D2C7 (scdsFv). The D2C7 (scdsFv) is connected to a truncated variant of Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide for improved intracellular transport (PE38KDEL). The binding affinity of D2C7 (scdsFv)-PE38KDEL for the EGFRwt and the EGFRvIII extracellular domain (ecd) was 6.3 x 10⁸ (mol/liter)^–1 and 7.8 x 10⁸ (mol/liter)^–1, respectively, as measured by surface plasmon resonance. Flow cytometry of EGFRwt-transfected (NR6W), EGFRvIII-transfected (NR6M), and parental NR6 cells, a mouse 3T3 fibroblast cell line that naturally lacks EGFR expression, further confirmed the dual specificity of D2C7 (scdsFv)-PE38KDEL for the two forms of transfected cells. The immunotoxin was highly cytotoxic, with an IC₅₀ of 1 ng/ml and 3 ng/ml, respectively, on cells expressing the EGFRwt (NR6W) and EGFRvIII (NR6M) receptors. There was no cytotoxic activity at 1,000 ng/ml on the parental NR6 cells. The cytotoxicity of D2C7 (scdsFv)-PE38KDEL on the pediatric GBM cell line D2159MG, expressing both the EGFRwt and EGFRvIII receptors, was 3 ng/ml. The combination of good affinity and cytotoxicity makes this immunotoxin a promising candidate for further evaluation for treatment of pediatric astrocytic tumors.

BIO 17. DUPLICATION OF 7Q34 IN PEDIATRIC LOW-GRADE ASTROCYTOMAS DETECTED BY HIGH-DENSITY SNP-BASED GENOTYPING

A.J. Sievert,¹ E.M. Jackson,² X. Gai,³ H. Hakonarson,⁴ A.R. Judkins,⁵ A.C. Resnick,² T.H. Shaikh,⁴ and J.A. Biegel⁴; ¹Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; ²Department of Neurosurgery, University of Pennsylvania School of Medicine, PA, USA; ³Center for Biomedical Informatics, The Children's Hospital of Philadelphia, PA, USA; ⁴Division of Human Genetics, The Children's Hospital of Philadelphia, PA, USA; ⁵Department of Pathology, The Children's Hospital of Philadelphia, PA, USA.

Pediatric low-grade astrocytomas are a heterogeneous group of tumors whose pathogenesis is not well understood. Histological diagnosis can be subjective. Moreover, the molecular characteristics of the tumors are not well described. A gain of chromosome 7 is the most commonly detected abnormality by cytogenetic analysis; however, this is found in only a minority of patient samples. More recently, studies utilizing comparative genomic hybridization have also failed to consistently find a unique genetic signature pattern for this set of tumors. We sought to identify novel genetic abnormalities such as duplications, deletions, loss of heterozygosity, and copy number variations, using high-density SNP-based microarray technology. DNA was extracted from snap-frozen specimens of 24 pediatric low-grade astrocytomas collected in our institution from 1998 through 2006. DNA samples were analyzed using the Illumina HumanHap 550K microarray platform. The microarray data were analyzed for copy number alterations using our Bioinformatics Core-Copy Number Analysis, Annotation, and Visualization tool. Results were compared to a database of known, common copy number variations seen in healthy controls. We found a consistent and unique duplication in the 7q34 region, previously not described. The 7q34 duplication was found in 13 of 15 (86.7%) juvenile pilocytic astrocytoma samples, one of which also had a whole chromosome copy number gain, and in 5 of 8 (62.5%) fibrillary astrocytoma samples. The sole astrocytoma, NOS sample did not have this duplication but did have a whole chromosome 7 copy number gain. Fluorescence in situ hybridization analysis using 7q and 7 centromere probes confirmed the 7q34 duplications. The 7q34 duplication was not identified in a set of 12 ganglioglioma, 20 ependymoma, and 3 anaplastic astrocytoma samples or a set of 3,000 normal control DNAs analyzed with the Illumina HumanHap 550K platform. We found >30 genes in the duplicated region using the UCSC genome browser; however, the homeodomain protein kinase 2 (HIPK2) gene seemed the most likely candidate given the recent body of literature describing its role not only in apoptosis but also in neuron cell survival. Mutation analysis of all 15 exons of HIPK2 yielded only known single nucleotide polymorphisms and alternative splice sites. Immunohistochemistry of 11 matched paraffin-embedded samples both with and without the 7q34 duplication using a commercially available HIPK2 antibody (Abcam) demonstrated normal nuclear staining. There was no cytoplasmic localization, which has been previously described with HIPK2 gene mutations. Real-time PCR and Western blot analysis did not show any differences in gene or protein expression between the different tumors. In summary, we have described a novel 7q34 duplication in a majority of pediatric low-grade astrocytoma samples that appears to be unique to this group of tumors. Our exploration of HIPK2 as a potential candidate gene failed to yield abnormalities by mutation analysis and/or gene expression; however, it is an intriguing gene as it has multiple transcriptional and p53 interactions. The 7q34 duplication is a novel genetic abnormality and further evaluation of genes in this region may help to explain the pathogenesis of juvenile pediatric astrocytomas and fibrillary astrocytomas.

BIO 18. EFFECTIVENESS OF A RODENT BRAINSTEM GLIOMA MODEL FOR THERAPEUTIC TESTING

Rintaro Hashizume,¹ Tomoko Ozawa,¹ Eduard Dinca,¹ Anuradha Banerjee,¹ Michael Prados,¹ C. David James,¹ and Nalin Gupta¹; ¹University of California, San Francisco, San Francisco, CA, USA.

Introduction: The prognosis for brainstem tumors remains extremely poor. Therapeutic testing of new agents and delivery strategies is limited by the lack of well-characterized model systems. In this study, we demonstrate the morphologic features and growth kinetics of an orthotopic brainstem tumor model in athymic rats. Tumor growth was quantitatively determined by in vivo bioluminescence imaging (BLI). Effectiveness of the model system was tested by comparing oral and intra-nasal delivery of temozolomide. Distribution studies for intranasal delivery using fluorescence-labeled liposomes were also performed.

Methods: U87MG glioblastoma cells were implanted into the pontine tegmentum of athymic rats. This cell line was modified to constitutively express luciferase by lentivirus infection. Survival time, location of the tumor, and time to development of symptoms were measured. In vivo BLI was performed using the Xenogen Lumina Imaging Station (Caliper Life Sciences) coupled to a data-acquisition PC running Livingimage software.

Results: Histopathologic analysis and BLI showed progressive tumor growth in all animals, with symptoms indicative of tumor burden between 26 and 31 days. Animals were euthanized at the onset of symptoms. BLI correlated with tumor volume calculated by three-dimensional measurements from serial histologic sections. For efficacy experiments, rats in one group received temozolomide delivered by oral gavage (50 mg/kg for 12 days), while the other group received intranasal delivery (10 mg/kg in 65 µl for 12 days). Animals treated with temozolomide, either by oral or intra nasal route, survived >50 days after implantation. Longitudinal BLI revealed a sustained decrease in luminescence of temozolomide-treated animals. Intranasal delivery with fluorescent liposomes showed accumulation of fluorescence in the pons 6 h after treatment.

Conclusions: This orthotopic brainstem tumor model system allows a reproducible assessment of survival defined by time to development of symptoms and should facilitate testing of preclinical therapeutic agents and novel delivery strategies. Tumor response to therapeutic agents can be noninvasively measured using BLI. Intranasal delivery may be an effective noninvasive method for the treatment of brainstem glioma.

BIO 19. EGFRVIII DELETION MUTATIONS IN PEDIATRIC HIGH-GRADE GLIOMA AND RESPONSE TO ERLOTINIB IN PEDIATRIC GLIOMA CELL LINES

Dorine Bax,¹ Nathalie Gaspar,² Lynley Marshall,¹ Suzanne Little,¹ Marie Regairaz,³ Lara Perryman,¹ Jorge Reis-Filho,⁴ Safa Al-Sarraj,⁵ Gilles Vassal,³ Andrew Pearson,⁶ Darren Hargrave,⁶ Paul Workman,² David Ellison,⁷ and Chris Jones¹; ¹Pediatric Oncology, Institute of Cancer Research, Sutton, Surrey, UK; ²Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK; ³Institut Gustave Roussy, Villejuif, Paris, France; ⁴Breakthrough Breast Cancer, Institute of Cancer Research, London, UK; ⁵Neuropathology, Kings College Hospital, London, UK; ⁶Paediatric Oncology, Royal Marsden Hospital, Sutton, Surrey, UK; ⁷St. Jude Children's Research Hospital, TN, USA.

Erlotinib (Tarceva, OSI-774) is a selective inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase and has shown activity in adult high-grade glioma (HGG) patients. A differential therapeutic response has been reported in these cases, with a variety of factors reported to be predictive for treatment efficacy, including activating mutations of EGFR and a wild-type PTEN. EGFR has been considered to play a less important role in pediatric glioma, although extensive data is lacking. We have sought to clarify the molecular pathology of EGFR in pediatric HGG and to assess the in vitro sensitivity of pediatric glioma cell line models to erlotinib. We retrospectively studied a total of 76 formalin-fixed, paraffin-embedded (FFPE) pediatric HGG specimens for EGFR overexpression, amplification, and mutation; 37% of cases demonstrated protein expression by immunohistochemistry. Gene amplification was detected by chromogenic in situ hybridization (CISH) in 18% cases, with a corresponding overexpression of the receptor. These cases had a shorter median overall survival time than those without amplification/overexpression (p < 0.05, log-rank test). None of the 76 samples contained mutations in either the extracellular (exons 2–8) or the tyrosine kinase domains (exons 18–21) of EGFR, as assessed by direct sequencing. By contrast, the exon 2–7 deletion mutation EGFRvIII was detected by RT-PCR and direct sequencing in 6 of 33 (18%) samples from which RNA was available. Seven pediatric glioma cell lines (three high grade: SF188, KNS42, UW479; two low grade: Res259/UW467, Res186/Res199) and two adult high-grade lines (U87MG, SF268) were assessed in triplicate for erlotinib efficacy in vitro by the MTS assay. Of note were the pediatric GBM lines—SF188 was sensitive to EGFR inhibition by erlotinib, with an IC₅₀ of 7 µM, while KNS42 was relatively resistant. Neither these, nor any other pediatric cell lines demonstrated appreciable EGFR protein expression by Western blot, and no activating mutations were found. We further created clones of U87MG (adult GBM, PTEN null) and SF188 (pediatric GBM, PTEN wild-type) stably transduced with either wild-type EGFR or EGFRvIII. Sensitivity to erlotinib was enhanced in clones containing the EGFRvIII construct in both cell lines, but not those overexpressing the wild-type receptor. These data identify an elevated frequency of EGFR gene amplification and EGFRvIII mutation in pediatric high-grade glioma than previously recognized and demonstrate the potential of treatments targeting the receptor in deletion mutant-positive cases.

BIO 20. EPIDERMAL PATHWAY GROWTH FACTORS IN CHILDHOOD EPENDYMOMA

Juliette Hukin,¹ Tami Yamashita,² Stephen Yip,³ Tamir Ailon,⁴ Michael Sargent,⁴ Ashtosh Singhal,⁴ Ruth Milner,⁴ Chris Fryer,⁵ Glenda Hendson,⁴ John Maguire,⁴ Cynthia Hawkins,⁶ and Sandra Dunn⁷; ¹University of British Columbia, Vancouver, BC, Canada; ²University of Kingston, Ontario, ON, Canada; ³MA, USA; ⁴BC, Canada; ⁵British Columbia's Children's Hospital, Vancouver, BC, Canada; ⁶ON, Canada; ⁷Departments of Pediatrics, Experimental Medicine, and Medical Genetics, Child and Family Research Institute, Vancouver, BC, Canada.

Introduction: The overall survival rate of childhood ependymoma is 30%–50%, inferior to that of adult ependymoma. Unfortunately, the current WHO grading system for intracranial ependymoma is not predictive of outcome. In other childhood malignancies, biologic characteristics of the tumors identify clinically relevant subsets of high-, intermediate-, or low-risk patients.

Hypothesis: Signaling components of the HER family: EGFR, HER-2, and YB-1 will provide a better risk stratification than the current histologically based classification system.

Methods: We performed a retrospective review of all children <17 years of age at diagnosis of ependymoma in British Columbia since 1982. The charts were reviewed, and the imaging and pathology were centrally evaluated. Tissue microarray (TMA) slides were constructed using cases in which the specimen was successfully retrieved. Each case was represented by triplicate (3) formalin-fixed, paraffin-embedded core biopsies on positively charged slides. TMA slides were immunostained for YB-1, EGFR, and HER-2. The TMA slides were incubated with either anti-YB-1 (1:250 dilution, Dr. Colleen Nelson from UBC), anti-EGFR (1:100 dilution, Stress-Gen Bioreagents) or anti-HER-2 (1:100 dilution, Lab Vision Corporation). For the YB-1 immunostain, each core was scored for both nuclear and cytoplasmic staining intensity and for EGFR and HER-2 generalized staining intensity on a scale from 0 to 3. Scores from the triplicate cores were averaged for each case. For YB-1 nuclear staining and HER-2, cases with an average score greater than 0 were considered positive. For YB-1 cytoplasmic and EGFR staining, cases with an average score 2 were considered positive. Kaplan-Meier overall survival was performed using SPSS software (Sutherland, Oncogene 2005).

Results: We reviewed the clinical data, centrally reviewed the pathology and radiology, and obtained cores for TMA in 46 of 59 patients diagnosed in this time period. The median age of this group is 5.3 years (range, 0.2–14.3). Three patients were WHO grade I, 29 grade II, and 14 grade III. The primary tumor location for 39 of 46 patients was the posterior fossa. The WHO grade was not predictive of survival. EGFR staining of 2 was considered positive. All patients with a positive EGFR (23 of 23) arose from the posterior fossa, 19 of 23 had at least a gross total resection (>95%), and two had evidence of dissemination at diagnosis. The median age of the patients with a positive result was 4.9 years (0.2–14.3), and 10 were female. Positive EGFR correlated with a worse 35% 5-year overall survival compared with 75% for those who were negative for EGFR (p < 0.05). No correlation was found relating positive immunostain for either nuclear or cytoplasmic YB-1 or HER-2 individually to overall survival.

Conclusion: This study suggests that positive immunostaining for EGFR in childhood ependymoma is a molecular prognostic indicator of poor survival. Positive EGFR may be limited to primary location in the posterior fossa, it is unrelated to age at presentation. There was no predictive value with positive YB-1 or HER-2 alone with respect to survival, however, this may be related to small numbers. Further studies are required to evaluate these and other molecular markers for their prognostic value.

BIO 21. ESTABLISHMENT AND CHARACTERIZATION OF CELL LINES AND TRANSPLANTABLE XENOGRAFTS FROM PEDIATRIC BRAIN TUMORS

Nidhi Srivastava,¹ Tracy Warr,² Charles Pegram,¹ Stephen Keir,¹ Ahmed Rasheed,¹ Roger Mclendon,¹ Chien-Tsun Kuan,¹ and Darell Bigner¹; ¹Pediatric Brain Tumor Foundation Institute, Duke University Medical Center, Durham, NC, USA; ²Institute of Neurology, London, UK.

Brain tumors are the leading cause of cancer-related mortality in children. Each year approximately 3,400 new cases are diagnosed in the United States alone. Although there are >120 different types of pediatric brain tumors, low-grade astrocytomas are the most common, accounting for approximately 40% of all pediatric cerebral tumors. Brain tumors in children are often located in the brainstem and cerebellum and are heterogeneous in regard to histology and clinical course. Improving survival rate in pediatric brain tumor patients continues to be a challenge, and for this we still need to understand the molecular pathways involved in the genesis, progression, and biological and clinical behavior of the pediatric brain tumors. Establishing stable cell lines and xenograft models provides access to continuous availability of representative tumors. We are establishing pediatric brain tumor cell lines and xenografts for various tumor types, including medulloblastomas, supratentorial primitive neuroectodermal tumors (PNET), and grade III and IV astrocytomas. Two glioblastoma multiforme (GBM) cell lines, D2159 MG and D2368 MG, and a grade II pleomorphic xanthoastrocytoma cell line are beyond passage level 60 in neural stem cell medium. We are also growing six new medulloblastoma cell lines, D2319 Med, D2341 Med, D2344 Med, D2377 Med, D2352 Med, and D2385 Med; one grade I desmoplastic infantile ganglioglioma cell line, D2378 DIG; one grade IV teratoid/rhabdoid cell line, D2376 ATRT; one grade I pilocytic astrocytoma cell line, D2384 GPA; one grade II pleomorphic xanthoastrocytoma cell line, D2388 PXA; and another GBM cell line, D2360 MG; all of these are in passage level 15–30 in neural stem cell medium. All of the above-reported cell lines are also growing into conventional zinc option (ZO) medium at different passage levels, except D2159 MG. Another GBM cell line, D456 MG has been established in ZO medium and carried out to passage number 84. Using athymic nude mice, we have established new subcutaneous xenografts with three different pediatric GBMs, D2159 MG, D2224 MG, D2234 MG; one xenograft from a grade IV intracranial sarcoma, D2339 SARC; one from a grade IV supratentorial primitive neuroectodermal tumor, D2216 PNET; and one from a grade II pleomorphic xanthoastrocytoma, D2363 PXA. Cells were used to establish the xenografts in most of the cases, except for D2363 PXA and D2339 SARC, where tissue was used for tumor transplantation. Subcutaneous tumor growth rates (reported in days to grow from inoculation to 1,000 mm³) were 32, 35, 24, 28, 120, and 47 days, respectively, for the above-named xenografts. Doubling time for xenograft D2159 MG was 7.43 days, and for xenograft D2224 MG it was 7.20 days. The consistent availability of these established cell lines and xenografts will aid both basic and preclinical pediatric brain tumor research.

BIO 22. EXPRESSION OF GLIOMA-ASSOCIATED ANTIGENS IN PEDIATRIC BRAINSTEM AND NONBRAINSTEM GLIOMAS

Ian Pollack,¹ Hideho Okada,¹ Keri Low,² and Ronald Hamilton¹; ¹University of Pittsburgh, Pittsburgh, PA, USA; ²Pittsburgh, PA, USA.

Background: The outcome for children with diffuse intrinsic pontine gliomas and incompletely resected nonbrainstem gliomas is poor with conventional therapies. In view of these discouraging results, there is a strong need for new therapeutic approaches that target those features of the tumor cell that distinguish it from surrounding normal cells. Vaccine strategies targeting glioma-associated antigens (GAAs) hold particular promise in that regard. In recent years, we have examined the applicability of a series of tumor cell-based immunization approaches for adult patients with gliomas. Although promising results have been achieved by us and others, a limitation of this strategy is the need for autologous tumor to generate a patient-specific vaccine, which delays instituting therapy and precludes use altogether in patients with unresectable lesions. In contrast, vaccines in the form of synthetic peptides encoding T-cell epitopes in GAAs would eliminate the need for autologous fresh glioma explants to generate clinical grade vaccines and facilitate timely vaccine production. To this end, we have focused attention on the identification and characterization of therapeutically applicable human GAA-derived cytotoxic T-lymphocyte (CTL) epitope targets.

Methods: We investigated the protein expression of three potential GAAs in pediatric brainstem glioma (BSG) and nonbrainstem glioma (NBSG) cases with a view to their possible use in immunotherapy. Expression of EphA2, IL-13R2 and Survivin were studied by immunohistochemistry on paraffin-embedded tissues using a series of 15 archival BSG cases and 12 NBSG cases.

Results: Thirteen of 15 BSGs and all 12 NBSGs overexpressed at least one of the GAAs. Seven of 15 BSGs were positive for EphA2, as were all NBGs; 10 of 15 BSGs and 7 of 9 NBSGs were positive for IL-13R2; and 8 of 15 BSGs and 8 of 12 NBSGs were positive for Survivin. In total, 7 of 15 BSGs and 9 of 12 NBSGs expressed at least two of these GAAs at significantly higher levels than nonneoplastic brain. There was no association between the tumor grade and levels of GAA expression. Although many cases demonstrated diffuse expression of GAAs throughout the tumor specimen, partial or patchy expression was noted in a small number of cases, supporting the need for targeting multiple GAAs in immunotherapy.

Conclusion: These results suggest that EphA2, IL-13R2 and Survivin are commonly overexpressed in pediatric malignant gliomas and constitute suitable targets for developing vaccine-based strategies for these tumors. A clinical trial to test this hypothesis has been developed and will be discussed.

BIO 23. EXPRESSION OF THE NOTCH PATHWAY EFFECTORS HES-1 AND HES-5 IS ASSOCIATED WITH FAVORABLE OUTCOME IN MEDULLOBLASTOMA

Mi Rim Choi,¹ Elisabeth Rushing,² Mary Rita Santi,¹ Robert Cornelison,³ and Tobey Macdonald¹; ¹Children's National Medical Center, Washington, DC, USA; ²Armed Forces Institute of Pathology, Washington, DC, USA; ³National Institutes of Health, Bethesda, MD, USA.

Background: Medulloblastoma (MB), an embryonal tumor of the cerebellum, is thought to arise from precursor cells of the cerebellar external granule layer. Signaling pathways such as Wnt, Hedgehog, PDGF, and Notch, which control the specification, proliferation, and survival of normal neuronal precursors of the developing cerebellum, are also aberrantly activated in MB. The role of Notch in tumorigenesis is less clear, but studies have demonstrated Notch overexpression in a variety of human cancers, including MB. However, reports in limited sample sizes of MB have described discrepant results in the expression patterns of two receptors, Notch-1 and Notch-2 (Cancer Res. 2004;64:7787; Cancer Res. 2004;64:7794). Due to the potentially important role of the Notch signaling pathway in MB and the limited existing information, we sought to determine the expression pattern of the Notch pathway using a robust MB sample size.

Methods: Immunohistochemistry analysis for the Notch receptors (Notch-1–4), the Notch ligands (Delta-1 and Jagged-1), the Notch antagonist (Numb) and the downstream targets (Hes-1 and Hes-5) was performed using tissue microarrays constructed from 205 childhood brain tumors, including 144 MB, with >95% of the specimens obtained at diagnosis prior to treatment. Protein expression of each marker was then compared to clinical and tumor characteristics.

Results: Of the evaluable specimens, 90% were positive for Notch-1 and 89% for Notch-2. Delta-1 and Jagged-1 were positive in less proportion (49% and 42%, respectively), while Hes-1 was positive in 34% and Hes-5 in 42% of the samples. The relative level of Notch pathway member expression did not correlate with any histologic subtype. All MB with documented metastatic disease at diagnosis (n = 11) were positive for Notch-1 (100%), while Notch-2 expression was seen in 90%. Similarly, all MB patients who had died of tumor progression (n = 20), had 100% Notch-1 positivity, while Notch-2 was seen in 93% of the samples. Interestingly, only 27% of all evaluable tumors were positive for both Hes-1 and Hes-5, but of these, 81% (22 of 27) are still alive, regardless of histology. Furthermore, the 20 specimens that were positive for both Hes-1 and Hes-5, as well as positive for either Notch ligand (Delta and/or Jagged) had a 95% survival rate (19 of 20) compared to 57% (4 of 7) of the samples that were positive for Hes-1 and Hes-5 and negative for both Notch ligands.

Conclusions: Our results represent the largest series of human MB investigated for the protein expression of Notch and demonstrate that although Notch-1 and Notch-2 are ubiquitously expressed at relatively high levels in MB, activity of the pathway, as evidenced by detectable expression of the downstream targets Hes-1 and Hes-5, is observed in a much smaller subset of tumors. Importantly, Notch pathway activation appears to be associated with a more favorable outcome independent of histology. This finding could have important implications for targeted therapeutics specifically directed against Notch in MB.

BIO 24. EXPRESSION PATTERN OF CHEMORESISTANCE-RELATED GENES IN PEDIATRIC BRAIN TUMORS

Iacopo Sardi,¹ Piergiorgio Modena,² Valentina Cetica,³ Gabriella Bernini,³ Maurizio Aricò,³ Maura Massimino,² and Lorenzo Genitori⁴; ¹Neurosurgery/Pediatrics Oncology, A. Meyer Children's Hospital, Florence, Italy; ²Pediatrics, National Tumor Institute, Milan, Italy; ³Pediatrics Oncology, A. Meyer Children's Hospital, Florence, Italy; ⁴Neurosurgery, A. Meyer Children's Hospital, Florence, Italy.

Chemotherapy in solid tumors is poorly effective, partly due to intrinsic or acquired drug resistance of tumor cells, which leads to unsatisfactory outcome. Resistance to chemotherapy in pediatric brain tumors, in particular, is complex and may involve multiple mechanisms. In this work we investigated the expression of some chemoresistance-related genes, alkylated repair protein alkB homolog 2 and 3 (ALKBH2, ALKBH3), O⁶-methylguanine-DNA methyltransferase (MGMT), P-glycoprotein (ABCB1/MDR), multidrug resistance-associated protein 1 (ABCC1/MRP), topoisomerase 2a (TOPO2a), and glutathione-S-transferase (GSTP1) in malignant pediatric brain tumors. We analyzed 58 bioptic samples identified histologically as 42 ependymomas, six medulloblastoma/PNETs, five low-grade gliomas, one glioblastoma, and four nonneoplastic brain tissues. Quantitative real-time PCR was performed on the corresponding cDNA synthesized from each sample and was repeated in triplicate. The different expression of the target genes normalized to GAPDH and HPRT housekeeping genes were calculated using Ct method. We used Universal Standard Reference RNA and brain nonneoplastic autoptic samples as calibrators. Expression levels of each of the analyzed genes were generally higher in brain tumors than in nonneoplastic brain tissues. ALKBH2 and ALKBH3 had a similar expression pattern in normal tissue while almost all the tumors showed higher ALKBH2 expression. There was a positive correlation among expression of ALKBH2, GSTP1, and ABCB1. The ABCC1/MRP gene was expressed at low level in controls; while tumor samples showed an increased expression. Anaplastic astrocytomas and medulloblastoma showed an increased expression of GSTP1 gene while TOPO2a presented a marked increment in the ependymoma, with suggestive statistical correlation with WHO grading. Our analysis showed a correlation between the absence of clinical response and the increased expression of chemoresistance-related genes. Additional correlations with clinical parameters will be investigated. This work represents a preliminary study on the expression pattern of genes possibly related to drug resistance in pediatric brain tumors; validation of these results on a higher number of samples, for each tumor histology, would contribute to define a better characterization of brain tumor chemo-responsive phenotype.

BIO 25. GANGLIOSIDE EXPRESSION IN PEDIATRIC BRAIN TUMOR XENOGRAFT LINES

Hailan Piao,¹ Charles N. Pegram,¹ Darell D. Bigner,¹ and Chien-Tsun Kuan¹; ¹Pediatric Brain Tumor Foundation Institute, Duke University Medical Center, Durham, NC, USA.

Gangliosides are component molecules of the outer leaflet of the plasma membrane that are believed to play a role in tumor formation and progression. These sialylated glycosphingolipids constitute the major class of glycoconjugates on neurons and carry the majority of the sialic acid within the CNS. They are involved in various cellular functions, including signal transduction, regulation of cell proliferation and differentiation, cell-cell recognition and adhesion, and cell death. Two gangliosides, 3'-isoLM1 and 3',6'-isoLD1, have been characterized and validated as molecular targets for the treatment of malignant gliomas, especially glioblastoma multiforme (GBM). These oncofetal gangliosides are attractive candidates because they are not expressed in the normal adult brain tissue or the CNS. We assessed the reactivity of previously prepared, highly specific monoclonal IgMs: SL50, specific for only 3'-isoLM1; DMAb 14, specific for both 3'-isoLM1 and 3',6'-isoLD1; and DMAb 21 and DMAb22, specific for only 3',6'-isoLD1. ELISA and BIAcore analyses showed that SL50 binds to 3'iso LM1 (K_d = 7.9 nM) and that DMab22 binds to 3',6'-isoLD1 (K_d = 56 nM). In this study, we found that gangliosides 3'-isoLM1 and 3',6'-isoLD1 were also expressed in pediatric brain tumor xenograft lines. Flow cytometry revealed that these IgMs reacted with several pediatric brain tumor cell lines and xenografts and showed cell-surface binding. Specifically, our results showed that the following pediatric brain tumor lines express 3'-isoLM1 and 3',6'-isoLD1 on the cell surface: DAOY, D341MED, D283MED, D324MED, D2159MG, and D2224MG. SL50 reacted with approximately 90% of the viable DAOY cell population and reacted with 20%–45% of the cell populations of the five other pediatric cell lines listed; DMAb22 was 80% positive with the DAOY line and 20%–30% positive with the other pediatric lines for cell-surface binding, as indicated by flow cytometry. The significant cell-surface expression of 3'-isoLM1 and 3',6'-isoLD1 on pediatric brain tumor cells, the low to no expression on normal brain cells, and the tumor antigen involvement in the migratory and invasive aspects of GBM cells demonstrate that these gangliosides are good candidates for antibody-based targeting. Elimination of cells expressing 3'-isoLM1 and 3',6'-isoLD1 should result in significant survival increases in pediatric brain tumor patients. In addition, since the IgM-type MAbs are unsuitable for therapy, we have begun to prepare high-affinity IgG MAbs against the gangliosides 3'-isoLM1 and 3',6'-isoLD1. We have successfully cloned the VH and VL fragment sequences from hybridomas SL50, DMAb21, and DMAb14 individually by isolation of mRNA and by RACE-PCR (rapid amplification of cDNA ends–PCR). We have constructed single-chain Fvs (scFvs) by connecting VH and VL sequences with a 15-amino-acid peptide linker (Gly4Ser)3 by PCR splicing technology. The scFv was expressed well in Escherichia coli BL21(DE3) cultures harboring the corresponding expression plasmid. After characterization of each scFv, the scFvs will undergo affinity maturation. The scFv with the highest affinity will then be converted into an intact human IgG1 with antibody-dependent cellular cytotoxicity or will be used to prepare PE38 immunotoxin for preclinical testing.

BIO 26. GENE EXPRESSION ANALYSIS OF SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL TUMORS (SPNETs)

Hazel Rogers,¹ Suzanne Miller,¹ James Lowe,² Keith Robson,² MarieAnne Brundler,³ Beth Coyle,¹ and Richard Grundy¹; ¹University of Nottingham, Nottingham, UK; ²Nottingham, UK; ³Birmingham, UK.

sPNETs are high-grade, predominantly pediatric tumors found within the cerebrum or suprasellar region of the brain and are composed of poorly differentiated neuroepithelial cells. Although relatively rare, sPNETs are highly malignant with a poor prognosis. Relatively little research has been undertaken to elucidate the molecular basis of sPNETs, and previous studies have often grouped them with the histologically similar tumor medulloblastoma. The aim of this study was to use global gene expression analysis of a set of sPNET samples to try to better understand their molecular basis. RNA was extracted from a cohort of histologically verified tumors that included 15 sPNETs, six medulloblastomas, and a fetal brain sample (Clonetech). The samples were hybridized to Affymetrix U133 plus two arrays. The expression data generated was initially analyzed using clustering methods, separating the tumors into distinct subgroups. Candidate gene lists distinguishing these groups were identified. The majority of the sPNETs divided into two groups, which were found to correlate with metastatic status. Genes differentially expressed included a number of extracellular matrix genes, such as type 3 and type 5 collagen, which have previously been linked to metastasis. These genes were up-regulated in the group of tumors where the majority had metastasized. Genes up-regulated in the nonmetastatic group included oligodendrocyte myelin glycoprotein (OMG), oligodendrocyte transcription factor 1 (OLIG1), and oligodendrocyte lineage transcription factor 2 (OLIG2), suggesting the presence of cells of the oligodendroglial lineage. A number of tumors clustered with fetal brain. Lower expression of genes involved in DNA replication and the cell cycle was seen in this group, suggesting these samples have a phenotype closer to normal brain. Hematoxylin- and eosin-stained smears of the frozen sections used confirmed this. Cell cycle genes significantly up-regulated in the tumors included topoisomerase (DNA) II alpha 170 kDa (TOP2A). Differential expression between sPNETs and medulloblastomas was also demonstrated, including genes linked to cerebellum development such as mab-21-like 1 (MAB21L1) and somatostatin receptor 2 (SSTR2). Putative tumor suppressors dachshund homolog 1 (DACH1) and early B-cell factor 3 (EBF3) were down-regulated in sPNETs only. Global gene expression analysis has enabled the identification of candidate genes and pathways that will help to elucidate the molecular basis of sPNETs. Differential expression has also been demonstrated between sPNET and medulloblastoma.

BIO 27. GENETIC AND GENOMIC ANALYSIS OF CHOROID PLEXUS TUMORS REVEALS DISTINCT MOLECULAR PHENOTYPES AND CAN BE TRACED TO THE GERMLINE

Uri Tabori,¹ Adam Shlien,² Berivan Baskin,² Sarah Levitt,² Jodi Lees,² Cynthia Hawkins,² and David Malkin²; ¹University of Toronto, Toronto, ON, Canada; ²ON, Canada.

Choroid plexus carcinomas (CPCs) and papillomas (CPPs) are rare pediatric brain neoplasms with variable clinical course. Insufficient understanding of the biological processes governing behavior of these tumors leads to inconsistent therapeutic approaches and poor outcomes. CPCs are frequently observed in Li-Fraumeni syndrome (LFS) families in which germline mutations in the TP53 tumor suppressor gene is associated with a high rate of multiple early onset cancers. In an attempt to determine the role of dysfunction in the TP53 pathway and genomic instability in choroids plexus tumors, we used genetic analysis of known polymorphisms in the pathway combined with high-throughput microarray platforms to examine complex molecular alterations. TP53 mutation and MDM2 SNP309 polymorphism analysis were performed on both tumors and paired germline (blood) samples. SNP array was applied to blood and available frozen samples using the Affymetrix 250K GeneChip to explore genomewide alterations. Twenty-two tumors and germline analysis revealed that all TP53 mutated tumors belonged to families with full LFS phenotype. All CPC and CPP patients without LFS phenotype were TP53 wild type in the germline. Interestingly, all tumors negative for TP53 mutation harbored the combination of the MDM2 SNP309 and TP53 codon 72 polymorphisms (p = 0.016) suggesting an alternative mechanism of p53 dysfunction. Single nucleotide polymorphism (SNP) analysis revealed extremely high copy number variation (CNV) in LFS-related tumors compared to wild-type tumors and CPP (p = 0.009). Strikingly, germline SNP analysis revealed the same pattern of very high CNV in CPC patients compared to controls (p = 0.04). Furthermore, CNV in tumors were not random but rather were extensions and propagations of germline CNV in these patients. In summary, our results revealed that while germline TP53 mutations was not consistently observed in CPC, alternative mechanisms of p53 functional abnormalities contributed to CPC tumorigenesis. CNV may possibly differentiate molecular and phenotypic subtypes in this family of tumors. The use of multiplex molecular genotyping may eventually lead to novel prognostic and therapeutic markers for choroid plexus tumors.

BIO 28. GENETIC REGULATION OF CNS STEM CELL DIFFERENTIATION AND MIGRATION: ROLE OF DLX HOMEOBOX GENES

Trung Le,¹ Molly Pind,² and David Eisenstat³; ¹Biochemistry and Medical Genetics, Winnipeg, MB, Canada; ²Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Winnipeg, MB, Canada; ³Pediatrics and Child Health, Human Anatomy and Cell Science, Manitoba Institute of Cell Biology, Winnipeg, MB, Canada.

Introduction: Stem cells are present in the developing and mature CNS as well as in neuroepithelial tumors. These stem cells divide symmetrically to produce two daughter stem cells and asymmetrically to repopulate the pool of stem cells or become committed CNS progenitors. These progenitors leave the cell cycle, commit to a neuronal or glial (astrocytic or oligodendroglial) cell fate, and migrate. This process is highly regulated by a combinatorial code of transcription factors, such as basic helix-loop helix (bHLH) or homeobox genes, expressed during defined developmental time periods and/or in specific neuroanatomical regions. Four Dlx homeobox genes (Dlx1, 2, 5, and 6) are expressed in the developing forebrain. In the Dlx1/Dlx2 double knockout mouse there is loss of tangential interneuron migration from the basal forebrain to the neocortex; these interneurons express the inhibitory neurotransmitter GABA. Furthermore, progenitors transplanted from Dlx1/Dlx2 mutant ventral telencephalon differentiate into myelinating oligodendrocytes, supporting a role for Dlx-mediated repression of oligodendrocyte precursor cell formation during forebrain development. In humans, Dlx2 is expressed in transit amplifying cells of the embryonic and adult subventricular zone (SVZ).

Methods: In order to understand the function of Dlx2 during CNS development, we have optimized chromatin immunoprecipitation (ChIP) technologies in embryonic tissues to identify Dlx2 transcriptional targets in vivo, including neuropilin-2, the receptor for Semaphorins 3A and 3F, that inhibit interneuron migration to the neocortex, and TrkB, the receptor for the neurotrophin BDNF. Subsequently, we have subcloned genomic DNA fragments bound to Dlx2 in E13.5 ganglionic eminences and generated a ChIP library of candidate Dlx2 targets (ChIP cloning). In addition, we have combined the ChIP assay with CpG island microarrays to identify putative Dlx2 transcriptional targets (ChIP-chip).

Results: The ChIP-cloning and ChIP-chip technologies may be complementary, with only partial convergence of data. To date, we have identified molecules associated with axonal guidance, the cytoskeleton, transcriptional regulation, G-protein signaling, neurotransmitter signaling, and ion channels. These candidate Dlx2 targets have been subjected to repeat ChIP analysis to confirm binding to Dlx2 in vivo, reporter gene assays to assess the functional significance of their regulation by DLx2 in vitro, as well as gene expression assays (quantitative PCR, in situ hybridization, and immunohistochemistry) in wild-type and Dlx1/Dlx2 mutant mouse forebrain. We are particularly focused on identification of oligodendroglial targets that may be repressed by Dlx2, including genes critical to myelination in the developing nervous system.

Conclusion: Before application of stem cell and/or novel differentiation therapies to CNS tumors of the developing nervous system, it will be necessary to understand the network of transcription factors and their target genes required for terminal differentiation and migration of committed CNS progenitors.

BIO 29. GENETICALLY ENGINEERED MOUSE MODEL OF BRAINSTEM GLIOMA

Oren Becher,¹ Dolares Hambardzumyan,² Timothy Gershon,¹ and Eric Holland³; ¹Memorial Sloan-Kettering Cancer Center, New York, NY, USA; ²Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY, USA; ³Cancer Biology and Genetics, Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Using the RCAS/tv-a technology for somatic cell gene transfer we have recently observed that we can generate brainstem gliomas (BSGs) by infecting nestin expressing neural stem cells in the posterior fossa of neonatal pups with PDGF, or by coinfection with Kras and AKT. As previously described for cortical gliomas, PDGF-induced BSGs are oligodendrglial in histology while Kras and AKT induced BSGs are astrocytic in histology. Cooperating genetic alterations such as INK4A-ARF loss can accelerate tumorigenesis (penetrance of >90% and latency of <8 weeks). These BSGs are highly aggressive, have high-grade histological characteristics such as microvascular proliferation and pseudopalisading necrosis, and can invade around the basilar artery. Interestingly, overexpression of Kras, AKT, as well as PDGFR has been noted in human samples of pediatric BSGs. This BSG model which recapitulates the genetic alterations of the human disease, and forms in its native environment may serve as an excellent preclinical model to better understand the biology of these tumors as well as to test novel agents for the treatment of pediatric brainstem gliomas.

BIO 31. GLOBAL ANALYSIS OF THE MEDULLOBLASTOMA EPIGENOME IDENTIFIES DISEASE SUBGROUP-SPECIFIC INACTIVATION OF COL1A2

Steven Clifford,¹ Jennifer Anderton,¹ Janet Lindsey,¹ Meryl Lusher,¹ Richard Gilbertson,² Simon Bailey,¹ and David Ellison²; ¹University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK; ²St. Jude Children's Research Hospital, Memphis, TN, USA.

Candidate gene investigations have indicated a significant role for epigenetic events in the pathogenesis of medulloblastoma, the most common malignant brain tumor of childhood. To assess the nature of the medulloblastoma epigenome more comprehensively, we undertook a genomewide investigation to identify genes that display evidence of methylation-dependent regulation. Expression microarray analysis of medulloblastoma cell lines following treatment with a DNA methyltransferase inhibitor revealed the methylation-dependent regulation of multiple transcripts (3%–6% of probes per cell line). Eighteen independent genes demonstrated >3-fold reactivation in all cell lines tested and were selected for detailed characterization. Bisulphite sequence analysis revealed dense CpG island methylation associated with transcriptional silencing for 12 of these genes. Analysis of the methylation status of these genes in primary tumors and the normal cerebellum revealed three major classes of epigenetically regulated genes in medulloblastomas: (1) normally methylated genes (DAZL, ZNF157, ASN), whose methylation in tumors reflects somatic patterns observed in the cerebellum; (2) X-linked genes (MSN, POU3F4, HTR2C), which show complex disruption of their normal sex-specific methylation patterns in tumors; and (3) tumor-specific methylated genes (COL1A2, S100A10, S100A6, HTATIP2, CDH1, LXN), which display enhanced methylation levels in tumors compared to the cerebellum. Detailed analysis of COL1A2 supports a key role in medulloblastoma tumorigenesis; dense biallelic methylation was observed in 46 of 60 cases (77%) and was associated with silencing of mRNA and protein expression. Moreover, COL1A2 status distinguished infant medulloblastomas of the desmoplastic histopathological subtype, indicating a distinct molecular pathogenesis may underlie these tumors and their more favorable prognosis. These data (1) reveal a more diverse and expansive medulloblastoma epigenome than previously understood; (2) identify novel molecular events in its pathogenesis; and (3) provide strong evidence that the methylation status of specific genes provide biological markers for the discrimination of specific disease subgroups.

BIO 32. HIGH-RESOLUTION GENOMIC ANALYSIS IDENTIFIES DISTINCT GROUPS OF PEDIATRIC SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL BRAIN TUMORS

Kyle Lee,¹ Meihua Li,² Charles Eberhart,³ Ching Lau,⁴ Scott Pomeroy,⁵ Peter Collins,⁶ Piergiorgio Modena,⁷ Amar Gajjar,⁸ Eric Bouffet,⁹ Michael Taylor,² Cynthia Hawkins,² and Annie Huang⁹; ¹University of Toronto, Toronto, ON, Canada; ²ON, Canada; ³Johns Hopkins University, Baltimore, MD, USA; ⁴Houston, TX, USA; ⁵Boston, MA, USA; ⁶UK; ⁷Pediatrics, National Tumor Institute, Milan, Italy; ⁸Memphis, TN, USA; ⁹Toronto, ON, Canada.

Primitive neuroectodermal tumors (PNETs) of the supratentorial region are rare, highly malignant embryonal brain tumors affecting young children. Although supratentorial PNETs (sPNET) are histologically indistinguishable from infratentorial PNETs/medulloblastomas, they are characterized by more aggressive clinical phenotypes that suggest sPNET represent distinct biological entities from medulloblastoma. In contrast to considerable progress in understanding the signaling pathways involved in medulloblastoma biology, little is known about which genes contribute to sPNET pathogenesis. Prior studies with low-resolution comparative genomic hybridization (CGH) techniques indicate sPNETs have frequent genomic imbalances and copy number aberrations (CNAs). We have utilized the ultrahigh resolution of the Affymetrix 500K single nucleotide polymorphism (SNP) array and gene expression profiling in order to map potential disease-related genes within CNAs and obtain a more comprehensive characterization of the molecular aberrations underlying sPNET development and tumor progression. Genomic DNA from 41 primary and 4 recurrent sPNET samples were analyzed using the Affymetrix 500K SNP genotyping microarrays. All tumors with available material were tested for expression of the INI1 protein to exclude tumors that may represent rhabdoid tumors. Preliminary data analysis reveals frequent CNAs across the sPNET genome, which encompasses both large and focal chromosome segments. Tumors can be segregated into at least two groups based on DNA copy number changes: tumors with frequent CNAs and tumors with relatively few or no CNAs detected. In addition, as reported in prior studies, we observed a lack of isochromosome 17q in all sPNET, an abnormality found in approximately a third of medulloblastomas. We observed large regions of copy number gains on 1q (27% of samples), as well as amplifications at 12q14.1 (5%) and 13 additional amplification events in distinct genomic loci. Frequent regions with DNA copy number losses included 10q and 13q (21% for each), 22q (17%), as well as a germline deletion at 21q21.1 and homozygous deletions at 13q14.2, 14q31.2, and 16q23.3. Correlations of specific CNAs with gene expression data indicate frequent aberrations of cellular adhesion pathways in sPNET. Further characterization and validation of these loci are underway and may direct future developments in therapy.

BIO 33. IDENTIFICATION AND CHARACTERIZATION OF A NOVEL DE NOVO GERMLINE P53 MUTATION WITH A FULMINANT CLINICAL COURSE

Mathew Schneiderjan,¹ Bahig Shehata,² Timothy Mapstone,³ Fredrick Barr,⁴ Robert Marcus,² Nadia Esiashvili,² Catherine Stockwell,² and Anna Janss⁵; ¹Neuropathology, Emory University, Atlanta, GA, USA; ²GA, USA; ³OK, USA; ⁴PA, USA; ⁵Emory University, Decatur, GA, USA.

Introduction: We present a case of a child with a unique germline p53 mutation who died with three concurrent malignancies.

Clinical Course: A 14-year-old male presented with escalating nausea and vomiting over 1 week and was found to have increased intracranial pressure due to a right frontal tumor and left intraventricular tumor. Neither lesion had been present in a brain CT scan performed 2 years prior. Staging showed no evidence of leptomeningeal dissemination. Pathologic diagnosis on the right frontal tumor was of a primitive neuroectodermal tumor (PNET). The second lesion, resected 1 month later, was a choroid plexus carcinoma. Identification of synchronous distinct brain tumors prompted genetic testing for predisposition to malignancy and systemic evaluation for cancer including bone scan and MRI of the chest, abdomen, and pelvis. Results were negative for other tumors. The child underwent craniospinal radiation (36 Gy) with boosts to primary tumors (20 Gy), followed by chemotherapy using cisplatin, vincristine, and CCNU. Within 5 months of presentation the child developed hip pain and subsequent surveillance imaging of his CNS revealed tumoral infiltration of the vertebral bodies, pelvis, hips, and liver. Staging procedures could not be performed due to hepatic failure with hyperammonemia and coagulopathy. The child died of respiratory failure 6 months after presentation. His systemic cancer was evaluated by autopsy.

Family History: The patient was survived by two parents, a younger brother, paternal grandparents, and maternal grandmother, all without history of malignancy. An older sibling was stillborn.

Pathology: Right frontal tumor—PNET, pleomorphic, poorly differentiated malignant neoplasm composed predominantly of small cells with scant cytoplasm, vesicular chromatin, and prominent nucleoli. There were multiple areas of necrosis and numerous tumor giant cells. Tumor cells stained positive for neurofilament, GFAP (focal), synaptophysin (focal), and p53 and negative for pan-cytokeratin (AE 1/3) and EMA. MIB-1 proliferation index was 80%–90%. Intraventricular tumor—choroid plexus carcinoma, pleomorphic epithelial cells with a high nuclear:cytoplasmic ratio and irregular, hyperchromatic nuclei arranged in papillary and solid patterns. Tumor cells were reactive for AE 1/3, GFAP (focal), and p53 and negative for CEA (monoclonal). Hepatic and bone tumor—alveolar rhabdomyosarcoma, clusters of large pleomorphic cells with hyperchromatic nuclei with high nuclear:cytoplasmic ratio arranged in an alveolar pattern. Tumor cells were reactive for myogenin and desmin and were negative for CD3, CD20, CD45, myeloperoxidase, AE 1/3, vimentin, lysozyme, EMA, synaptophysin, GFAP, and S-100. Cytogenetic analysis demonstrated a translocation, t(2;13)(q35;q14), characteristic of alveolar rhabdomyosarcoma.

Genetic Analysis: Sequencing of patient DNA showed a previously undescribed p53 allele with a premature stop codon in the oligomerization/nuclear export signal (NES) domain (R342ter). The patient's brother tested negative for p53 mutations. The patient's parents are untested for p53 mutations but have no malignancy to date.

Conclusions: This case identifies a novel germline p53 mutation and shows a more fulminant course than typical cases of Li-Fraumeni syndrome. We discuss potential causes for the unusually aggressive course of this patient's illness and suggest that the aggressive course may be due to the nature of the mutation.

BIO 34. IDENTIFICATION OF ANTIGENS ON CANCER STEM CELLS FOR BRAIN TUMOR THERAPY

Vidyalakshmi Chandramohan,¹ Charles N. Pegram,¹ Stephen T. Keir,¹ Chien-Tsun Kuan,¹ and Darell D. Bigner¹; ¹Pediatric Brain Tumor Foundation Institute, Duke University Medical Center, Durham, NC, USA.

Approximately 26,000 children have brain tumors and >3,000 new pediatric brain tumor cases are diagnosed every year in the United States alone. Brain tumors are the leading cause of cancer death for children <20 years, and there are >120 different types of childhood brain tumors. Recent findings in human leukemia and breast cancer strongly implicate cancer stem cells as the undercurrent that fuels the growth of many human cancers, and the concept of cancer stem cells has been extended to brain tumors. The discovery of brain tumor stem cells (BTSCs), which have biological properties that are distinct from the bulk of tumor cells, has identified new targets for brain tumor treatment. In the present study, we demonstrated screening of pediatric brain tumors for the presence of BTSCs and further analyzed the expression of known brain tumor antigens on these cancer stem cells. BTSCs are known to proliferate and generate multipotent clones of cells in vitro, termed neurospheres, in the presence of growth factors such as epidermal growth factor and basic fibroblast growth factor 2. Neurospheres were isolated from the pediatric xenografts D456MG, D2159MG, and D2224MG and were found to be positive for the expression of the stem cell marker CD133. The CD133-positive BTSCs were further screened for the expression of known brain tumor–associated antigens: wild-type epidermal growth factor receptor (EGFRwt), mutant EGFR variant III (EGFRvIII), chondroitin sulfate proteoglycan (CSPG), interleukin 13 receptor alpha 2 (IL13R2), glycoprotein nonmetastatic melanoma protein B (GPNMB), 3',6'-iso-LD1, 3'-iso-LM1, and multidrug resistance protein 3 (MRP3). The D2159MG cells that were positive for the tumor antigens EGFRwt (20%), EGFRvIII (35%), CSPG (17%), and MRP3 (8%) were also positive for CD133. The overexpression of EGFRwt (24-fold) and MRP3 (22-fold) mRNA was confirmed by Q-PCR in D2159MG neurospheres. Analysis revealed that the EGFRvIII mRNA was expressed only in the D2159MG neurospheres and not in the normal human neural progenitor population, confirming it as a tumor specific antigen. The expression of EGFRwt and EGFRvIII proteins in the D2159MG neurospheres was further demonstrated by Western blot analysis. Next, 13% of the D456MG cells were found to express both CD133 antigen and the tumor antigen CSPG. Finally, analysis of the D2224MG cells revealed the coexpression of CD133 and the tumor antigens 3',6'-iso-LD1 (85%), 3'-iso-LM1 (94%), CSPG (91%), and IL13R2 (71%). Thus, using specific immunotoxins or radioimmunotherapy to target the identified tumor antigens expressed by BTSCs should result in significant survival increases in pediatric brain tumor patients.

BIO 35. IDENTIFICATION OF MEDULLOBLASTOMA SUBTYPES WITH DISTINCT GENETIC PROFILES, PATHWAY SIGNATURES, AND CLINICOPATHOLOGICAL FEATURES

Marcel Kool,¹ Jan Koster,¹ Jens Bunt,¹ Dirk Troost,² Netteke Schouten-Van Meeteren,³ Bauke Ylstra,⁴ Wieslawa Grajkowska,⁵ Wolfgang Hartmann,⁶ Torsten Pietsch,⁶ David Ellison,⁷ Steven Clifford,⁸ and Rogier Versteeg¹; ¹Human Genetics, Academic Medical Center, Amsterdam, Netherlands; ²Neuropathology, Academic Medical Center, Amsterdam, Netherlands; ³Pediatric Oncology, Academic Medical Center, Amsterdam, Netherlands; ⁴Pathology, VU University Medical Center, Amsterdam, Netherlands; ⁵Pathology, Children's Memorial Health Institute, Warsaw, Poland; ⁶Neuropathology, University of Bonn, Bonn, Germany; ⁷St. Jude Children's Research Hospital, Memphis, TN, USA; ⁸Northern Institute for Cancer Research, Newcastle-upon-Tyne, UK.

Medulloblastoma is the most common malignant brain tumor in children. We established gene expression profiles of 62 medulloblastomas using Affymetrix HG-U133 plus 2.0 GeneChips, and 52 of them were also analyzed by comparative genomic hybridization (CGH) using 30K oligonucleotide arrays. Five molecular subtypes were identified, characterized by WNT pathway activation (type A, 9 cases), sonic hedgehog pathway activation (type B, 15 cases), neuronal differentiation genes (types C and D, 16 and 11 cases, respectively) and photoreceptor genes (types D and E, both 11 cases). Activation of the WNT pathway in type A tumors was in all nine cases caused by mutations in the beta-catenin gene. No beta-catenin mutations were found in tumors of other subtypes. PTCH1 mutations, which lead to activation of the sonic hedgehog pathway, were identified in 4 of 15 type B tumors, but not in tumors of the other subtypes. Also, several fully or partly subtype-specific chromosomal aberrations were found, such as loss of chromosome 6 only in type A tumors, loss of 9q sequences in type B tumors, or loss of chrom 8 and gain of 17q in type C and D tumors. RNA expression levels for genes located on these chromosomes faithfully reflected the identified chromosomal copy number changes. We also found several striking associations between the molecular subtypes and clinicopathological parameters. Metastatic disease at diagnosis was associated with subtypes C and D and most strongly with subtype E (p = 0.005). Also, age at diagnosis was significantly different among the five subtypes (p = 0.006). All patients <3 years of age had either type B or DE tumors (p = 0.0006). Type B contained most desmoplastic cases (p = 0.046). We validated and confirmed these molecular subtypes and the associated clinicopathological parameters using data of a previously published series of 46 medulloblastomas (Thompson et al. J Clin Oncol. 2006;24). Medulloblastoma patients have a poor prognosis. Despite recent improvements in cure rates, survivors suffer from serious side-effects caused by the intensive therapy. Recent data showed that patients with WNT-activated tumors respond very well to current therapies (Ellison et al. J Clin Oncol. 2005;23; Gaijar et al. Lancet Oncol. 2006;7), suggesting that these patients can be treated less severely, thereby reducing the side effects. These results demonstrate the need of a correct classification of medulloblastoma patients and a detailed knowledge of oncogenic signaling pathways in each subtype. The medulloblastoma subtypes presented in our study will therefore be of great importance for a better understanding of this heterogeneous disease and for a better selection of patients for future clinical trials of new molecular-targeted therapies. Ultimately, our results may lead to more individualized therapies with improved cure rates and a better quality of life.

BIO 36. IDENTIFICATION OF NOVEL CHROMOSOMAL ALTERATIONS SPECIFIC TO PEDIATRIC HIGH-GRADE ASTROCYTOMAS

Karine Jacob,¹ Huiqi Qu,² Olivier Delattre,³ Alexandre Montpetit,² Lauren Solomon,⁴ Peter Hauser,³ Miklos Garami,³ László Bognár,⁵ Zoltan Hansely,³ Constantin Polychronakos,⁶ Cynthia Hawkins,⁴ and Nada Jabado⁶; ¹Human Genetics, Montreal Children's Hospital, Montreal, QC, Canada; ²QC, Canada; ³France; ⁴ON, Canada; ⁵Neurosurgery, University of Debrecen, OEC, Debrecen, Hungary; ⁶Montreal, QC, Canada.

Background: Brain tumors are the largest group of solid neoplasms in children and are currently the leading cause of cancer-related mortality and morbidity in the pediatric years. Pediatric glioblastoma (pGBM) is a rare but devastating brain tumor. Several studies suggest that pGBMs may be biologically distinct from their adult counterparts, despite being histologically identical. In contrast to GBM in adults (aGBM), relatively little is known in children about the molecular mechanisms underlying its development and progression, and the low number of clinical samples available has resulted in very few studies of this cancer. Available data on genetic events in pediatric high-grade astrocytomas (pHGAs) are scarce. This has traditionally been a major impediment to understanding the pathogenesis of this tumor and to developing ways for more effective management. The aim of this study is to chart DNA copy number aberrations (CNAs) in pHGAs and get insight into genetic pathways involved in gliomagenesis in children.

Methods: Using the Illumina Infinium Human1 bead chip array (100K SNPs), we genotyped 19 pediatric and 6 adult HGAs. Results were compared to BAC-array profiles harvested on 16 of the same pHGAs to an independent data set of 9 pHGAs analyzed on Affymetrix 204K SNP arrays and to existing data sets on HGA. CNAs were additionally validated by real-time quantitative PCR in a set of genes in pHGA.

Results: Our results show an unexpected low frequency of genetic amplification and complete deletions and high frequency of loss of heterozygozity in a high-grade rapidly dividing tumor. The most common copy number alterations were LOH on chromosome 10q22, 15q15, 17p13, and 22q12. Despite commonalities, most CNAs in pHGAs were distinct from CNAs in aHGAs. Novel genomic regions identified with nonrandom clustering of CNAs, suggest that alterations in tumor suppressors and genes involved in the regulation of RNA processing and the cell cycle are major events in the pathogenesis of pHGA. To further characterize this tumor, integrative studies comparing those genomic results with previous results obtained through a transcriptional analysis are now being performed.

Conclusions: This first complete profiling of the tumor cell genome using high-resolution SNP arrays fills an important gap in studies on pHGA and may ultimately guide mapping of oncogenic networks unique to pHGA. It further shows that pHGA and aHGA are two diametrically different disease contexts. Our findings suggest that alterations in tumor suppressor genes involved in the regulation of the cell cycle are major events in pHGA pathogenesis and may ultimately lead to a search for more specific molecular therapeutic targets.

BIO 37. IDENTIFICATION OF PATHWAYS INVOLVED IN THE PATHOGENESIS OF CHOROID PLEXUS PAPILLOMAS

Martin Hasselblatt,¹ Sonja Mertsch,¹ Astrid Jeibmann,¹ Barbara Riesmeier,¹ Heike Stegemann,² Brigitte Wrede,³ Johannes Wolff,⁴ and Werner Paulus¹; ¹Institute of Neuropathology, University Hospital Muenster, Muenster, Germany; ²Integrated Functional Genomics, IZKF, University Hospital Muenster, Muenster, Germany; ³Department of Pediatric Oncology, University of Regensburg, Regensburg, Germany; ⁴The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

The pathogenesis of choroid plexus papillomas, intraventricular papillary neoplasms predominantly occurring in children and young adults, remains uncertain. In order to identify genes differentially expressed in choroid plexus papillomas, gene expression profiles obtained from laser-microdissected choroid plexus papilloma cells (n = 7) were compared to those of normal choroid plexus epithelial cells laser-microdissected from human autopsy tissue (n = 8). On DNA microarray data analysis, a total of 55 probe sets were found to be differentially expressed in choroid plexus papilloma tumor cells (>7-fold), up-regulation of TWIST1, TRPM3, BCLAF1, AJAP1, WIF1, and TAC1; down-regulation of IL6ST and SPACL1 was also confirmed using quantitative RT-PCR. Knockdown of TWIST1 gene expression in the rat choroid plexus epithelial cell line Z310 reduced proliferation as assessed by MTT assay, whereas cell migration was not significantly affected. To conclude, TWIST1 is among the genes differentially expressed in choroid plexus papillomas and promotes proliferation in vitro. The functional role of other identified genes is currently being examined.

BIO 38. IMMUNOMODULATORY EFFECTS OF GLIOBLASTOMA CELLS: INFLUENCE ON MONOCYTE EXPRESSION OF THE GRANZYME B INHIBITOR PROTEINASE INHIBITOR 9 (PI-9)

Carl Classen¹; ¹University Children's Hospital Rostock, Rostock, Germany.

Glioblastomas are known to influence the immune system in many ways; this is of particular interest since vaccine strategies increasingly show promising results in glioblastoma therapy. Proteinase inhibitor 9 (PI-9)— the only known endogeneous natural antagonist of the lymphocyte protease granzyme B (GrB)—is an intracellular serpin expressed in lymphocytes and in monocyte-derived cells. By intracellular flow cytometry, we have previously shown that ex vivo stimulation by lipopolysaccharides (LPS) leads to up-regulation of PI-9 within 24 h in the monocyte, but not the lymphocyte fraction; this can be inhibited by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Here, we studied the influence of the glioblastoma cell lines GMS-10 and U-138-MG on the spontaneous and LPS-induced expression of PI-9 in monocytes in a coincubation assay. We found that the presence of glioblastoma cells in a 10:1 and even a 1:100 ratio with PBMC lead to PI-9 up-regulation in monocytes, similar to the up-regulation induced by LPS. Combination of coincubation with LPS did not further enhance PI-9 expression. It seems that the effect does not require cell-cell contact, since the supernatant of GMS-10 cells was sufficient to induce PI-9 up-regulation. Since it has been shown that PI-9 overexpression in antigen presenting cells (e.g., dendritic cells) leads to an enhanced immune response by protection of cells from activation-induced or bystander kill, our findings may be relevant for specific immune therapy. Understanding the regulation of PI-9 expression in monocyte-derived cells in glioblastomas might help to optimize the strategies in dendritic cell-based vaccine therapies.

BIO 39. INDUCIBLE CRE RECOMBINASE ACTIVITY IN MOUSE MATURE ASTROCYTES AND ADULT NEURAL PRECURSOR CELLS: DEVELOPING NOVEL ANIMAL MODELS FOR GLIOMA

Lionel Chow,¹ Junyuan Zhang,¹ and Suzanne Baker¹; ¹St. Jude Children's Research Hospital, Memphis, TN, USA.

Astrocytes are the most abundant glial cell type in the mammalian brain and play important roles in all aspects of the developing and mature organ. They are proposed to be at the root of numerous pathological processes and may be the cell of origin for the most common primary brain tumor, glioma. The availability of the appropriate molecular tools to manipulate gene expression in vivo in this specific cell type is therefore critical to further our understanding of gliomagenesis. Many genetically engineered models for human disease currently employ the bacteriophage P1 creatine (Cre) recombinase-LoxP system, which enables gene manipulation in a tissue- and cell-specific manner. A large number of brain-specific Cre mouse strains have been developed, including those using the glial fibrillary acidic protein (GFAP) promoter to drive astrocyte-specific expression of Cre. Because Cre-mediated recombination is irreversible, embryonic expression in neural precursor cells directed by the GFAP promoter and/or aberrant expression resulting from transgene insertion effects have resulted in substantial Cre-mediated recombination in mature neurons as well as glial cells in all GFAP-Cre mice characterized to date. Therefore, the ability to restrict Cre activity during embryogenesis could greatly mitigate neuronal Cre-mediated recombination in GFAP-Cre mice and allow for the study of gene function in mature astrocytes without disruption of function in neurons. Cre activity can be regulated in a temporal fashion by engineering a fusion protein with the ligand-binding domain of a steroid hormone receptor, such as the estrogen receptor. Furthermore, a specific point mutation within the ligand-binding domain termed ERTM abolishes the binding to endogenous steroid hormones while retaining its interaction with synthetic estrogen analogs such as tamoxifen. The fusion protein, CreERTM, is inactive while sequestered in the cytoplasm by heat shock protein complexes. Binding of tamoxifen releases CreERTM from this complex allowing ligand-dependent translocation to the nucleus where the fusion protein is active and directs recombination between loxP sites. Two transgenic mouse lines expressing an inducible form of the Cre recombinase (CreERTM) under the control of the human GFAP promoter have been generated and characterized. In adult mice, expression of the fusion protein is largely confined to astrocytes in all regions of the CNS. Minimal spontaneous Cre activity was detected and recombination was efficiently induced by intraperitoneal administration of tamoxifen in adult mice. The pattern of recombination closely mirrored that of transgene expression. The percentage of astrocytes undergoing recombination varied from region to region ranging from 35% to 70% while a much smaller portion (<1%) of oligodendrocytes and neural precursor cells showed evidence of Cre activity. The degree of recombination varied directly and consistently with the dose of tamoxifen administered to mice. Using these new Cre mouse strains, we have also engineered several novel models for spontaneously arising gliomas.

BIO 40. MAGIC ANGLE SPINNING METABOLITE PROFILES CHARACTERIZE GLIAL AND PRIMITIVE NEUROECTODERMAL TUMORS

Martin Wilson,¹ Marie-Anne Brundler,² Carmel Mcconville,³ Richard Grundy,⁴ and Andrew Peet⁵; ¹University of Birmingham, Edgbaston, Birmingham, West Midlands, UK; ²Pathology, Birmingham Children's Hospital NHS Foundation Trust, Birmingham, UK; ³Birmingham, Western Australia, Australia; ⁴Children's Brain Tumour Research Centre, Queens Medical Centre, Nottingham, Nottinghamshire, UK; ⁵Academic Paediatrics and Child Health, University of Birmingham, Edgbaston, Birmingham, West Midlands, UK.

Introduction: ¹H high-resolution magic angle spinning NMR spectroscopy (MAS) is a technique that measures concentrations of multiple metabolites in small pieces of tissue leaving the sample remains intact. The metabolite profiles provide a powerful characteristic of tumor tissue and reflect tumor biology. The metabolic pathways implicated in specific tumors provide targets for new agents. Small sample size, minimal sample preparation, and the potential for rapid analysis also makes the technique a novel candidate for rapid intraoperative diagnosis. In this study, MAS is combined with automated analysis to explore differences between glial and primitive neuroectodermal tumors (PNET) in children.

Methods: Forty tissue samples were collected from a total of 29 children at diagnostic and therapeutic operations. Each sample was obtained prior to the patient receiving adjuvant treatment. The PNET group comprised nine medulloblastomas, one supratentorial PNET, and seven neuroblastoma cases. The glial tumors comprised 10 juvenile pilocytic astrocytomas and 2 ependymomas (grade II). Biopsy tissue (5–20 mg) was snap frozen in liquid nitrogen shortly after resection and stored at –80°C. MAS was performed on a Varian 600-MHz spectrometer using a 4-mm gHX nanoprobe. The probe temperature was set to 0.1°C, giving a sample temperature of 6.7°C, and the sample was spun at 2,500 Hz. A standard pulse and acquire sequence was used with water presaturation giving a total acquisition time of 28 min. The TARQUIN algorithm was used to fit the metabolite components of the signal, automatically quantifying a range of metabolites. Metabolites were normalized to the real part of their fitted spectral contribution between 0.5 and 4.5 ppm. A principal component analysis was performed on the standardized metabolite quantities and a two-tailed t-test performed to determine significant differences between the metabolite quantities in glial tumors versus PNETs and medulloblastomas versus neuroblastomas.

Results: Glial and PNET tumors formed two distinct groups in the principal component analysis showing that the two tumor groups have characteristic MAS metabolite profiles. The principal component analysis also showed that the medulloblastomas formed a group that was distinct from neuroblastomas. Statistically significant differences (p < 0.05) between PNET and glial tumor specimens were found in 7 of the 17 metabolites analyzed. Taurine was found to be significantly elevated in PNETs, and the ratio of phosphocholine:glycerophosphocholine was higher in PNETs. Significant differences were also found in 8 of the 17 metabolites between medulloblastoma and neuroblastoma specimens.

Conclusion: MAS combined with automated analysis can provide rapid information on the biochemistry of childhood tumor tissue. MAS metabolite profiles are a powerful characteristic of tumor type for PNETs and glial tumors and can detect significant differences between medulloblastomas and neuroblastomas despite their similar morphology demonstrating the potential of the technique as a rapid diagnostic aid. Key differences are seen in taurine and choline metabolism in PNETs compared with glial tumors, in keeping with in vivo magnetic resonance spectroscopy findings and demonstrating that MAS can identify key molecular pathways in these tumors.

BIO 41. MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF PEDIATRIC HIGH-GRADE GLIOMA CELL LINES AS MODELS FOR PRECLINICAL DRUG DEVELOPMENT

Dorine Bax,¹ Lynley Marshall,¹ Nathalie Gaspar,² Lara Perryman,¹ Suzanne Little,¹ Marta Viana-Pereira,³ Gilles Vassal,⁴ Rui Reis,³ Sue Eccles,² Paul Workman,² Andrew Pearson,⁵ David Ellison,⁶ Darren Hargrave,⁵ and Chris Jones¹; ¹Paediatric Oncology, Institute of Cancer Research, Sutton, Surrey, UK; ²Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK; ³Life and Health Science Research Institute (ICVS), University de Minho, Portugal; ⁴Institut Gustave Roussy, Villejuif, Paris, France; ⁵Paediatric Oncology, Royal Marsden Hospital, Sutton, Surrey, UK; ⁶Neuropathology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Although pediatric high-grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. Understanding and exploiting these similarities and differences is an important strategy for the development of new targeted therapies. One important factor hampering this is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumors. We have carried out a detailed molecular and phenotypic characterization of a series of pediatric high-grade glioma cell lines in comparison to routinely used adult lines. The panel comprised two patients with pediatric glioblastoma multiforme (GBM) (SF188: 8 years old, male; KNS42: 16 years old, male); one with pediatric anaplastic astrocytoma (UW479: 13 years old, female); and six with adult GBMs (LN229, A172, U118MG, U138MG, U87MG, SF268). All lines proliferate as adherent monolayers with doubling times of 26–48 h and express glial fibrillary acidic protein (GFAP), S100 protein, synaptophysin, and/or vimentin. Classical cytogenetics revealed highly complex karyotypes for all three pediatric lines, underpinned by copy number profiling using Affymetrix 500K SNP and Agilent 44K CGH arrays. SF188 harbored multiple high-level amplifications including MYC, CCND1, and CDK4, which were confirmed by fluorescence in situ hybridization on metaphase spreads. KNS42 contained numerous low-level gains, such as at 3q26 harboring the PIK3CA oncogene, and losses including 13q13-q14, encompassing the BRCA2 and RB1 loci. UW479 was highly rearranged and harbored a homozygous deletion of the CDKN2A locus, among others, as well as extensive gene promoter hyper methylation, as assessed by methylation-specific MLPA. Constitutive protein expression by Western blot analysis showed Akt pathway activation in all pediatric and adult lines, although the pediatric lines were all PTEN wild-type. This was in contrast to a lack of phosphorylated GSK3beta and S6 kinase in the pediatric lines compared with the majority of the adult lines. KNS42 demonstrated high levels of phospho-ERK, not observed in SF188 and UW479. Expression profiling using Affymetrix U133 Plus2.0 arrays revealed 301 differentially expressed genes that we were able to distinguish between the adult and pediatric high-grade cell lines. These included overexpression in pediatric lines of kinases of the Src family, including YES1 and LYN, and low levels of the receptor tyrosine kinase AXL, highly expressed in adult lines. All three pediatric lines were successfully grown as subcutaneous xenografts in nude mice. These data demonstrate that high-grade glioma cell lines derived from pediatric patients show key molecular differences to those from adults, some of which are well known, while others may provide novel targets for evaluation in primary tumors. We thus provide the rationale and demonstrate the practicability of using pediatric high-grade glioma cell lines for in vitro and in vivo preclinical and mechanistic studies.

BIO 42. MOLECULAR INVERSION PROBES (MIPS) IDENTIFY NOVEL COPY NUMBER CHANGES IN PEDIATRIC ASTROCYTOMAS

Joshua Schiffman,¹ Scott Vandenberg,² Paul Fisher,¹ James Ford,¹ Hanlee Ji,¹ and Graeme Hodgson²; ¹Stanford University, Palo Alto, CA, USA; ²University of California, San Francisco, San Francisco, CA, USA.

Background: Childhood brain tumors (CBTs) are the most common solid pediatric cancer and the leading cause of pediatric cancer mortality. More than half of all CBTs are gliomas, but little is known about the genetic events that contribute to the development and progression of these tumors. To address this, we used molecular inversion probes (MIPs) to identify recurrently amplified or deleted genomic loci in advancing stages of pediatric astrocytomas (PAs). This study takes important steps toward a molecular classification of PAs, which is critical for the development of biomarkers and patient-specific therapeutic approaches for disease treatment.

Methods: DNA was extracted from 14 flash-frozen PA biopsies (WHO grade II [n = 3, mean age, 7 years]; grade III [n = 2, mean age, 6 years], grade IV [n = 9, mean age, 12 years]). DNA was also extracted from five adult-gliosis biopsies to use as nonneoplastic controls. The MIP assay was run using 37 ng genomic DNA sample on a customized Affymetrix 24K Cancer Panel representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes. Copy number (CN) changes were identified by comparing probe signal intensity between tumors and controls.

Results: We observed the full spectrum of genomic CN aberrations including regions of single copy gain and loss, homozygous deletions, and high-level focal amplifications. Grade III and IV tumors showed more CN alterations relative to grade II tumors. Regions of high-level amplification (CN>5) involved chromosome bands: 7q34–36 (10.8 Mb) and 12p13 (4.7 Mb) in one grade III tumor; 1q32 (3.1Mb), 3q13 (2.8 Mb), and 8q24 (1.1 Mb) in one grade IV tumor; and 4q12 (0.65 Mb) in two grade IV tumors. At least 218 known genes map within these amplicons, including well-established oncogenes such as MYCC (8q24), PDGFRA, KIT (4q12), and CCND2 (12p13). Other amplified genes include those that have been previously implicated in glioma development such as the Forkhead transcription factor FOXM1, and those that are known to regulate Wnt-signaling (WNT5B) and p53 activity (MDM4). Further studies will help delineate the extent to which these genes contribute to PA development, and the utility of these genes as biomarkers and therapeutic targets for PA treatment.

Conclusions: The MIP platform enabled the identification of low-level (1–4 copies) and high-level (>5 copies) copy number aberrations in pediatric astrocytomas from relatively small amounts of genomic DNA. Pediatric astrocytomas displayed marked heterogeneity in genomic copy number, suggesting that a comprehensive assessment of copy number aberrations will be required to enable accurate molecular subclassification of PAs. The MIP platform is well suited for such large-scale analyses because of its compatibility with formalin-fixed paraffin-embedded specimens.

BIO 43. MOLECULAR PORTRAITS OF EPENDYMOMA RECURRENCE

Matthieu Peyre,¹ Frederic Commo,¹ Carmela Dantas-Barbosa,¹ Stephanie Puget,² Ranjeev Bhangoo,¹ Ludovic Lacroix,¹ Francoise Drusch,¹ Pierre Varlet,³ Veronique Scott,¹ Philippe Dessen,¹ Christian Sainte-Rose,² Gilles Vassal,¹ and Jacques Grill¹; ¹Institut Gustave Roussy, Villejuif, France; ²Necker Sick Children's Hospital, Paris, France; ³Saint Anne Hospital, Paris, France.

Purpose: Recurrence is a frequent phenomenon in intracranial childhood ependymomas. To gain new insights into this process and identify pathways associated with recurrence, we compared the genomic and gene expression profiles of local recurrences with the corresponding initial tumors.

Experimental Design: We studied 27 relapses in 17 patients by comparing their gene expression profile to the one obtained at diagnosis in the same child. Recurrences analyzed occurred after surgery alone in seven cases, surgery plus chemotherapy in 10 cases, and any treatment plus radiotherapy in 11 cases. For each recurrence, gene expression levels were compared in relation to the gene's corresponding initial tumor by dual color competitive hybridization on Agilent 44K gene expression microarrays. Statistical analysis was performed after initial filtering which retained only sequences differentially expressed (p = 0.05) in at least 50% of experiments. Filtering criteria as well as the research of a common signature in recurrences and group comparisons (tumor location, treatment, delay between diagnosis and recurrence) were performed with Rosetta Resolver software. Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry were next performed on genes of interest. In parallel, genomic profiling at diagnosis and at recurrence was performed using Agilent 44K oligo-CGH DNA microarrays, and expression signatures were compared according to gene copy number changes.

Results: A subset of 298 sequences appeared to be significantly associated with recurrences (p < 0.01). Forty-eight of them did so in 60% or more of the studied recurrences. Among genes up-regulated at recurrence, some belonged to the Wnt (SFRP1, SFRP2, FZD2, FZD8, and WNT10B) and Notch (DLL1) pathways. In addition, several genes involved in proliferation were up-regulated (Ki67, 4 genes of the G2/M checkpoint and 10 genes of the kinetochore and mitotic spindle). Metallothioneins were the genes most frequently down-regulated at recurrence (60%–85% depending on the isotype). RT-PCR and IHC data on selected genes confirmed the microarray results. Most frequent copy number changes were 3p and 6p losses and 9q and 1q gains and were present in at least 20% of patients. Copy number changes increased at recurrence compared with diagnosis in nine patients and remained stable or decreased in eight patients. Comparison of microarray data with genomic imbalances revealed that most of the modifications in gene expression were not related to copy number changes. We identified one patient with amplifications at 2p (containing the N-myc gene that was overexpressed) and 11p both at diagnosis and at recurrence.

Conclusion: The most frequent events associated with ependymoma relapse include increased proliferation associated with overexpression of kinetochore proteins and down-regulation of metallothioneins. Targeting of these pathways may offer novel therapeutic options in these refractory brain tumors.

BIO 44. MOLECULAR UNDERSTANDING OF SENSITIVITY TO CHEMOTHERAPEUTIC AGENTS IN MEDULLOBLASTOMA

Daniel Meley,¹ David Spiller,¹ Michael White,¹ Heather Mcdowell,² Barry Pizer,² and Violaine See¹; ¹Centre for Cell Imaging, University of Liverpool, Liverpool, Merseyside, UK; ²Oncology, Royal Liverpool Children's Hospital, Liverpool, UK.

Introduction: Neuronal proliferation, differentiation, and migration are coordinated during cerebellar development. Disruption of these processes can lead to medulloblastoma (MB), the most common malignant pediatric brain tumor. Despite recent advances in understanding the pathogenesis of MB, the specific genetic alterations involved in the majority of these tumors are still ill defined. Here, we elucidated the specific tuning of NF-kB– and p53-dependent transcription upon chemotherapeutic treatment in MB and its correlation to resistance/sensitivity to the treatment.

Methods and Results: (1) We first determined the sensitivity of different cell lines to different chemotherapeutic agents. We used four different MB cell lines (DAOY, D283-MED together with MHH-Med-1 and MEB-Med-8A, kindly provided by Prof. Torsten Pietsch) and compared their resistance to chemotherapeutic drugs. We tested the viability of these cell lines with a time and dose response to cisplatin, methothrexate, and etoposide. The four cell lines had different sensitivity to chemotherapy. The D283-MED and DAOY cells were more sensitive than the MHH-Med-1 and MEB-Med-8A. (2) To explain these different sensitivities, we decided to further explore the activity of key transcription factors involved in death/survival balance in basal conditions and on exposure to chemotherapeutic drugs. We looked at the activity of NF-kB– and p53-dependent pathways at basal levels in the different cell lines and their activation in response to etoposide. We showed that the four cell lines displayed different sensitivity to etoposide depending on the status of both p53 and NF-kB molecules. One interesting finding was that, in all cell lines that were not p53 mutated, we observed an induction of CD95 (also called Fas) upon exposure to etoposide. (3) We then investigated the activation of CD95, a death receptor that might be responsible for the observed etoposide-induced delayed NF-kB activation. We confirmed the p53-dependent transcription of CD95 using a p53 inhibitor, Pifithrin a. The D283-MED cells were very sensitive to etoposide and displayed a proapoptotic delayed NF-kB upon etoposide treatment. The Med-1 cells activated CD95 transcription, but the NF-kB pathway was impaired in this cell line resulting in a medium sensitivity to etoposide. Conversely, the Med-8 cells had a normal NF-kB signaling, but no activity was observed upon etoposide treatment, probably due to a mutation in p53 response and the absence of CD95 activation. This was associated with a high level of resistance to chemotherapeutic agents.

Conclusion: We have demonstrated that depicting the mutation status of key transcription factors in MB could lead to enhancement of response to chemotherapy. Elucidation of etoposide-induced genes in cell lines that are not mutated in p53 may allow to target them directly and independently of p53 using specific agents and therefore improve treatment of MB that are p53 mutated.

BIO 45. NOTCH 1 MUTATIONS IN PEDIATRIC POSTERIOR FOSSA EPENDYMOMAS

Carmela Dantas-Barbosa,¹ Ludovic Lacroix,¹ Matthieu Peyre,¹ Stephanie Puget,² Ranjeev Bhangoo,¹ Patrick Saulnier,¹ Felipe Andreiuolo,¹ Alexander Valent,¹ Pascale Varlet,³ Christian Sainte-Rose,² Gilles Vassal,¹ and Jacques Grill¹; ¹Institut Gustave Roussy, Villejuif, France; ²Necker Sick Children's Hospital, Paris, France; ³Sainte Anne Hospital, Paris, France.

Purpose: A comparative genomic hybridization (CGH) array study of pediatric ependymomas revealed a significant increase of 9q34 gain in recurrent tumors compared to diagnostic tissue. Up-regulation of one of the candidate genes located on 9q34 (Notch-1) was found in most of the samples. The implications of Notch 1 mutations in others tumors such as T-ALL and breast cancer prompted us to analyze mutations in Notch pathway. In addition, we also studied the tumor suppressor gene Fbxw7/hCDC4 mutated in several cancers and involved in Notch-1 ubiquitin-dependent proteolysis.

Experimental Design: Notch sequencing of exons 26, 27, and 34 was performed in 72 ependymoma samples from 42 patients: 33 diagnosis and 39 recurrences as well as Fbxw7 sequencing of exons 5, 6, 7, 8, and 9. The tumor location was posterior fossa in 45 samples and supratentorial in 20 and 7 other localizations. 9q.34 gain was confirmed by fluorescence in situ hybridization (FISH) and measure of expression levels of Notch pathway genes, such as HES-1 by RT-PCR, and immunohistochemistry.

Results: Notch1 mutation was found in six samples from three distinct patients (8.3% of the whole samples and 20% of the samples with 9q34 gain on CGHarray); for two patients, it was found at diagnosis and recurrence; for the other, in two subsequent recurrences. All Notch-1 mutations were found among posterior fossa ependymomas samples, 6 of 45 (13.3%). Both samples from the same patient presented the same mutation: in the HD domain for one patient (V1671I) and TAD domain for the two others (G2152R and A2279V). 9q gain region was observed in 21 samples (29.2%). FISH confirmed increased copy number of the 9q34 at relapse compared to diagnosis. For the patient for whom we disposed only of relapses, 30% of cells from the first and 100% in the second recurrence presented 9q gain. Functional analysis of Notch pathway revealed Notch 1 overexpression as well as downstream Notch-regulated genes such as HES1. The Fbxw7 gene analysis did not show any mutations.

Conclusion: The presence of Notch-1 mutation at diagnosis, even in the absence of detectable 9q34 gain in CGHarray suggest that it is an early event in posterior fossa oncogenesis. The overexpression of Notch-1 was not due to this increased half-life as a consequence of FBXW7 mutations, a negative regulator of the Notch-1 by mediating its ubiquitin dependent proteolisis, since none mutation on this gene was observed. On the other hand, the Notch-1 activation can be explained, at least in the patient that presents the HD mutations, since alterations in this domain render the protein susceptible to be cleaved in a ligand-independent manner that generally results in Notch-1 activation. This is the first report of recurrent mutations in oncogene in pediatric ependymomas. The frequency of Notch-1 mutations in posterior fossa ependymomas is comparable to PTCH mutations in posterior fossa PNET/medulloblastoma.

BIO 46. P53 INDEPENDENT ABROGATION OF G1 ARREST IN MURINE BRAIN TUMOR–DERIVED STEM LIKE CELLS AFTER IONIZING RADIATION

Andrew Foy,¹ Hong Ye,² and Cynthia Wetmore³; ¹Mayo Foundation for Medical Education and Research, Rochester, MN, USA; ²Mayo Clinic, Rochester, MN, USA; ³Rochester, MN, USA.

Treatment and cure of medulloblastoma continues to be disappointing. Approximately 30% of cases recur, and even when a cure is achieved, the treatment is highly morbid to the developing brain. Recent evidence suggests that many solid tumors are sustained by a population of cells with stem cell–like properties that have extensive capacity for self-renewal. Tumor stem cells (TSC) are also thought to have increased radio- and chemoresistance and may be responsible for treatment failures. Mechanisms of DNA damage response have not been explored previously in neural stem cells. Identifying novel therapeutic targets that spare normal neural stem cells while eradicating the tumor-derived stem cell population is a long-term goal. Additionally, insight into mechanisms of DNA damage repair in the population of self-renewing neural stem cells will allow for an improved understanding into how the nervous system maintains its molecular integrity. TSCs were isolated from medulloblastomas that spontaneously arose in mice haplo-insufficient for Patched (Ptc), a component of the sonic hedgehog receptor, and cultured in serum-free media as free-floating spheres. Neural stem cells were derived from the early postnatal hippocampus and cerebellum of wild-type and Ptc+/– mice. Cultures were exposed to 2 Gy of ionizing radiation, and cell cycle progression was characterized with flow cytometery. We found that normal neural stem cells had a similar phenotype with sustained accumulation in G1 within 2 h of irradiation. In contrast, we found that TSCs failed to arrest in G1 and showed a marked but unsustained G2/M arrest 8 h after radiation. By 48 h after irradiation, TSCs had reentered the cell cycle. The level of apoptosis was unchanged compared to controls. We repeated these experiments at 10 Gy of radiation and in the TSCs found a large increase in apoptosis over time. Accumulation of TSCs in G2/M was again noted at 8 h, however the G2/M arrest was sustained at 24 and 48 h after irradiation. Preliminary analysis shows that p53 is wild-type in TSCs, suggesting that the abrogated G1 arrest is not due to mutation or deletion of p53. We have characterized the cell-cycle and DNA damage response of murine NSCs and found that this population of undifferentiated cells undergo cell cycle arrest in G1 after DNA damage. This is in contrast to embryonic stem cells that lack a G1 checkpoint and do not initiate cell cycle arrest and undergo apoptosis after exposure to ionizing radiation. Additionally, we found that TSCs have escaped the G1 checkpoint in a p53-independent manner and proceed to replicate their DNA without adequate repair that likely contributes to genomic instability and propagation of new therapy-resistant clones. These data suggest that TSC and normal neural stem cells have distinct responses to ionizing radiation and DNA damage. Studies are ongoing to determine the molecular basis for the distinct phenotype observed in response to radiation in normal and neoplastic stem cells and to specifically increase the sensitivity of TSCs to apoptosis after DNA damage. Supported by Neurosurgery Research and Education Foundation (A.F.), Sontag Foundation (C.W.), and Waterman Foundation for Cancer Genetics (C.W.)

BIO 47. PRIMARY TUMOR-BASED ORTHOTOPIC XENOGRAFT MOUSE MODELS OF PEDIATRIC EPENDYMOMA ARE CLINICALLY RELEVANT AND PRESERVE CANCER STEM CELL POOL

Litian Yu,¹ Eastwood Leung,¹ Adekunle Adesina,¹ Tsz-Kwong Man,¹ Qin Shu,¹ Jack Su,¹ Patricia Baxter,¹ Lazlo Perlaky,¹ Murali Chintagumpala,¹ Ching C. Lau,¹ Susan M. Blaney,¹ Pulivarthi H. Rao,¹ and Xiao-Nan Li¹; ¹Baylor College of Medicine, Houston, TX, USA.

Animal models play crucial roles in both biological and preclinical studies of human cancers. For ependymomas, which are the second most common malignant brain tumors of childhood, the availability of tumor models that reliably recapitulate the biology of this neoplasm is extremely limited. To create mouse models that would faithfully mimic histopathological, immunophenotypical, and genetic characteristics of human ependymomas, we injected fresh surgical specimen from an anaplastic ependymoma into cerebrum of SCID mice. The developed xenografts have since been serially passaged in mouse brains for three generations while maintaining constant tumorigenicity rate (100%) with as few as 25,000 cells. Detailed characterization of the xenograft tumors revealed that they shared nearly identical histological features with the original tumor and expressed broadly similar immunohistochemical profiles. Although the xenograft tumor formed a clear-cut edge toward the adjacent nervous tissue, isolated tumor cells invading into mouse brain were detected with immunohistochemical staining using human specific antibodies. Gene expression profiling revealed strong similarity between xenograft tumor and the original patient tumor. To determine the presence of cancer stem cells, we utilized antibodies against CD133 to detect and isolate CD133+ cells with FACS. We found that the CD133+ cells only constitute a very small fraction (<1%) of tumor population. In the presence of EGF and FGF, the isolated CD133+ cells formed neurospheres that have be maintained in vitro for >6 months, although spontaneous differentiation as evidenced by the growth of attached cells was occasionally seen. Expression of glial and neuronal markers was detected in the attached cells induced to differentiation. In summary, we have developed a novel orthotopic xenograft ependymoma model that accurately replicated the histopathological, genetic, and behavioral features of anaplastic ependymoma in children.

BIO 48. PROGENITOR CELLS IN THE FLOOR OF THE IV VENTRICLE ARE SUSCEPTIBLE TO TRANSFORMATION BY ACTIVATING MUTATIONS IN BETA CATENIN (CTNNB1) RESULTING IN THE FORMATION OF A DISTINCT SUBGROUP OF MEDULLOBLASTOMA

Paul Gibson,¹ Yiai Tong,¹ Margaret Thompson,¹ David Ellison,¹ and Richard Gilbertson¹; ¹St. Jude Children's Research Hospital, Memphis, TN, USA.

Evidence suggests that subgroups of histologically similar brain tumors arise from different populations of neural progenitor cells (NPCs). We have shown that medulloblastomas (MBs) comprise at least three subgroups that display distinct clinical and molecular characteristics (Thompson et al. J Clin Oncol. 2006). Tumors containing mutations in the sonic hedgehog (SHH) pathway that occur in younger patients frequently display a desmoplastic histology, have a relatively poor outcome, and most likely arise from cerebellar granule neuron precursors (GNPCs). In contrast, tumors that contain activating mutations in CTNNB1 display monosomy 6, classic histology, and excellent clinical outcome and tend to occur in older patients. We hypothesized that CTNNB1-mutant medulloblastomas are clinically and molecularly distinct from SHH-mutant tumors because they arise from progenitor cells outside the GNPC lineage. In support of this hypothesis, we used in situ hybridization to show that signature genes of human SHH pathway-mutant medulloblastoma (n = 71 genes) are expressed in the rhombic lip and GNPC of the developing cerebellum, while CTNNB1-mutant tumor signature genes (n = 72 genes) are expressed in the primary germinal layer and floor of the IV ventricle. Using transgenic mouse technology we show further that Cre-dependent activation of Ctnnb1 in neural progenitor cells of the developing mouse results in the formation of tumors that mimic the human CTNNB1-mutant disease. These tumors arise from the floor of the IV ventricle and primary germinal zone but not the GNPC lineage. Indeed, we show that transgenic expression of mutant Ctnnb1 in GNPC under the Math 1 promoter does not induce medulloblastoma. Our data provide strong evidence that subgroups of medulloblastoma are distinct diseases with different cellular and molecular origins. Of particular note we show for the first time that medulloblastomas can arise outside the GNPC linage and provide the first mouse model of CTNNB1-mutant medulloblastoma.

BIO 49. RHABDOID TUMORS: GENE EXPRESSION PATTERNS PROVIDE CLUES TO THE CELL OF ORIGIN, PATHOGENESIS, AND POTENTIAL THERAPEUTIC TARGETS

Simone Sredni,¹ Samantha Gadd,¹ Ching-Chiang Huang,¹ and Elizabeth Perlman¹; ¹Northwestern University, Feinberg School of Medicine and Robert H. Lurie Cancer Center, Chicago, IL, USA.

Background: Rhabdoid tumors (RTs) are highly aggressive tumors first described in the kidney of young children. Although they can arise in a variety of extrarenal sites, the most frequent location is the CNS where they are called atypical teratoid/rhabdoid tumors (AT/RTs). More than 90% of the patients with AT/RT are <5 years of age at diagnosis, and 70% present with high-stage disease. The highly aggressive nature of these tumors associated with the absence of effective treatment results in a very poor overall survival of no more than 20% at 3 years. Although the etiology of these tumors is unknown, RT at all sites have a common genetic abnormality: the mutation or deletion of the hSNF5/INI-1 gene located at chromosome 22q11.

Aims: This study seeks to clarify the pathogenesis of RT and to uncover potential therapeutic targets.

Methods: Gene expression (GE) analysis was performed using oligonucleotide arrays that include >22,000 probe sets (affymetrix U133A). The GE of 10 RTs was compared with the GE of 43 pediatric tumors (12 cellular mesoblastic nephromas, 16 clear cell sarcomas of the kidney, and 15 Wilms tumors). Verification methods included immunohistochemistry, immunoblotting, and quantitative RT-PCR.

Results: One hundred fourteen genes were differentially expressed in RT (p < 0.001, fold change >2 or <0.5). INI-1 and genes previously reported to be associated with INI-1 were down-regulated (ATP1B1, PTN, DOCK4, SPARC, PLOD1, PTP4A2, and PTPRK). CDKN1A, CDKN2A, CDKN1C, and CCND1 showed low to no expression in RTs, and MYC was up-regulated, suggesting that RTs gain a proliferative advantage through loss of cyclin-dependent kinase inhibition and up-regulation of c-MYC (both of which may be directly due to INI-1 loss). Twenty-three of the 114 top genes were involved with neural development and were all sharply down-regulated in RT (including PTN, CDH2, FYN, HES1, NOTCH2, NCAM1, SOX11, PTPRK, and LEF1); additionally, RT showed Nestin expression by immunohistochemistry. These findings strongly suggest that RT may arise in neural crest stem cells during a critical developmental window. Differentially expressed genes associated with tumor suppression, invasion, and metastasis were documented. These included up-regulation of SPP1, MMP12, TFRC1, SERPINF1, LGALS1, NCOA3, and ZNF217 and down-regulation of COL18A1 (endostatin), PTPRK, SELENBP1, and DOCK4.

Conclusions: RT may arise within neural crest stem cells. During a crucial developmental stage, loss of INI-1 in these cells can result in striking repression of neural development. The inactivation of INI-1 may also be the cause of the loss of the cyclin-dependent kinase inhibition, which gives a proliferative advantage to the affected cells. The identification of abnormal regulation of several genes involved in aggressive behavior provides promising therapeutic targets.

BIO 50. ROLE OF SNX3 ON ENDOSOMAL TRAFFICKING AND SIGNALING OF RECEPTOR TYROSINE KINASES IN GLIOBLASTOMA CELL LINES

Takrima Haque,¹ Carol Nahn,² Damien Faury,³ and Nada Jabado³; ¹McGill University, Montreal, QC, Canada; ²QC, Canada; ³Montreal, QC, Canada.

Background: Pediatric glioblastomas (pGBMs) have dismal prognosis and, unlike adult GBMs (aGBMs), little is known about molecular events driving oncogenesis in children. We previously established in primary pGBM samples that there are at least two subtypes of pGBM; both were molecularly distinct from aGBM: one was associated with Ras/Akt-activation and a poor prognosis and the other with no obvious Ras/Akt activation and a better outcome. Aberrant Ras activation in GBM is driven by upstream events including aberrant signaling through receptor tyrosine kinases (RTKs) that are amplified, mutated, or rearranged in a large proportion of aGBMs. However, unlike primary aGBMs, which harbor amplification of EGFR in approximately two-thirds of tumors, pGBMs show no genetic amplification/mutation of RTKs. Data from our microarray analysis identified Sorting Nexin 3 (SNX3) as one of the genes to be exclusively overexpressed in the pGBM subset associated with Ras activation. SNX3 belongs to a family of proteins involved in the regulation of intracellular trafficking of membrane receptors. It is associated with early endosomes through a novel motif (PX domain) following its interaction with phosphatidylinositol-3-phosphate. Overexpression of SNX3 in A431 cells has been shown to delay EGFR degradation prolonging signaling of EGF-EGFR complexes from within the endosomes. This study investigates the role of SNX3 in endosomal trafficking of RTKs in GBM. Our working hypothesis is that overexpressed/overactivated SNX3 prevents/delays RTK degradation in a subset of pGBMs thus generating prolonged RTK-signaling, aberrant Ras activation, and increased cell proliferation/survival/invasion.

Methods: Overexpression of SNX3 was validated by qRT-PCR and immunohistochemistry on the same pGBM samples. We stably overexpressed MYC-tagged SNX3 in pGBM cell lines (SF188 and SJ-G2) and transiently in an adult GBM cell line (U87), which had similar endogenous levels of SNX3 and active PI3K-pathway. In parallel, we transiently knocked-down SNX3 in SF188 cells. Effects of overexpression and silencing of SNX3 on cell signaling (Ras and Akt pathways), RTK expression (EGFR/MET/PDGFR), and cell proliferation and invasion were investigated.

Results: Myc-tagged-SNX3 localized similar to the endogenous protein within early endosomes in all cell lines. SNX3 overexpression led to a more sustained and stronger activation of Ras pathway and to delayed degradation of EGFR following EGF stimulation in all cell lines as compared to empty-vector (EV) transfectants. No effect was observed on the Akt pathway. Silencing SNX3 in SF188 decreased EGFR baseline level and Ras pathway activation. Preliminary experiments indicate that cell proliferation rate correlates with the level of EGFR expression and Ras pathway activation following SNX3 overexpression or silencing.

Conclusion: RTKs are major oncogenes particularly in GBM. Our results suggest that SNX3 modulates EGFR signaling by preventing/delaying its degradation, thus helping sustain Ras activation in GBM cell lines. We are currently studying the role of SNX3 in the endosomal trafficking of other receptors including PDGFR, FGFR, or MET to ascertain that the effect we observe on EGFR targets multiple RTKs or if it is focused to specific membrane receptors. We also are investigating the role of aberrant PI3K activation in sustaining SNX3 function and delay in lysosomal degradation of these RTKs.

BIO 51. THE CSF PROTEOME OF MEDULLOBLASTOMA

Meena Rajagopal,¹ Yetrib Hathout,¹ Tobey Macdonald,¹ and Brian Rood¹; ¹Children's National Medical Center, Washington, DC, USA.

Identification of medulloblastoma biomarkers in readily accessible body fluids like cerebrospinal fluid (CSF) would be useful for diagnosis, treatment monitoring, and recurrence detection. CSF is an attractive potential source of brain tumor biomarkers given the intimate association between the CSF compartment and brain tumors. In this pilot study, cerebrospinal fluid samples from patients diagnosed with medulloblastoma and control individuals were collected and subjected to two-dimensional gel electrophoresis. Seventy-five micrograms of total CSF protein from each sample were loaded using the criterion 2-DE. Gel analysis was carried out using Ludesi software, and proteins were identified after in-gel trypsin proteolysis using matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). Twenty-one protein spots were found to be altered 2.5-fold in CSF from medulloblastoma patients (p < 0.01). Unsupervised hierarchical clustering was able to differentiate the tumor samples from control samples indicating a potential use in diagnostics and recurrence detection. We also found a significant alteration of the isoform profile of prostaglandin D2 synthase (PGD2S), a brain specific glycoprotein that is synthesized by the choroid plexus. Five isoforms of PGD2S including three acidic, one neutral, and one basic isoform were all at least twofold down-regulated in tumor samples. Our findings further suggest the importance of posttranslational modifications as a source of potential biomarkers.

BIO 52. THE EXPRESSION PROFILE OF THE MULTIDRUG-RESISTANCE GENES DIFFERS BETWEEN PEDIATRIC BRAIN TUMOR CELLS AFTER IN VITRO SHORT-TERM EXPOSURE TO VINBLASTINE

Elvis Terci Valera,¹ Maria Angelica Abdalla De Freitas Cortez,² Fabio Morato Oliveira,² Nada Jabado,³ Damien Faury,³ Michael S. Bobola,⁴ Helio Rubens Machado,⁵ Carlos Alberto Scrideli,¹ and Luiz Gonzaga Tone¹; ¹Pediatric Oncology, University of São Paulo, Ribeirão Preto, São Paulo, Brazil; ²Department of Genetics, University of São Paulo, Ribeirão Preto, São Paulo, Brazil; ³Department of Pediatrics, Montreal Children's Hospital Research Institute/McGill University Health Center, Montreal, QC, Canada; ⁴Department of Neurological Surgery, University of Washington, Seattle, WA, USA; ⁵Surgery and Anatomy, Division of Neurosurgery, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.

Background: Drug resistance is a major cause of treatment failure in the pediatric brain cancer setting. The multidrug resistance (MDR) phenotype can be mediated by the superfamily of adenosine-triphosphate binding cassette (ABC) transporters. The dynamics of expression of the MDR genes after exposure to chemotherapy, especially the comparison between pediatric brain tumors of different histologies, are poorly described.

Objectives: To compare the mRNA expression profile of the ABCB1, ABCC1, and ABCG2 genes from neuroepithelial pediatric brain tumors prior and following short-term culture with vinblastine (10 and 60 nM for 24 and 72 h) by real-time PCR.

Methods: Immortalized cell lines from pylocitic astrocytoma (R286), anaplasic astrocytoma (UW467), glioblastoma (SF188), and medulloblastoma (UW3) were cultured at 37°C in a 5% CO₂ atmosphere using HAM-F10 media supplemented with 10% fetal bovine serum, 1% L-glutamine, and 5% antibiotic-antimycotic solution. Cells were used between passage numbers of 20–40. After subculture at 90% confluence, vinblastine sulphate was added at final concentration of either 10 or 60 nM. Cells were harvest after 24 or 72 h of vinblastine exposure by trypsine (0.25%)/EDTA. Total RNA was extracted from each cell line using the TRIzol reagent. cDNA were obtained with the High Capacity kit. The RQ-PCR was performed with the SYBR Green PCR Master MIX kit. mRNA levels were normalized by GUS-β housekeeping gene. Relative amounts of expression were compared between cell lines and normal human brain tissue.

Results: As expected, the mRNA levels of expression for ABCB1 gene have augmented in parallel of increasing concentration and time of exposure to vinblastine for R286, UW467, and UW3 cell lines. Interestingly, the SF188 glioblastoma cell line has diminished the ABCB1 expression when exposed to vinblastine. Following chemotherapy, ABCB1 expression has lowered in all cell lines other then glioblastoma; actually, ABCB1 expression of SF188 increased after 24-h exposure to 60 nM of vinblastine. The expression of ABCG2 gene was not influenced by vinblastine exposure.

Conclusions: Pediatric glioblastoma cell lines show a different pattern of mRNA expression of MDR genes when stimulated by vinblastine. To validate these findings, evaluation of ABC transporters at proteic and functional levels is ongoing. The resistance to chemotherapy frequently observed in pediatric glioblastoma might be mediated by other genetic or epigenetic events other than ABCB1 overexpression. These preliminary findings may be useful in determining novel strategies of treatment for neuroepithelial pediatric brain tumors. Supported by FAPESP grant 2007/04065-9.

BIO 53. THE GENE EXPRESSION, AMPLIFICATION, AND PROMOTER METHYLATION OF NOTCH2 IN EPENDYMOMAS

Hehuang Xie,¹ Veena Rajaram,² Tange Yuichi,³ Chris D. Mccabe,⁴ Wendy Stellpflug,⁵ Stewart Goldman,⁶ Tadanori Tomita,⁷ and Marcelo Bento Soares³; ¹Northwestern University, Chicago, IL, USA; ²Plaza, IL, USA; ³IL, USA; ⁴Molecular Diagnosis, CMH, IL, USA; ⁵Chicago, IL, USA; ⁶Hematology/Oncology, Children's Memorial Hospital, Chicago, IL, USA; ⁷Pediatric Neurosurgery, Children's Memorial Hospital, Chicago, IL, USA.

Based on the origins of tumors, ependymomas are classified as supratentorial, infratentorial, and spinal ependymomas. Recent studies suggest these three subclasses of ependymomas have distinct genetic features and gene expression patterns. Some studies have shown distinct genes/pathways associated with these subclasses. IFG-1 and HOX homeobox genes were shown to be overexpressed in infratentorial ependymomas; while Notch signaling pathway was associated with the development of supratentorial ependymomas. Our in-house microarray data demonstrated that genes in Notch signal pathways were also up-regulated in infratentorial ependymomas. In this study, we examined Notch2 expression, gene amplification, and promoter methylation status in a subset of fresh frozen tissues of infratentorial ependymomas. Notch2 expression was determined by quantitative RT-PCR, the gene dosage by quantitative PCR, and the methylation status of Notch2 promoter was determined with pyrosequencing technology. We observed Notch2 overexpression in five infratentorial ependymomas examined. Amplification of Notch2 gene was observed in two out of seven infratentorial ependymomas. Notch2 promoter was found to be hypo-methylated in both normal brain tissues and ependymomas.

Conclusion: Our preliminary data suggested Notch signaling pathway may play a role in the development of infratentorial ependymoma in addition to supratentorial ependymomas demonstrated in previous studies. The mechanism of Notch2 overexpression could not be fully explained by gene amplification. In addition, compared to normal human brain, no significant methylation alteration on Notch2 promoter was observed for ependymomas. Further studies for Notch2 amplification by other techniques such as fluorescence in-situ hybridization and expression studies on additional tumor samples from all locations may help understand the role of Notch signaling pathway in the pathogenesis of ependymomas. This study was supported by The Everett/O'Connor Charitable Trust; Dr. Ralph and Marian C. Falk Medical Research Trust; Gus Foundation; The Maeve McNicholas Memorial Foundation; and Medical Research Institute Council.

BIO 54. THE INTEGRATION OF "OMICS" DATA ON BRAIN TUMORS

Min Wang,¹ Hehuang Xie,² Wendy Stellpflug,² Jared Bischof,² Maria De Fatima Bonaldo,² Chris Mccabe,² Veena Rajaram,³ Stewart Goldman,⁴ Tadanori Tomita,⁵ and Marcelo Bento Soares²; ¹Northwestern University, Chicago, IL, USA; ²IL, USA; ³Plaza, IL, USA; ⁴Hematology/Oncology, Children's Memorial Hospital and Northwestern University, Chicago, IL, USA; ⁵Pediatric Neurosurgery, Children's Memorial Hospital, Chicago, IL, USA.

The identification of molecular markers for early diagnosis and better prognosis has been a focus for recent brain tumor studies. Numerous genes and pathways have been associated with brain tumor genesis and progression. In spite of many large-scale gene expression studies, underlying molecular mechanisms leading to the malfunction of these genes and associated networks are still far from clear. Global hypomethylation and localized hypermethylation are two concurrent phenomena frequently observed in tumors that can significantly affect transcription of cellular genes. However, little is known with respect to epigenomic alterations in brain tumors and their impact on the transcriptome. To assist in the identification of epigenetic and genetic factors that contribute to altered gene expression in brain tumors, an integrated brain tumor epigenomics/genomics at Children's Memorial Hospital database (BTECH) was designed and implemented. Using molecular-based integration, the genomics, transcriptomics, and epigenomics data on brain tumors are combined in this database. On top of the database, a genome browser was developed to allow synchronized visualization of gene expression, methylation, and various annotated features. This integrated platform facilitates a systematic understanding of the interactions of various genetic and epigenetic factors associated with brain tumor development. This study was supported by The Everett/O'Connor Charitable Trust; Dr. Ralph and Marian C. Falk Medical Research Trust; Gus Foundation; The Maeve McNicholas Memorial Foundation and Medical Research Institute Council.

BIO 55. THE ONCOGENIC ROLE OF OTX2 IN MEDULLOBLASTOMA PATHOGENESIS AND ITS CLINICAL IMPLICATIONS AS A TUMOR MARKER AND THERAPEUTIC TARGET

Hai Yan,¹ Cory Adamson,² Qun Shi,² Chunhui Di,² Chris Duncan,¹ Roger Mclendon,³ and Darell Bigner²; ¹Duke University, Durham, NC, USA; ²NC, USA; ³Durham, NC, USA.

OTX2 is a developmentally regulated homeodomain-containing transcription factor that plays extraordinarily important roles in early morphogenesis of the CNS. We previously identified OTX2 amplification in medulloblastoma, one of the most aggressive pediatric brain tumors. However, the mechanism of genetic activation of OTX2, its clinical implication, and its role in medulloblastoma pathogenesis remain unknown. In our studies, we demonstrated that OTX2 amplification and overexpression in medulloblastoma associated with tumor anaplasia and worse survival. We have shown that knockdown of OTX2 by small interfering RNA reduced medulloblastoma cell growth, and that the cells with high levels of OTX2 expression were sensitive to treatment with retinoic acid (RA). Exogenous retinoids, when exogenously applied, have been shown to displace or repress OTX2 expression in the embryonic nervous system and also in embryonal carcinoma cells through cis-acting elements of the OTX2 promoter. In our studies, we observed that RA abrogated cell proliferation in every OTX2-expressing cell line in a dose-dependent manner. These studies lay the conceptual framework for clinical trials of retinoids in the treatment of a commonly lethal pediatric brain tumor. Furthermore, we have investigated the OTX2 molecular signaling pathway and demonstrated that another medulloblastoma oncogene c-Myc was transcriptionally regulated by OTX2. We expect the studies to advance our knowledge on the pathogenesis of medulloblastoma, which could make significant contributions to the therapeutic intervention in this frequently lethal cancer.

BIO 56. THE SMO/SMO MOUSE MODEL: SHH-INDUCED MEDULLOBLASTOMA WITH 90% INCIDENCE AND LEPTOMENINGEAL SPREAD

Beryl Hatton,¹ Elizabeth Villavicencio,¹ and James Olson¹; ¹Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Toward the goal of generating a mouse medulloblastoma (MB) model with increased tumor incidence, we developed a homozygous version of our ND2:SmoA1 model. MBs form in 93% of homozygous Smo/Smo mice by 2 months of age. Tumor formation is thus predictable by age, prior to the symptomatic appearance of larger lesions. This high incidence and early onset of tumors is ideal for preclinical studies because mice can be enrolled before symptom onset and with a greater latency period before late-stage disease. Smo/Smo tumors also display leptomeningeal dissemination of neoplastic cells to the brain and spine, which occurs in many human cases. Despite an extended proliferation of granule neuron precursors (GNPs) in the postnatal external granular layer (EGL), the internal granular layer (IGL) formed normally in Smo/Smo mice and tumor formation occurred only in localized foci on the superficial surface of the molecular layer. Thus, tumor formation is not simply the result of overproliferation of GNPs within the EGL. Moreover, Smo/Smo MBs were transplantable and serially passaged in vivo, demonstrating the aggressiveness of tumor cells and their transformation beyond a hyperplastic state. The Smo/Smo model is the first mouse medulloblastoma model to demonstrate leptomeningeal spread. The adherence to human pathology, high incidence, and early onset of tumors thus make Smo/Smo mice an efficient model for preclinical studies. Results from initial drug testing studies will be presented. We recently published a manuscript describing "Tumor Paint," a bioconjugate of chlorotoxin and Cy5.5 that binds to brain cancer cells and illuminates them so that surgeons can distinguish brain tumors from normal tissue (Cancer Res. 2007;67:6882). Studies are currently underway to determine the limits of detection in Smo/Smo mice and these data will also be presented.

BIO 57. UP-REGULATION OF WW DOMAIN-CONTAINING OXIDOREDUCTASE WOX1 IN CERTAIN TYPES OF NERVOUS SYSTEM TUMORS

Ming-Fu Chiang,¹ Shur-Tzu Chen,² and Nan-Shan Chang³; ¹Neurosurgery, Mackay Memorial Hospital, Taipei, Taiwan; ²Department of Cell Biology and Anatomy, Nation Cheng Kung University, Tainan, Taiwan; ³Institute of Molecular Biology, Institute of Molecular Biology, Tainan, Taiwan.

Human WWOX gene, located on a chromosomal fragile site FRA16D, encodes a tumor suppressor WW domain-containing oxidoreductase WOX1, also known as WWOX or FOR. Homozygous deletion of human WWOX gene has been observed in several different cancers, indicating the chromosome fragile WWOX gene plays an important role in tumorigenesis. WWOX mRNA expression profile in epithelial ovarian cancer also supports the WWOX variant 1 as a tumor suppressor. Down- or up-regulation of WWOX expression has been detected in various tumor tissues, such as hematopoitic malignancies, stomach cancer, and squamous cell carcinoma. The goal of these studies is to evaluate WOX1 protein expression levels in brain tumors and further investigate whether WWOX expression in certain types of meningomas and glioblastromas is functionally colocalized with neurofibromatosis type 2 (NF2). In this study, we have demonstrated that the differential expression of WWOX appears in normal controls and different types of brain tumors. By immunohistochemistry, WWOX expression is low in normal cortical neurons, mainly distributed on the axon fibers, whereas moderate immunoreactivity is present in the cytosol of small fibroblast-like cells of meningioma. In the microcystic meningoma case, WWOX is highly expressed in moderate to large cells. By in situ hybridization, the staining pattern and intensity of WWOX mRNA in different types of meningioma resembles that of WWOX expression. Notably, electron microscopical immunohistochemistry indicates WOX1 and NF2/merlin colocalized in certain tumors. Further, astrocytoma stained moderately to strongly positive for WOX1 protein. WOX1 expression is present in small condense cells and neuritis. Although WWOX is located at a common fragile region, FRA16D, we are unable to detect the loss of heterozygosity in our collected cases (n = 3). In conclusion, we have demonstrated that the differential expression of WOX1 on different types of meningoma, astrocytoma amd glioblastoma. Alterative expression of WOX1 is possibly to correspond to tumor development and metastsis.

	BRAINSTEM GLIOMA

Top ATRT BIOLOGY BRAINSTEM GLIOMA CHOROID PLEXUS TUMORS CASE REPORTS CRANIOPHARYNGIOMA EPENDYMOMA EPIDEMIOLOGY GERM CELL TUMOR HIGH-DOSE CHEMOTHERAPY HIGH-GRADE GLIOMA NEURO-IMAGING INFANT TUMORS LATE EFFECTS LEPTOMENINGEAL AND DISSEMINATED... LOW-GRADE GLIOMA NURSING AND MULTIDISCIPLINARY... NEUROFIBROMATOSIS AND ASSOCIATED... NEUROPATHOLOGY NEUROSURGERY OUTCOME STUDIES PNET/MEDULLOBLASTOMA QUALITY OF LIFE RADIATION ONCOLOGY RARE TUMORS THERAPEUTIC AND NEW DRUG...

 
BSG 1. A CANADIAN MULTICENTER PILOT STUDY OF EXTENDED LOW-DOSE TEMOZOLOMIDE AND RADIATION THERAPY IN PEDIATRIC BRAINSTEM GLIOMA

Diana Stempak,¹ Janet Gammon,¹ Derek Stephens,² Anne Feltis,¹ Ute Bartels,¹ Chris Fryer,³ Juliette Hukin,³ David Eisenstat,⁴ Donna Johnston,⁵ Yvan Samson,⁶ Eric Bouffet,¹ and Sylvain Baruchel¹; ¹Division of Hematology/Oncology, Hospital for Sick Children, Toronto, ON, Canada; ²Child Health Evaluative Sciences Program, Hospital for Sick Children, Toronto, ON, Canada; ³British Columbia's Children's Hospital, Vancouver, BC, Canada; ⁴Cancer Care Manitoba, Winnipeg, MB, Canada; ⁵Children's Hospital of Eastern Ontario, Ottawa, ON, Canada; ⁶CHUQ (Centre Hospitalier Universitaire de Québec), Quebec, QC, Canada.

Background: Brainstem gliomas (BSGs) constitute 10% of all pediatric CNS tumors and have an extremely poor prognosis, with 90% of children succumbing within 18 months of diagnosis. Temozolomide is an orally administered cytotoxic alkylating agent with activity against primary brain tumors, including high-grade gliomas. We evaluate here which, in combination with radiation therapy, is the standard therapy for the treatment of pediatric BSGs.

Objectives: (1) To evaluate the survival rate in newly diagnosed pediatric BSG patients when treated with the combination of concomitant extended low-dose oral temozolomide and radiation therapy. (2) To evaluate the antiangiogenic activity of this regimen by analyzing soluble plasma proteins as surrogate markers and to correlate these biomarkers with response to metronomic temozolomide and radiation

Methods: This is a Canadian multicenter nonrandomized pilot study. Temozolomide (85 mg/m² daily for 6 weeks) was administered in combination with standard radiotherapy followed by maintenance therapy of temozolomide alone (6 weeks/cycle with 1 week rest between cycles). Blood was collected pretreatment and before each new cycle for the analysis of angiogenic proteins in the plasma as potential surrogate markers of angiogenesis. bFGF, VEGF, VCAM-1, ICAM-1, and endoglin were measured using commercially available ELISA kits.

Results: Between April 2005 and July 2007, 15 patients were enrolled in five centers (6 males, 9 females; median age, 6.4 years; range, 2.4–12.3 years). A median of three cycles was received (range, 1–5). The median progression-free survival (PFS) was 5 months, and the median overall survival (OS) of these patients was 11 months. Dose-limiting thrombocytopenia led to treatment discontinuation in three patients. Of the potential surrogate markers evaluated, plasma bFGF, VCAM-1, and ICAM-1 did not demonstrate any statistically significant changes over time. While the patient numbers are too small to reach statistical significance, several interesting trends were observed for plasma VEGF and endoglin. Both tended to decrease during the first two cycles of therapy, after which VEGF tended to rise in those patients who remained on therapy, while endoglin concentrations appeared to plateau. Further analysis demonstrated that patients with a longer PFS tended to have a greater decrease in VEGF concentrations than patients with a shorter PFS, but no other correlations between VEGF or endoglin concentrations and PFS or OS were observed.

Conclusions: This combination therapy does not appear to improve the PFS or OS of BSG patients when compared to historical controls. With respect to the surrogate markers, there were several limitations including small sample size and high interpatient variation, however the initial decrease in both VEGF and endoglin during the combination phase of therapy (radiation + temozolomide) may suggest antiangiogenic activity.

BSG 2. ANAPLASTIC TECTAL GLIOMA IN A 6-YEAR-OLD GIRL: A CASE REPORT

Howard Chang,¹ Reuben Cuison,² Renuka Gera,³ Elna Saah,³ and Ajovi Scott-Emuakpor³; ¹Neurology and Ophthalmology, Michigan State University, East Lansing, MI, USA; ²Laboratories, Sparrow Health System, Lansing, MI, USA; ³Pediatrics and Human Development, Michigan State University, East Lansing, MI, USA.

Tectal glioma is a topographical diagnosis consisting of a heterogeneous group of gliomas in children, with relatively benign clinical course in most previous case reports. We present a case of a tectal glioma with histological features of anaplastic mixed oligoastrocytoma that continues to progress despite chemotherapy and radiation therapy.

Case report: A 6-year-old African American girl presented with severe headache for several days associated with vomiting, neck stiffness, fever, and unsteady gait. Physical examination was otherwise unremarkable. She was recently treated for otitis media approximately 1 week ago. Mother reported that the patient had frequent headaches and incontinent of bowel and bladder during the past year. A lumbar puncture showed high CSF protein but no signs of infection. CT and MRI of the brain showed obstructive hydrocephalus with a 2.3 cm nonenhancing pineal region mass attached to the tectum. An MRI of the spinal cord at this time was negative. She underwent craniotomy with biopsy and partial resection of the tumor. The histopathology revealed a moderately hypercellular tumor with small cells containing scant cytoplasm and round to oval nuclei with fine chromatin, embedded in a loose myxoid fibrillary matrix. The cells have long thin processes. No definitive eosinophilic granular bodies or Rosenthal fibers are seen. There are no significant mitoses. Immunocytochemistry shows that most tumor cell nuclei are positive for S-100. The matrix has many GFAP-positive processes but the cell bodies appear negative for GFAP. Many neurofilament protein and synaptophysin positive processes are also found in the background. Immunostain for Ki67 shows generally low labeling index, but it is positive in up to 20% of tumor cells focally. The overall features are suggestive of an anaplastic mixed oligoastrocytoma. Fluorescence in situ hybridization study, however, showed no evidence of chromosomes 1p and 19q deletions. The slides were reviewed by an external expert consultant who indicated that this could be a low-grade astrocytoma most consistent with a "low-grade tectal glioma."

Clinical Course: The patient had no neurological deficits including ataxia, headache, nausea or vomiting at the time of initial discharge. She was initially treated with oral temozolomide but showed progression as she developed back pain and had several episodes of urinary incontinence about 2 months after the surgery. MRI revealed extensive coating of the cauda equina and the distal thecal sac consistent with drop metastases. The residual original tectal lesion appeared unchanged. She then received fractional radiation therapy involving the whole brain (3,600 cGy), a boost to the tumor bed (5,400 cGy), cervical spine (4,600 cGy), and thoracic-lumbar spine (4,600 cGy). When it was noted that she continued to progress on this treatment, chemotherapy was resumed with one cycle of PCV. However, she continues to deteriorate at 6 months after initial presentation with MRI evidence of leptomeningeal gliomatosis.

BSG 3. DIFFUSE INTRINSIC PONTINE GLIOMAS OF CHILDHOOD: HIROSHIMA UNIVERSITY EXPERIENCE

Kazuhiko Sugiyama¹; ¹Department of Neurosurgery, Hiroshima University, Hiroshima, Japan.

To evaluate the overall survival of pediatric patients with diffuse intrinsic potine gliomas treated with conventional radiotherapy concurrently with ACNU-vincristine chemotherapy followed by oral etoposide. Patients between 5 and 20 years of age with typical diffuse intrinsic potine gliomas on MRI/MRS or histologically proven grade III/IV astrocytoma in pons from 2003 to 2007 were eligible. Induction treatment consisted of ACNU (80 mg/m² x days 1 and 36) and vincristine (1.5 mg/m² x days 1, 8, 15, 22, 29, 36, and 43) chemotherapy combined with conventional 54-Gy radiotherapy. After this treatment, patients received monthly oral etoposide chemotherapy consisting of daily dose of 25 mg/m² (days 1–10) for 12 months or until performance status deterioration. Twelve patients were enrolled (five males and seven females; median age, 6 years). Surgical procedure included three biopsies. The most frequent adverse effects were gastrointestinal and hematologic. There were no toxic deaths. The response to combined treatment included no complete response, three partial response, four stable disease, and five partial response, four stable disease, and five progressive disease. Ten tumors progressed locally, and these 10 patients died; two patients with stable disease are alive. The overall median survival was 332 days, ranging from 135 to 818 days. Our treatment to diffuse intrinsic potine gliomas of childhood did not improve survival. The treatment was well tolerated and should be evaluated for more chemosensitive pediatric malignancies.

BSG 4. PATTERNS OF FAILURE FOR DIFFUSE INFILTRATING BRAINSTEM GLIOMA: NEW GUIDELINES FOR RADIOTHERAPY PLANNING

Andrew Chang¹ and Thomas Merchant¹; ¹St. Jude Children's Research Hospital, Memphis, TN, USA.

Introduction: Diffuse infiltrating brainstem glioma (DIBG) is a uniformly fatal diagnosis for the pediatric patient. Radiation therapy remains an integral component of current treatment regimens that seek to improve symptoms and test new agents in a combined modality approach. To make progress and compare results among treatment regimens requires uniform and consistent guidelines for irradiation. Because there is not agreement on target volume definitions for conformal treatment planning, we undertook a review of all patients treated at St. Jude Children's Research Hospital after CT-based treatment planning to determine patterns of failure with respect to the targeted volume.

Methods: Between 1994 and 2006, 84 patients with median age of 6.4 years (range, 1.5–14 years) and diagnosis of DIBG were treated with radiation therapy and subsequently progressed and died. Fifty-nine of these patients had postirradiation MRI available for the pattern of failure analysis. Postirradiation MRI was performed at an average of 2.2 months (range, 1–4) until death. The first MRI to demonstrate treatment failure was imported into the treatment planning system for registration with the original three-dimensional treatment plan. First failures were recorded as local, regional, or distant. The definition for local failure was tumor progression within the contoured gross tumor volume (GTV). Regional failure was defined as any component of tumor progression beyond the confine of the contoured GTV. Distant failure was defined as neuraxis metastastic disease.

Results: T2-weighted MR imaging was used to demonstrate treatment failure in all cases and showed the propensity of DIBG to extend along neuronal tracts in all dimensions. Regional failure involved superior spread into the cerebral peduncles and thalamus. Axial spread involved posterior extension into the cerebellar peduncles; there were no cases where the tumor progressed beyond the confines of the normal anatomy. Median progression-free and overall survivals were 7.0 and 12.2 months, respectively. At the time of first failure, 29 had local failure, 25 had regional failure, two had regional and distant failure, and three had distant failure alone. Considering the 27 patients with a component of regional failure, 21 had a component of superior progression, four a component of inferior progression, and 18 a component of lateral progression. The average distances from the contoured GTV were determined: superior, 1.1 cm (0.3–2.75 cm); inferior, 0.4 cm (0.1–0.6 cm); lateral, 1.1 cm (0.3–2.0 cm). Superior failure occurred in 13 patients from 0.3 to 1 cm, five patients from 1.0 cm to 2.0 cm, and two patients >2.0 cm.

Conclusions: For DIBG, we recommend new target volume guidelines. The GTV should include identifiable tumor on T1- (postcontrast) and T2-weighted MRI. The CTV should include a 2-cm anatomical expansion in the transverse and inferior dimensions and 3-cm anatomical expansion in the superior dimension. Tumors confined to the pons should have the CTV expanded superiorly to cover the midbrain. PTV margins should be individualized (0.3–0.5 cm) but not less than the MR image section thickness used for treatment planning. Treatment planning MRI should include the upper cervical spinal cord.

BSG 5. PEDIATRIC BRAINSTEM GLIOMA: HYPOFRACTIONATED ACCELERATED IRRADIATION, A SINGLE INSTITUTION'S EXPERIENCE

Christelle Dufour,¹ Christine Levy-Piedbois,² Jacques Grill,¹ Jean-Louis Habrand,¹ Chantal Kalifa,¹ and Frederic Dhermain¹; ¹Institut Gustave Roussy, Villejuif, France; ²Institut de Radiotherapie, Bobigny, France.

Purpose: To prospectively evaluate efficacy and side effects of hypofractionated irradiation in children affected with brainstem glioma.

Methods and Materials: Between April 1996 and January 2004, 22 patients with brainstem glioma were enrolled in this study after radiological diagnosis of rapidly progressing, diffusely infiltrating pontine lesions. They underwent radiotherapy at once-daily fraction of 3 cGy, achieving the total dose of 45 cGy. Treatments were delivered over a 3-week period using five daily fractions per week.

Results: Median age at diagnosis was 5.9 years. Median duration of irradiation was 22 days (range, 8–63 days). Acute toxicity was limited to increased signs of intracranial hypertension in eight patients. This was corrected by increasing the steroid dose, but five patients received a modified therapy that consisted of conventional fraction (1.5 cGy) instead of 3 cGy. One child died during radiotherapy, probably due to progressive disease. The median time to disease progression was 5.6 months (range, 1.4–16 months). At time of report, 17 patients died of disease, one due to intratumoral hemorrhage (15 months after end of radiotherapy), and four patients were lost at follow-up. The median duration of survival for 18 patients was 7.38 months.

Conclusion: Hypofractionated radiotherapy can be used for children with brainstem glioma to reduce conventional radiotherapy overall treatment time. This treatment seems not to increase toxicity.

BSG 6. PHASE II TRIAL OF TIPIFARNIB IN CHILDREN WITH NEWLY DIAGNOSED INTRINSIC DIFFUSE BRAINSTEM GLIOMAS

Daphne Haas-Kogan,¹ Anuradha Banerjee,¹ Mehmet Kocak,² Michael Prados,¹ Russell Geyer,³ Maryam Fouladi,² Tracy Mcknight,¹ Tina Young Poussaint,⁴ Alberto Broniscer,² Susan Blaney,⁵ James Boyett,⁶ and Larry Kun⁶; ¹University of California, San Francisco, San Francisco, CA, USA; ²St. Jude Children's Research Hospital, Memphis, TN, USA; ³Seattle, WA, USA; ⁴Department of Radiology, Dana Farber, Boston, MA, USA; ⁵Houston, TX, USA; ⁶Memphis, TN, USA.

Background: Signals that emanate from growth factor receptors such as epidermal growth factor receptor (EGFR) activate Ras, a small guanosine triphosphatase (GTPase) that requires FTase-mediated posttranslational modifications for its activity. FTase inhibitors (FTIs) impede Ras functions, including promotion of oncogenesis and radiation resistance. FTIs sensitize human cancer cells to radiation, supporting the hypothesis that FTIs will augment tumor response to radiation. We performed a phase II study to assess the efficacy of tipifarnib (Tarceva), a farnesyltransferase (FTase) inhibitor, administered concurrently with radiation therapy (RT) in children with newly diagnosed intrinsic diffuse brainstem gliomas (BSG).

Patients and Methods: Children from 3 through 21 years of age with newly diagnosed nondisseminated intrinsic diffuse BSG were treated with concurrent tipifarnib and RT, followed by adjuvant tipifarnib. Tipifarnib was taken orally twice daily, 125 mg/m²/dose, continuously, for the entire duration of RT, followed by a 2-week break. Post-RT tipifarnib, 200 mg/m²/dose, was administered twice daily for 21 consecutive days, in 28-day cycles.

Results: In the phase I portion of this Pediatric Brain Tumor Consortium (PBTC) study, the MTD of tipifarnib with concurrent RT was established at 125 mg/m²/dose, twice daily. Forty eligible patients were treated at the MTD dose of 125 mg/m²/dose of tipifarnib (six treated during the phase I portion of the trial are included with 35 treated on the Phase II component; one patient had been declared ineligible). Median age was 5.5 years (range, 3.3–16.5); there were 25 females and 15 males. The progression-free and overall survival at 1 year will be compared to the PBTC contemporary BSG experience. Toxicities that were at least grade 3 in severity and were considered related to tipifarnib consisted of one grade 5, seven grade 4, and 37 grade 3 events: one grade 5 CNS hemorrhage; one grade 4 seizure, one grade 4 encephalopathy, one grade 4 low-serum bicarbonate, one grade 4 leukopenia, one grade 4 lymphopenia, and two grade 4 neutropenias; among grade 3 toxicities the majority were hematologic but also included three transaminase elevations, two hypokalemia, two febrile neutropenias, two infections with normal ANC, and one of each of the following: urinary obstruction, hyponatremia, hypomagnesemia, skin rash, and nausea/vomiting.

Conclusion: The MTD of tipifarnib with concurrent radiation (125 mg/m²/dose, administered twice daily) was tested in a phase II trial of efficacy with comparison to progression-free and overall survival in the contemporary PBTC BSG experience. When administered concurrently with radiation, tipifarnib exhibited significant toxicities, involving primarily neurologic, hematologic, and metabolic systems.

BSG 7. PHASE II TRIAL THALIDOMIDE AND CARBOPLATIN FOR THE TREATMENT OF BRAINSTEM GLIOMA (CNS1099)

Stewart Goldman,¹ Tadanori Tomita,² Maryanne Marymont,³ Stephanie Hannon,¹ Monica Newmark,³ Joanna Weinstein,¹ Anne Bendel,⁴ Michael Etzl,⁵ Lauren Evans,¹ Molly Fouts,¹ and Jason Fangusaro⁶; ¹Children's Memorial Hospital, Chicago, IL, USA; ²Pediatric Neurosurgery, Children's Memorial Hospital, Chicago, Armed Forces Middle East, USA; ³IL, USA; ⁴Minneapolis, MN, USA; ⁵AZ, USA; ⁶Northwestern University, Chicago, IL, USA.

Brainstem gliomas account for approximately 10% of pediatric CNS tumors. Multiple clinical trials of radiation with standard cytotoxic regimens have failed to increase survival. This protocol incorporates an antiangiogenic agent (thalidomide) with carboplatin and radiation therapy to increase PFS.

Materials and Methods: Forty-five patients with pontine glioma age 3.1–19.8 years (mean, 7.7 years) were treated on a protocol, which included thalidomide and carboplatin. Thirty-eight patients were enrolled in the study at the time of diagnosis with seven entered after first progression. The newly diagnosed patients received 54–60 Gy irradiation concurrent with the start of this protocol. Demographics for the 45 patients revealed 24 females, 21 males (28 Caucasian, 9 Hispanic, 3 African American, 2 Native American, and 3 other) have been enrolled. The lesions were intrinsic in all with eight having partially extrinsic tumors. Carboplatin was administered at a dose of 560 mg/m²; thalidomide dosed at 300 mg/m² escalating to 700 mg/m² daily.

Results: Best response data are available for 38 of 45 patients. Of the 34 evaluable for response treated at diagnosis: 2 complete response, 12 partial response, 17 stable disease, and 3 progressive disease. Best response for those treated with progression: four stable disease and two progressive disease. Two newly diagnosed patients remain progression free at 320 and 2,914 days. Mean time to progression (TTP) for newly diagnosed: 343 days (median, 185 days); 1- and 2-year projected overall survival is approximately 50% and 11%, respectively. Of the six with recurrent tumor mean TTP of 118 days. Three patients progressed with disseminated disease while maintaining local control. One patient was ineligible, two inevaluable, four were removed from study without progressive disease per family wishes, and six went off study with toxicity. Toxicities included Pseudomonas exotoxin (1), Stevens-Johnson syndrome (1), TIA/CVA (1), and colitis (1). Rash, constipation, and somnolence was seen in most patients but easily controlled with prophylactic regimens and titration of dose.

Conclusions: This outpatient regimen is feasible and generally well tolerated. This regimen compares favorably with historical controls and could be incorporated into future trials.

BSG 8. POSTTHERAPEUTIC METABOLIC CHANGES IN DIFFUSE INTRINSIC BRAINSTEM GLIOMA: INITIAL EXPERIENCE

Ashok Panigrahy,¹ Jonathan Finlay,² Girish Dhall,² Mark Krieger,³ Marvin Nelson,⁴ and Stefan Bluml²; ¹Childrens Hospital Los Angeles, Los Angeles, CA, USA; ²Los Angeles, CA, USA; ³Neurosurgery, Childrens Hospital Los Angeles, Los Angeles, CA, USA; ⁴CA, USA.

Background and Purpose: Among pediatric brain tumors, diffuse intrinsic brainstem gliomas (DIBSGs) carry the worst prognosis. These tumors are inoperable and highly resistant to chemo- and radiation therapy. Thus, without effective treatment, the majority of the patients die within 6–18 months after diagnoses. Noninvasive MR imaging—considered the gold standard for diagnoses of DIBSG—is often of limited value to assess disease progression or response to therapy. This lack of appropriate monitoring tools compromises clinical research and there has been no improvement in mean survival time over the last several decades. The goal of this study was to compare metabolic changes in patients that undergo a newly developed therapy with changes observed in previous patients that underwent the standard therapy.

Methods: A trial to evaluate a novel treatment (radiation therapy, daily carboplatin, and etoposide followed by temodar, irinotecan, and avastin = TRnew) for DIBSG has commenced at our institution. Thirteen prospective MR spectroscopy studies performed in four subjects prior to and following therapy were evaluated. Data were compared with changes observed in five subjects (17 studies) that were treated with standard therapy (radiation therapy followed by temodar and irinotecan = TRstd) (Panigrahy et al. Neuro-Oncology, e-pub Nov. 14, 2007). All spectra were acquired using a single-voxel PRESS sequence (repetition time = 1.5 s, echo time = 45 ms). LCModel software (S. Provencher Inc.) was used for processing and absolute concentrations of metabolites and metabolite concentrations ratios were determined. Diffusion tensor and perfusion MR imaging was also performed.

Results: An increase of N-acetyl-aspartate (NAA) concentrations and NAA/tCho (tCho = total choline) relative to baseline levels was observed in four of four newly enrolled patients in the first posttreatment study. This was observed only in one of five subjects treated with TRstd.

Discussion and Conclusion: The observed changes in newly enrolled subjects could be interpreted as a decreased density of neoplastic tissue within the region of interest and thus more effective therapy compared to the cohort of patients treated with the standard therapy. Follow-up and correlation with survival times are necessary to determine whether metabolic changes observed immediately after initial therapy are of predictive value for clinical course. If confirmed, MRS could serve as an early surrogate for the effectiveness of therapy and speed-up clinical trials significantly.

BSG 9. PSEUDOPROGRESSION IN PEDIATRIC DIFFUSE PONTINE GLIOMA TREATED WITH RADIATION AND CONCOMITANT TEMOZOLOMIDE

Lucie Lafay-Cousin,¹ Jason Feniak,² Beverly Wilson,³ Xingchang Wei,⁴ Carmen Rotaru,⁴ Eric Bouffet,⁵ and Douglas Strother⁴; ¹University of Calgary, Calgary, AB, Canada; ²Calgary, AB, Canada; ³Edmonton, AB, Canada; ⁴Calgary, AB, Canada; ⁵Toronto, ON, Canada.

Background: The concept of pseudoprogression (PsP) has been recently described in adult glioblastoma multiforme (GBM) treated with a combination of radiation (RT) and temozolomide (TMZ). It is defined by evidence of progressive disease on imaging early after RT, with or without supporting clinical signs, subsequently followed by improvement or stabilization of initial findings. PsP has often been mentioned in pediatric high-grade glioma (HGG) trial using RT and TMZ, however the incidence in children is unknown.

Aim of the Study: Estimation of the incidence of PsP in children with DPG treated with RT ± chemotherapy. Secondary objective was to identify potential predisposing factors for PsP (e.g., TMZ).

Methods: Retrospective review of clinical and radiological responses of pediatric DPG diagnosed in the province of Alberta from 1995 to 2007. MRIs from diagnosis, early after completion of RT and every 3 months thereafter were reviewed. Initial clinical symptoms, clinical response at similar time points of MRI evaluation, use of steroids, and survival data were collected separately.

Results: Thirteen cases (7 females) were evaluated in the study. Median age at diagnosis was 6.7 years (range, 4.3–16.4). Six patients received RT and TMZ, three had RT and other chemotherapy, and four received RT alone. Median times to clinical and radiological progression from end of RT were 5.5 months (0.7–14.7) and 5.7 months (0.7–15.6), respectively. Median survival time from completion of RT was 10.5 months (1.5–20.2). After completion of RT, six patients showed decrease in size of their lesions, three had stable disease, and four (31%) showed evidence of MRI and clinical progression at a mean time of 1.1 months from RT (early progression). Three of the six patients treated with TMZ showed sign of early progression compared to one of seven who did not receive TMZ. Two of these four patients with early progression remained clinically stable at 4.9 and 4.3 months post-RT with a documented partial response on MRI in one patient. None of the patients were on steroids at the time of clinical stabilization. The two patients later showed clinical and radiologic progression at 5.9 and 6.0 months post-RT. These two cases were felt to be in keeping with PsP. The occurrence of two PsP led to TMZ discontinuation in one case.

Conclusion: In this small series of DPG, 4 of 13 (31%) patients presented signs of early progression. Among them, 50% were in keeping with the entity of pseudoprogression. However given the short progression-free survival time of DPG, PsP may not be as recognized as in adult GBM and may require different definition criteria. Our two cases were associated with the use of TMZ. A larger, multiinstitution study extended to all pediatric HGG is ongoing.

BSG 10. RADIOLOGICAL CHANGES FOLLOWING COMBINED TEMOZOLOMIDE AND RADIATION IN CHILDREN WITH DIFFUSE BRAINSTEM GLIOMAS

Eric Bouffet,¹ Lucie Lafay-Cousin,² Annie Huang,³ Uri Tabori,⁴ Sylvain Baruchel,⁴ Darren Hargrave,⁵ and Ute Bartels⁴; ¹Haematology/Oncology, The Hospital for Sick Children, Toronto, ON, Canada; ²University of Calgary, Calgary, AB, Canada; ³Toronto, ON, Canada; ⁴University of Toronto, Toronto, ON, Canada; ⁵Sutton, UK.

Background: Evaluation of response in children with diffuse pontine gliomas is based on repeated clinical assessment, evaluation of steroid requirements, and radiological changes. At the end of the radiotherapy period, previous studies have reported clinical improvement in 70%–90% of patients, discontinuation of steroids in 60%, and radiological response in 40%–45% of the patients, with 45% stabilization and 15%–20% worsening (Hargrave et al. Lancet Oncol. 2006). The aim of this study was to compare radiological changes observed after radiation combined with temozolomide (TMZ) with those observed in a cohort of patients treated without TMZ.

Method: Retrospective clinical and radiological review of data in patients with diffuse pontine glioma seen at the Hospital for Sick Children between 2000 and 2007 who had two MRI evaluations (i.e., one baseline and one evaluation postradiation). Comparison of radiological changes on FLAIR and T2 images in TMZ versus non-TMZ patients.

Results: Twenty-five patients were identified (12 males and 13 females; median age, 6.6 years; range, 3.1–11.5), and three were excluded (no postradiation evaluation). Ten patients received radiation with TMZ, eight received radiation only, and four received radiation combined with motexafin gadolinium. The postradiation MRI was performed at 39 days (range, 9–66) after completion of radiation in the non-TMZ group versus 34 days (7–44) in the TMZ group. In the non-TMZ group, nine patients experienced clinical improvement, two had no change, and one had evidence of clinical progression. In the TMZ group, six patients experienced improvement, two had no change, and three had clinical deterioration. Radiological measurement showed a 48% median decrease (range, –85% to 256%) in 3D dimension in the TMZ group compared to a median 16% increase (range, –71% to 366%) in 3D dimensions in the TMZ patients. Radiological progression was observed in five patients in the TMZ group versus one patient in the non-TMZ group. The median survival time between the two groups did not significantly differ (10.3 vs. 10 months in TMZ vs. non-TMZ patients, respectively).

Conclusion: Half of the patients treated with TMZ and radiation show evidence of radiological progression on FLAIR/T2 on the early postradiation MRI. However, these changes are not correlated with clinical response and do not seem to predict a shorter survival span.

BSG 11. SPONTANEOUS REGRESSION OF BRAINSTEM GLIOMA IN AN INFANT

Monica Koehn¹; ¹Department of Neurology/Pediatric Neurology, Marshfield Clinic, Marshfield, WI, USA.

A 40-day-old term female presented with neonatal onset of neurologic symptoms including feeding difficulties, failure to thrive, and progressive irritability. Examination demonstrated a cachectic neonate with 10% weight loss since birth, uncoordinated swallow, stridorous respirations with regurgitation upon feeding, profound irritability, bifacial and tongue weakness, and suck-swallow incoordination. Infectious and traumatic evaluations were unremarkable. CT head demonstrated a nonenhancing brainstem mass extending throughout pons and medulla. MRI brain and cervical spine showed a large 3.3 x 3.1 cm, well-defined, completely intrinsic, nonenhancing mass extending from pons through C1 levels with signal intensities decreased on T1 and increased on T2-weighted images, all features consistent with brainstem glioma. The mass produced mild compression of the fourth ventricle without hydrocephalus. No brainstem biopsy was felt appropriate. No chemotherapy or radiation initated. Tube feeds resulted in weight gain. Dexamethasone 2 mg/kg/day initiated. Over the subsequent 6 weeks, dexamethasone was tapered very slowly to 0.5 mg/kg/day with dosage based on balancing perceived adverse effects of steroid-induced excessive irritability and clinical response of improved lower cranial nerve dysfunction. Follow-up MRI 7 weeks following the first study demonstrated a dramatic reduction in the size of the nonenhancing brainstem mass with no alteration in imaging features when compared to the initial study, no ventricular obstruction, and no new abnormalities. Steroid taper continued very slowly. Transition to exclusive oral feeding was successful. Neurologic examination and developmental assessment at 4 months of age was normal. This case reiterates the importance of considering the possibility of spontaneous regression in the presentation of brainstem glioma in a young infant. Although the dramatic improvement in neuroimaging and clinical features in this case is unusual relative to the typical clinical course of steady progression of neurologic decline in this diagnosis, spontaneous regression should be considered a rare but real possibility when discussing the very limited treatment options in this age group with a mass lesion involving this inoperable location.

BSG 12. THE ROLE OF RADICAL HYPOFRACTIONATION RADIOTHERAPY FOR DIFFUSE INTRINSIC BRAINSTEM GLIOMA IN CHILDREN: A PILOT STUDY

Geert Janssens,¹ Corrie Gidding,² Erik Van Lindert,³ Foppe Oldenburger,⁴ Corrie Erasmus,⁵ Antoinette Schouten-Van Meeteren,⁶ and Johannes Kaanders¹; ¹Department of Radiation Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; ²Department of Paediatric Oncology, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands; ³Department of Neurosurgery, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands; ⁴Department of Radiation Oncology, Academic Medical Centre Amsterdam, Amsterdam, Netherlands; ⁵Department of Paediatric Neurology, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands; ⁶Department of Paediatric Oncology, Academic Medical Centre Amsterdam, Amsterdam, Netherlands.

Background and Purpose: Despite aggressive approaches in many clinical trials done over the last three decades, the prognosis for children with diffuse intrinsic brainstem glioma remains dismal, and most will die within 1 year of diagnosis. As an alternative to the standard conventional radiotherapy schedule (30 fractions of 1.8 Gy over 6 weeks), we propose a radical hypo-fractionation schedule, 13 fractions given over 3 weeks, to limit outpatient department transfers and overall treatment time.

Materials and Methods: Between July 2004 and October 2007, nine children, ages 3–13 years, were treated in the radiotherapy departments of Nijmegen (seven patients) and Amsterdam (two patients) by a hypofractionation schedule consisting of 13 fractions of 3 Gy (eight patients) or 6 fractions of 5.5 Gy (one patient) given over 3 weeks (range, 18–22 days). All patients include, had symptoms for <3 months (mean, 6.3 weeks) and at least two signs of the neurological triad (long tract signs, ataxia, cranial nerve deficit). On MR imaging, there was bilateral involvement of the pons in eight of nine patients, encasement of the basilary artery in seven of nine patients and extension into the cerebellar peduncle in six of nine patients. Because of doubt, a stereotactic biopsy was obtained in four patients, all confirming a WHO grade IV astrocytoma.

Results: Symptom improvement occurred in all patients within 2 weeks after start of radiotherapy. Median time to progression (TTP) from diagnosis was 4.9 months. At a mean follow-up time of 15 months (range, 3–41 months), seven patients have died. Six out of seven patients died of local disease progression; one patient died of CNS infection but with tumor progression as proven by MRI. Median time to death (OS) from diagnosis was 8.6 months. Median time to death after progression was 3.6 months. These results are not different from what is reported in the literature. According to a recently published review of clinical trials, the median TTP, OS, and time between progression and death ranged from 5 to 8.8, 7 to 16, and 1 to 4.5 months, respectively, with conventional, accelerated or hyperfractionated schedules and other experimental treatment combinations (Hargrave et al. Lancet Oncol. 2006;7:241).

Conclusion: With this radical hypofractionation schedule, the median TTP, OS, and time to death after progression are within the range of published data. In our opinion, 13 fractions of 3 Gy, given over 3 weeks, are a good alternative to the conventional radiotherapy schedules of 54 Gy in 30 fractions over 6 weeks for diffuse intrinsic brainstem glioma. This treatment offers patients a quick symptom relief and a temporary improvement of neurological signs and symptoms with minimal toxicity and overall treatment time and maximal escape from outpatient department transfers and/or hospital stay.

BSG 13. USE OF IMAGING IN RESPONSE CRITERIA FOR PEDIATRIC PATIENTS WITH DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

Robert Hayward,¹ Robyn Bent,² Emilie Steffen-Smith,² Jonathan Wiest,² and Katherine Warren³; ¹NIH, Bethesda, MD, USA; ²MD, USA; ³National Institutes of Health (NIH), Bethesda, MD, USA.

Background: DIPGs are devastating tumors that represent 80% of brainstem gliomas in the pediatric population. Standard treatment includes the use of radiation therapy. These patients are diagnosed and followed by magnetic resonance imaging (MRI). Significant decrease in the size of DIPGs (e.g., 50% as per definition of response using 2D criteria), even after radiation, is not typical. Because of their dismal prognosis with median survival <1 year, many patients enter investigational trials. Investigational trials determine the activity of new agents by a reduction in tumor size, as defined by MRI. It has been demonstrated previously that measurement of DIPG by MRI is complicated because definition of tumor boundaries and distinguishing active tumor from radiation effects is difficult. In addition, methods of measuring can differ among radiologists and institutions.

Objective: To assess the variability in practice in the methods for MRI measurements and definition of response for patients with DIPG.

Methods: A sample of pediatric oncologists and neuro-oncologists in North America were asked to answer 10 questions by electronic survey regarding importance and use of imaging in the clinical management of pediatric patients with DIPG. Subjects were asked to rate likelihood or importance based on a 1–5 scale. In addition, the definition of response for clinical trials for which patients with DIPG were eligible over the past decade were reviewed.

Results: Fifty-nine responses were obtained from the survey. Eighty-five percent of respondents have treated >10 patients with DIPG. Significant variability in responses was noted for use and importance of MRI in different clinical situations. For comparing MRI scans, 51% chose the scan immediately prior to current scan as most important, while 38% chose the best response scan and 11% chose the diagnostic scan. Twenty-five percent of respondents were not likely to use the definition of progression by imaging to remove a DIPG patient from protocol. The majority of respondents do not routinely use FDG-PET studies to follow patients with DIPG and only 14% rated FDG-PET as important for clinical decision making. A total of 34 investigational studies for which DIPG patients were eligible over the past decade were reviewed (phase I, n = 19; phase II, n = 11; phase I/II, n = 4). Definitions of response varied and included RECIST, standard two-dimensional criteria only, standard two-dimensional criteria with instructions on how to measure, use of steroids, and requirement for response to be maintained over a specific length of time. No study included quality of life as a measure of response.

Conclusion: Patients with DIPG frequently enter clinical trials and are followed for response by MRI. The method of tumor measurement and definition of response is variable. The importance of MRI scans and sequences in clinical decision making is variable. The role of MRI in the management of patients with DIPG should be reviewed. At minimum, standard criteria for response for patients with DIPG enrolled on clinical trials need to be defined, and additional measurements of response, such as quality of life measures, should be included.

BSG 14. YOUNG AGE PREDICTS A BETTER OUTCOME FOR CHILDREN WITH DIFFUSE PONTINE GLIOMA

Alberto Broniscer,¹ Fred Laningham,² Robert Sanders,² Larry Kun,³ David Ellison,³ and Amar Gajjar²; ¹St. Jude Children's Research Hospital, Memphis, TN, USA; ²TN, USA; ³TN, USA.

Background: Since diffuse pontine glioma (DPG) is rare among young children, the outcome of affected patients is unknown.

Methods: We reviewed clinical and radiological characteristics of all children <3 years with diffuse pontine glioma (DPG) evaluated at our institution. Inclusion followed standard magnetic resonance imaging criteria for diagnosis of DPG.

Results: Median age at diagnosis in ten patients was 2.2 years (range, 0.8–2.7 years). Median interval from onset of symptoms and diagnosis was 2.5 months. All patients presented with cranial nerve palsy with (n = 7) or without (n = 3) other neurological deficits attributable to brainstem involvement. All patients had pons-based tumors involving >50% of this brainstem segment. Histological confirmation was attempted in two patients who had atypical radiological features at diagnosis. Original diagnosis was low-grade astrocytoma in both cases. Four patients were initially observed only. All patients have required therapy consisting of radiation therapy (RT; n = 2), RT and chemotherapy (n = 6), or chemotherapy only (n = 2). Median dose of RT dose was 54 Gy (range, 50.4–55.8 Gy). Three patients were younger than 2 years at the time of start of RT. Four patients have died of tumor progression after a median of 0.7 years (range, 0.5–3.7 years). Six patients have survived for a median of 2.3 years (range, 0.6–8 years). The 3-year progression-free and overall survival were 60% ± 22% and 69% ± 19%, respectively.

Conclusion: Children <3 years with DPG can potentially fare better than older patients with the same diagnosis despite use of similar therapy. Our results suggest that DPG in younger children may be biologically distinct from similar tumors in older age groups.

	CHOROID PLEXUS TUMORS

Top ATRT BIOLOGY BRAINSTEM GLIOMA CHOROID PLEXUS TUMORS CASE REPORTS CRANIOPHARYNGIOMA EPENDYMOMA EPIDEMIOLOGY GERM CELL TUMOR HIGH-DOSE CHEMOTHERAPY HIGH-GRADE GLIOMA NEURO-IMAGING INFANT TUMORS LATE EFFECTS LEPTOMENINGEAL AND DISSEMINATED... LOW-GRADE GLIOMA NURSING AND MULTIDISCIPLINARY... NEUROFIBROMATOSIS AND ASSOCIATED... NEUROPATHOLOGY NEUROSURGERY OUTCOME STUDIES PNET/MEDULLOBLASTOMA QUALITY OF LIFE RADIATION ONCOLOGY RARE TUMORS THERAPEUTIC AND NEW DRUG...

 
CPT 1. ABERRANT MGMT (O⁶-METHYLGUANINE-DNA METHYLTRANSFERASE) PROMOTER METHYLATION IS A FREQUENT EVENT IN CHOROID PLEXUS TUMORS

Martin Hasselblatt,¹ Joerg Muehlisch,² Brigitte Wrede,³ Birgit Kallinger,² Astrid Jeibmann,¹ Ove Peters,³ Johannes Wolff,⁴ Werner Paulus,¹ and Michael Fruehwald²; ¹Institute of Neuropathology, University Hospital Münster, Münster, Germany; ²Department of Pediatric Hematology and Oncology, University Children's Hospital, Münster, Germany; ³Department of Pediatric Oncology, University of Regensburg, Regensburg, Germany; ⁴University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

Promoter methylation of the O⁶-methylguanine-DNA methyltransferase (MGMT) DNA-repair gene is a surrogate marker for response to chemotherapy with alkylating agents (e.g., temozolomide) in the treatment of malignant gliomas. Because a role of temozolomide in the treatment of relapsing choroid plexus tumors is currently being considered, MGMT promoter methylation status was assessed in a retrospective series of 16 choroid plexus papillomas, 12 atypical choroid plexus papillomas, and seven choroid plexus carcinomas using combined bisulfite restriction analysis. MGMT promoter methylation was detected in 81% of choroid plexus papillomas, 58% of atypical choroid plexus papillomas, and 43% of choroid plexus carcinomas. Upon a median follow-up of 34 months, relapses had occurred in one of the choroid plexus papillomas, two of the atypical choroid plexus papillomas, and three of the choroid plexus carcinomas. Even though MGMT promoter methylation status tended to be associated with a higher probability of recurrence on univariate analysis, histopathological grading was the only independent predictor of outcome (p = 0.018). In conclusion, MGMT promoter methylation is frequent in choroid plexus tumors. Determination of MGMT promoter methylation status is warranted for stratification of patients for alklylator-based treatment in prospective future clinical trials.

CPT 2. CLINICAL FEATURES OF THE NEW WHO ENTITY OF ATYPICAL CHOROID PLEXUS PAPILLOMA: A SUBGROUP ANALYSIS OF THE INTERNATIONAL RANDOMIZED CPT-SIOP-2000 STUDY

Brigitte Wrede,¹ Ove Peters,¹ Peter F. Thall,² Georg Ebetsberger,³ Michaela Nathrath,⁴ Norbert Jorch,⁵ Martin Hasselblatt,⁶ Adela Cañete,⁷ and Johannes Wolff⁸; ¹Pediatric Oncology/Hematology, University of Regensburg, Regensburg, Germany; ²Department of Biostatistics and Applied Math, M.D. Anderson Cancer Center, Houston, TX, USA; ³Pediatrics, Landes-Frauen- und Kinderklinik, Linz, Austria; ⁴Pediatric Oncology, University Children's Hospital Munich, Munich, Germany; ⁵Pediatric Oncology, Ev. Krankenhaus, Bielefeld, Germany; ⁶Institute of Neuropathology, University Hospital Münster, Münster, Germany; ⁷Pediatric Oncology, Hospital Infantil La Fe, Valencia, Spain; ⁸Pediatric Neuro-Oncology, M.D. Anderson Cancer Center, Houston, TX, USA.

Introduction: Atypical choroid plexus papilloma (APP) is a novel entity proposed by the World Health Organization in 2007 with intermediate features between low-grade choroid plexus papilloma (CPP) and high-grade choroid plexus carcinoma (CPC). APP are histologically defined by increased mitotic activity compared to CPP, but their clinical features have not been elucidated in detail. We report on an APP subgroup analysis of the CPT-SIOP-2000 study, an international randomized trial for choroid plexus tumors (CPT), initially designed to identify risk factors and to determine the effectiveness of carboplatinum versus cyclophosphamide treatment in CPT.

Methods: A worldwide registration of children and adults with CPT and a randomized trial for patients requiring postoperative treatment were started in 2000. For APP, maximal surgical resection was performed. Patients with completely resected tumors were observed, whereas incompletely resected or metastasized APP were treated with six chemotherapy courses (VP16 100 mg/m², d1–5, vincristine 1.5 mg/m², d1, and either carboplatinum 350 mg/m², d1,2, or cyclophosphamide 1,000 mg/m², d1,2), and additional irradiation after the second course for patients older than 3 years of age.

Results: Until January 2008, 21 nations had registered 123 patients (45 CPC, 32 APP, 46 CPP). Of these, 49% were male. Patients with APP had a median age of 0.7 years and were thus significantly younger than patients with CPP (2.3 years) or CPC (2.7 years). 84% of APP were located in the lateral ventricles. Primary metastases were observed in 13% of the patients (5% in CPP, 19% in CPC). Complete resection was achieved in 69% of APP patients. In the observational group, no death was seen. In the APP group receiving further treatment, early response was similar to CPC with partial response or stable disease in seven of eight patients. The APP patients had an intermediate 5-year event-free survival of 70% ± 14 SD (CPP, 95% ± 4 SD; CPC, 45% ± 14 SD). 14% of the APP patients had a relapse (5% CPP, 33% CPC) and two of those showed transformation to CPC.

Conclusion: The average age of patients with atypical choroid plexus tumors was lower than that of patients with other choroid plexus tumors. In other aspects, the intermediate position of APP defined by histology was also clinically reflected by the rate of metastases, relapse, and survival between CPP and CPC. Radiochemotherapy of incomplete resected APP may yield similar results compared to CPC, but more patients are required.

CPT 3. GENETIC ASPECTS OF CHOROID PLEXUS TUMORS (CPTS): THE EXPERIENCE AT A SINGLE INSTITUTION IN ITALY

Maria Luisa Garrè,¹ Gilberto Fronza,² Claudia Milanaccio,¹ Liliana Varesco,³ Alessandro Raso,⁴ Samantha Mascelli,⁴ Andrea Rossi,⁵ Salvina Barra,⁶ Guia Hanau,¹ Paolo Nozza,⁷ Alessandro Consales,⁴ Armando Cama,⁴ and Valeria Capra⁴; ¹Haemato-Oncology, Giannina Gaslini Children's Research Hospital, Genoa, Italy; ²Molecular Mutagenesis and Translational Oncology, National Cancer Research Institute (IST), Genoa, Italy; ³Genetics, National Cancer Research Institute (IST), Genoa, Italy; ⁴Neurosurgery, Giannina Gaslini Children's Research Hospital, Genoa, Italy; ⁵Neuroradiology, Giannina Gaslini Children's Research Hospital, Genoa, Italy; ⁶Radiation Oncology, National Cancer Research Institute (IST), Genoa, Italy; ⁷Pathology, Giannina Gaslini Children's Research Hospital, Genoa, Italy.

Choroid plexus tumors (CPTs) are rare CNS tumors; prognosis of the malignant type, choroid plexus carcinoma (CPC), remains poor. CPTs have been reported as tumors occurring in Li-Fraumeni syndrome (LFS), although the frequency of occurrence of LFS in large series of prospectively treated patients with CPTs is not reported yet. We have investigated the clinicopathological characteristics, genetic profile, treatment, and outcome of 21 consecutive cases of CPTs diagnosed and treated at a single large institution in Italy in the period 1990–2006. Eleven were males, 10 females; age at diagnosis ranged from 2 months to 15 years (median, 26 months). There were 10 choroid plexus papillomas (CPP), 3 atypical CPP (ACPP), 9 CPC; lateral ventricles were involved in 17 cases (80%); IV and III ventricles were involved in 2 cases each (10%). Six cases (30%) had metastasis at diagnosis (five CPC, one APP). Treatment consisted of surgical approach with radical intent in all cases; preoperative chemotherapy in four cases (2 CPC, 2 APP); postoperative chemotherapy in 10 cases (9 CPC, 1 ACPP); 2 patients with CPC were irradiated. No relapses were observed in the CPP and ACPP subgroups. Outcome was very poor in CPC: six of nine dead of disease (66%). One patient with ACPP developed a second brain tumor (glioblastoma) at 5 years after discontinuation of treatment, and at that time this patient was found to present constitutional P53 mutation. Concerning the frequency of LFS, it was clearly present in one-third of cases with CPC (three of nine cases) and ACPP (one of three cases). One case in the CPP was reported to have Aicardi syndrome.

Conclusions: CPC and ACPP are frequently (30% of cases) associated with LFS in our study. Therefore, cases of CPC should probably be investigated at diagnosis for constitutional mutations of TP53, at least in families with positive history; genetic counseling should be offered to the families. Cases cured for CPTs should be followed for a prolonged period for occurrence of other cancers, and family history frequently updated. Impact of up-front genetic evaluation on treatment strategy of CPC should also be discussed in the plan of large international cooperative studies.

CPT 4. WHAT IS THE DEVELOPMENTAL FUTURE OF YOUNG CHILDREN WITH CHOROID PLEXUS PAPILLOMAS?

Silvia Gatscher,¹ Dianne Gumley²; ¹Neurosciences, Great Ormond Street Hospital, London, UK; ²Pediatric Psychology, Great Ormond Street Hospital, London, UK.

Introduction: Psychometric evaluation is nowadays part of routine follow-up in children who have been treated with radiotherapy for a CNS tumor. Regular developmental and psychological assessment has provided us with enormous insight regarding long-term side effects. This has sparked our interest in assessing the developmental progress in children who have only undergone a surgical resection for their brain tumor and to look at the consequences of a major neurosurgical procedure, in particular at an early age. We decided to carry out developmental assessments in children with choroid plexus papillomas (CPP), since CPPs usually present at an early age and surgery is the curative treatment.

Methods: Between September 2005 and March 2007, 10 children were admitted to Great Ormond Street Hospital with CPP and underwent a complete surgical resection. Eight children were less then 3 years old at the time of presentation; two were 11 and 14, respectively, at the time of diagnosis. One child was lost to follow-up. Seven children were <5 years of age when assessed and were evaluated with the Vineland Adaptive Behavior Scale and the Mullen scales. In the two older children, 12 and 15 at the time of follow-up, we used the Wechsler Intelligence Scales for Children Fourth UK Edition and the Children's Memory Scales. We also asked the parents to complete the Social Development Questionnaire and the Behavior Rating Inventory of Executive Function.

Results: We evaluated social and emotional development in both patient groups and executive function skills in the older group. The outcome from the initial assessment of the younger group was particularly encouraging. Developmental progress was within average range in five of seven patients, and five were making good progress with social and adaptive skills. The two older children also performed as expected for their age group.

Conclusion: We feel it is important to continue monitoring these children, to see whether their cognitive profile continues to progress as expected, and to observe their emotional and behavioral development.

	CASE REPORTS

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CR 1. A HIGHLY UNDIFFERENTIATED TUMOR IN THE POSTERIOR FOSSA IN A YOUNG CHILD WITH CUTANEOUS PIGMENT LESIONS

Joris Verlooy,¹ Caroline Van Den Broecke,² Edward Baert,³ Tom Boterberg,⁴ Valerie Meersschaut,⁵ Els Vandecruys,¹ Yves Benoit¹; ¹Pediatric Oncology/Hematology, Ghent University Hospital, Ghent, Belgium; ²Department of Pathology, Ghent University Hospital, Ghent, Belgium; ³Neurosurgery, Ghent University Hospital, Ghent, Belgium; ⁴Radiotherapy, Ghent University Hospital, Ghent, Belgium; ⁵Radiology, Ghent University Hospital, Ghent, Belgium.

We describe the case of a boy presenting at 7 months of age with signs of intracranial hypertension due to a posterior fossa tumor. Two months earlier three truncal large cutaneous pigment lesions had been removed and microscopy revealed several deep penetrating nevi, without evidence of malignancy. The boy underwent a macroscopically total resection confirmed by postoperative MRI. Microscopical examination revealed a highly undifferentiated tumor with small cells having a hyperchromatic nucleus and very little cytoplasm. There were no rosettes to be found, and no signs of glial or neuronal differentiation. Immunohistochemistry showed no positive markers for medulloblastoma (CD 56 and synaptophysin negative). GFAP, CD 99, desmin, keratins, and LCA were all negative. INI 1 staining of tumor cell nuclei was normal. Because of the atypical clinical picture of an intracranial mass and cutaneous lesions, markers for melanocytic lineage were also performed on the CNS tumor: Melan A and NKI-C3 were negative. HMB45 showed a dotlike staining pattern in the cytoplasm of some tumor cells. Staining with MITF-1 was, however, clearly positive. Based on this staining pattern, the diagnosis of a highly undifferentiated tumor with evidence of melanocytic differentiation was put forward. Genetic analysis for nevoid basal cell carcinoma syndrome was negative, and no other genetic anomalies were found. The boy was treated with a chemotherapy regimen consisting of vincristine, carboplatin, cyclophosphamide, and etoposide (SIOP PNET 3), but showed a massive regrowth of the tumor under this treatment at the age of 12 months. A second total resection was performed and because of the melanocytic aspect of the disease he was treated with a combination of irradiation on the posterior fossa, temozolomide, and dendritic cell vaccination. During this therapy he developed a significant number of new cutaneous lesions. MRI examination after irradiation revealed several spinal drop metastases. Therapy was intensified with intrathecal etoposide, but the spinal lesions grew massively and all treatment was ceased. In this child the combination of dermal nevi and CNS melanocytic lesions is suggestive of a neurocutaneous melanosis refractory to a combination of treatments.

CR 2. BONE MARROW TRANSPLANTATION (BMT) FOR APLASTIC ANEMIA SECONDARY TO TEMOZOLOMIDE THERAPY IN A LONG-TERM SURVIVOR WITH GLIOBLASTOMA MULTIFORME (GBM)

Brannon Morris,¹ Ulrike Reiss,² Kimberly Kasow,² and Alberto Broniscer¹; ¹St. Jude Children's Research Hospital, Memphis, TN, USA; ²St. Jude Children's Research Hospital, TN, USA.

Introduction: Radiotherapy plus concomitant/adjuvant temozolomide is standard treatment for glioblastoma multiforme (GBM) in adults. Although myelosuppression is a recognized dose-limiting toxic effect, temozolomide is generally considered to be well tolerated. More recently, several patients with irreversible bone marrow aplasia after treatment with temozolomide have been reported. We report the unusual case of an adolescent patient with GBM who underwent bone marrow transplantation (BMT) for treatment of presumed temozolomide-related aplastic anemia.

Case History: This patient was an otherwise healthy 16-year-old girl until she presented with a left frontoparietal intraparenchymal hemorrhage in April 2004. The source of hemorrhage was presumed to be an arterialvenous malformation, and she was followed until a second bleed occurred in July 2004. At this time, she underwent a biopsy and was subsequently referred to our institution for subtotal resection of a tumor that proved to be GBM. She was enrolled in August 2004 on CNS126, a phase II study utilizing cranial irradiation with concurrent temozolomide. By day 24 of therapy she became pancytopenic and temozolomide was discontinued. Radiation therapy continued, and the patient received a total dose of 59.4 Gy. The patient's pancytopenia worsened and ultimately she became red blood cell dependent and platelet transfusion dependent with an absolute neutrophil count (ANC) of zero. Bone marrow aspirate at this time revealed a hypocellular marrow with no dysplasia, infection, or neoplasm. A diagnosis of severe aplastic anemia was made, and she received filgrastim therapy from December 2004 to March 2005. Although her ANC responded with consistent levels >500, she remained transfusion-dependent with weekly red cell infusions to keep her hemoglobin count >8 g/dl and biweekly platelet transfusions to maintain her counts >20,000/mm³ due to frequent epistaxis. A bone marrow aspirate in September 2005 revealed cellularity <5% and no morphologic evidence of microorganisms or malignancy. Cytogenetics was not completed because no metaphases were available for analysis. Subsequent chromosome breakage analysis (diepoxybutane-induced) was within normal range and paroxysmal nocturnal hemoglobinuria screen was negative. In March 2006 she was diagnosed with significant iron overload (serum ferritin >5,000 ng/ml and liver iron concentration 30 mg/g). All the while, surveillance neuroimaging demonstrated stable residual tumor. In June 2006, 26 months after her sentinel bleed and 22 months after diagnosis of GBM, she began intensive immunosuppression therapy with antithymocyte globlulin, cyclosporine, and corticosteroids. Unfortunately, she did not have an adequate response to this therapy. In February 2007, after >30 months of stable neuroimaging, the patient underwent a matched unrelated donor BMT with the hope of improving her quality of her life by becoming transfusion independent. Unfortunately, despite studies showing her donor status to be 100% (T-cell chimerism = 98%), her graft function remained quite poor. In July 2007, she underwent additional immunosuppression followed by another infusion of her original donor stem cells. Currently, this patient is back at home with stable neuroimaging. Although she still requires blood and platelet transfusions two to three times per month, she has enrolled in her local community college with a reduced load of classes.

CR 3. CASE REPORT: GERMINOMA PRESENTING AS GRANULOMATOUS INFLAMMATION IN THE PINEAL GLAND

Veena Rajaram,¹ Nicholas Slimack,¹ and Arthur Dipatri¹; ¹Children's Memorial Hospital, Chicago, IL, USA.

Germ cell tumors, more specifically germinomas, are the most frequently encountered neoplasms in the pineal region in adolescents. In the absence of elevated serum and/or cerebrospinal fluid markers for alpha-fetoprotein and human chorionic gonadotrophin, tissue diagnosis is necessary in many cases before initiating therapy. Biopsy finding of a granulomatous inflammation generates a wide differential including infection, Langerhans cell histiocytosis, and juvenile xanthogranuloma. Germinomas presenting as a granulomatous inflammatory process have been reported and if this entity is not considered in the differential it may lead to the institution of inappropriate therapy.

Case Report: A previously healthy 15-year-old boy presented with a 1-week history of vertical diplopia, blurred vision, and intermittent headaches. An MRI of the brain revealed a heterogeneously enhancing mass originating in the pineal region with acute hydrocephalus. An endoscopic third ventriculostomy was performed. Tumor within the posterior portion of the third ventricle could not be visualized, so a biopsy was not obtained. Serum and cerebrospinal fluid (CSF) alpha-fetoprotein and human chorionic gonadotrophin levels were all in the normal range. Cytological examination of the CSF was negative for malignant cells. The patient's visual symptoms improved and the hydrocephalus resolved. Ten days later a craniotomy was performed for biopsy. At surgery the mass appeared to arise in the pineal region and extend superiorly along the posterior surface of the splenium until it collided with the falx. It then continued along the lateral surfaces of the falx. Multiple tissue samples were taken from the superficial and deep parts of the lesion. Histologically, sections showed extensive inflammation with lymphocytes and numerous multinucleated giant cells admixed with clusters of small blue cells with extensive crush artifact. Some of these cells had large nuclei and scant cytoplasm. Immunohistochemistry studies showed focal positivity in the crushed area and in rare single tumor cells for placental alkaline phosphatase with diffuse positivity in the cell clusters for CD117 (C-KIT). The multinucleated giant cells were positive for CD68, which also stains the admixed histiocytes. Synaptophysin and GFAP were negative. Special stains for microorganisms (acid-fast bacilli and fungus) were negative. The histology, background, and immunostaining pattern was most consistent with a germinoma with an extensive reactive, granulomatous response. We present this case to increase awareness of an atypical presentation of this entity.

CR 4. CASE REPORT: PAPILLARY MENINGIOMA (PM) PRESENTING AS LOSS OF MILESTONES IN A TODDLER

Stewart Goldman,¹ Robin Bowman,² Wendy Stellpflug,³ Tara Goei,³ and Veena Rajaram¹; ¹Children's Memorial Hospital, Northwestern University, Chicago, IL, USA; ²Neurosurgery, Children's Memorial Hospital, Northwestern University, Chicago, IL, USA; ³Children's Memorial Hospital, Chicago, IL, USA.

Introduction: Papillary meningioma (PM) is a rare malignant variant of meningioma with a propensity for intracranial recurrence and pulmonary metastasis. PM was first described by Cushing and Eisenhardt in 1938. Meningioma accounts for 13%–19% of intracranial tumors of adulthood; papillary meningiomas make up only 1%–2.5% of meningiomas in adulthood and have been rarely reported in children.

Case Report: We report a 20-month-old who presented to her MD with changes in balance and decrease in her ability to walk; she developed emesis and lethargy followed by acute decompensation. Emergency MRI revealed hydrocephalus and a large post fossa mass. She underwent gross total resection of her left cerebellar hemispheric mass. On histological examination, the tumor showed sheets of discohesive cells with large vesicular nuclei, occasional nucleoli, and moderate amount of eosinophilic cytoplasm arranged as clusters and perivascular papillary/pseudorosette-like structures. Occasional intranuclear pseudoinclusion was identified. Mitosis was easily identified and there were areas of tumor necrosis. Immunohistochemical studies showed focal weak positivity for epithelial membrane antigen and rare tumor nuclei positivity for progesterone receptor. Ultrastructural studies showed tumor cells with numerous highly interdigitating cellular processes, occasional intercellular junctions consistent with desmosomes, and no zipperlike junctions, ciliary bodies, or microvilli. The ultrastructural findings are suggestive of meningothelial differentiation and the histology was that of a papillary meningioma. Six weeks postoperative she had local recurrence. Workup following a second resection prior to focal proton beam therapy revealed multiple pulmonary and liver metastases. She underwent chemotherapy with ifosfamide, carboplatin, and etoposide (ICE) with significant decrease of her systemic metastasis after the first cycle and obtained complete response after 2 courses. Further cycles of ICE alternating with cycles of doxorubicin, vincristine, and cytoxan were given over 6 months; she then completed high-dose chemotherapy and peripheral-blood stem cells with a carboplatin, thiotepa, and etoposide preparatory regimen; presently she remains with no evidence of disease with short follow-up. This case is presented to increase awareness of this extremely rare entity in young children.

CR 5. CLINICAL AND RADIOGRAPHIC CHARACTERIZATION OF RADIATION RECALL IN THE DEVELOPING CNS

Anna Janss,¹ Nadia Esiashvili,² Mark Kieran,³ and Susan Chi⁴; ¹Emory University, Decatur, GA, USA; ²GA, USA; ³MA, USA; ⁴Boston, MA, USA.

Radiation recall describes an acute inflammatory reaction in previously irradiated areas after the administration of inciting systemic agents which include several cytotoxic drugs. The skin is the tissue where the phenomenon was first described, but it has been reported in other organ systems. Radiographic and clinical features of radiation recall in the CNS have not been well described. We present a case report of radiation recall affecting the CNS during treatment protocol for children with newly diagnosed CNS atypical teratoid/rhabdoid tumor (AT/RT), DFCI 02-294. This 51-week treatment regimen includes the following systemic agents: vincristine, cisplatin, etoposide, cyclophosphamide, doxorubicin, dactinomycin, and temozolomide. Intrathecal therapy (methotrexate, cytarabine, and hydrocortisone) is administered throughout the regimen. Radiation therapy is given during weeks 7–12; for this patient, conformal 54 Gy in 180 cGy fractions was delivered.

Case: A 3-year-old girl with a subtotally resected pineal AT/RT received induction chemotherapy, chemoradiation (conformal RT), and consolidation therapy, with complications limited to constipation, anemia, and neutropenia. Evaluation performed at week 19 showed tumor regression on imaging and a normal neurological examination. On week 24 of therapy she developed partial seizures featured by right arm shaking and altered mental status, in the absence of systemic illness or metabolic abnormality. Seizures were controlled with benzodiazepines, then levetiracetam. Neuroimaging revealed stable tumor in the pineal gland and dorsal midbrain with new T2 signal abnormality in the posterior mesiotemporal lobes, thalami, mid-brain, and medial cerebellum. This distribution closely matched the high-voltage regions in her radiation dosimetry maps. After this episode the child developed ataxia, nystagmus, vertical skew, tremor, and right hemiparesis. These symptoms gradually improved but did not completely resolve. Imaging over the ensuing 6 months revealed regression of the T2 signal abnormality, replaced by transient patchy enhancement. Dactinomycin, doxorubicin, and intrathecal medications were held or reduced for the remainder of her therapy. The child remains on anticonvulsants without evidence of recurrence 6 months after completion of therapy. This case highlights an example of the radiographic features and prolonged neurological consequences of radiation recall in the developing nervous system, contrasting with the more classic and temporary findings typically seen in radiation recall. Recognition of radiation recall within the brain and differentiation from other treatment-related changes will require a better understanding of their presentation and etiology.

CR 6. EXTRACRANIALLY METASTATIC PNET PRESENTING WITH ABDOMINAL PAIN

Arja Harila-Saari,¹ Hannu Tuominen,² Sakari Knuutila,³ Merja Mottonen,¹ Ritta Herva,² and Satu Lehtinen¹; ¹Department of Pediatrics, Oulu University Hospital, Oulu, Finland; ²Department of Pathology, Oulu University Hospital, Oulu, Finland; ³Department of Pathology, Laboratory of Cytomolecular Genetics, Helsinki, Finland.

A 6-year-old boy was admitted into our hospital because of a 2-week history of abdominal pain. He was febrile and unable to walk because of the pain, but his physical examination was otherwise unremarkable. CRP was high (120 mg/liter), as were also ferritin (1,890 µg/liter) and LD (4,216 U/liter). Subtle anemia (Hb 9.0 g/dl), leukocytosis (22 x 10⁹/liter), thrombocytopenia (94 x 10⁹/liter), and clear eosinophilia (45%) were found. Splenomegaly and enlarged abdominal and hilar thoracal lymph nodes were observed in CT. Bone marrow was nearly filled with nonleukemic small blastlike malignant cells. Immunohistochemisty was not compatible with Ewing's sarcoma, and the test for translocation t(11;22) was negative. The disease progressed and the patient's condition deteriorated so rapidly that a course of chemotherapy (VAC) had to be started before the final diagnosis. FDG-PET scan showed increased glucose uptake in the bone marrow, lungs, pleura, abdomen, and brain. MRI revealed a huge frontal brain tumor. No malignant cells were shown in CSF, and the patient did not have any neurological symptoms or findings. The tumor was operated as soon as the patient's condition improved. PAD was best compatible with atypical PNET. Molecular caryotyping showed gene amplifications in the entire chromosome 18 and chromosome 19, especially in the 19q arm. The chromosome 9p gene CDKN2A was deleted. These findings fit the diagnosis of PNET. Despite two additional chemotherapy courses, the disease progressed, and the patient died 3 months later. At autopsy, the tumor was found to have metastasized diffusely within the CNS and all over the body. Such an aggressive brain PNET with disseminated disease at diagnosis is extremely rare and seems to be associated with rapid progression and poor prognosis

CR 7. HIGH-GRADE CONGENITAL PRIMITIVE NEUROEPITHELIAL TUMOR: A CASE STUDY

Wendy Beaudoin,¹ Diane Arndt,² Vivek Mehta,² and Lothar Resh²; ¹Stollery Children's Hospital, Edmonton, AB, Canada; ²AB, Canada.

A 9-day-old female infant was admitted after a 2-day history of nonbilious projectile vomiting and dehydration. Head circumference at that time was 37.5 cm. The infant was born of a 30-year-old mother at 40 weeks gestational age via scheduled C-section. Apgars were 8 at 1 min and 10 at 5 min. Two prenatal ultrasounds had been done at 22 weeks and 34 weeks gestation. Both were reported to be unremarkable. On day 2 of admission a head ultrasound was completed, revealing a very large heterogeneous mass within the right cerebral hemisphere. A CT and MRI of the head were obtained and revealed a mass that was centered within the right frontal lobe but crossed midline and extended into the right middle cranial fossa. The tumor measured 8.0 x 6.6 x 6.0 cm. At 13 days of age, a right frontal craniotomy with drainage of cyst and tumor biopsy was performed. Intraoperative frozen section was described to be consistant with astrocytoma. On postoperative day 1, the patient developed tongue fasciculations and lip smacking and was started on phenobarbital; CT scan showed some improvement in size of the cyst and less mass effect on the right lateral ventricle. A family conference was held day 13 postoperatively to review pathology, and palliative chemotherapy was offered. The family declined any intervention, and on postoperative day 17 the child was discharged home on phenobarbital and tube feeds with home care and the palliative care team was involved. The patient died peacefully at home 3 months later. The incidence of congenital brain tumors is rare. The definition is controversial. The two main criteria for classifying a brain tumor as congenital are (1) the age of the patient at presentation of clinical manifestations and (2) the histopathological characteristics of the tumor. These tumors account for only 0.5%–1.5% of all childhood brain tumors, but for 5% of all neonatal deaths.

CR 8. IDIOPATHIC INTRACRANIAL PACHYLEPTOMENINGITIS IN A CHILD TREATED FOR ATYPICAL TERATOID/RHABDOID TUMOR (ATRT)

Lidija Kitanovski¹ and Martina BüRger Lazar¹; ¹Department of Hematooncology, University of Ljubljana, University Children's Hospital Ljubljana, Ljubljana, Slovenia.

A now 5-year-old boy was diagnosed with multifocal atypical theratoid rhabdoid tumor (ATRT) at the age of 2 years. The infratentorial tumor was surgically removed. He was treated intensively according to German ATRT-ZNS 2004 protocol for 14 months, including chemotherapy, intraventricular methotrexate, and local radiotherapy. During induction treatment he experienced persistent nausea and vomiting with ventricular dilatation. Diamox had no effect. VP drainage was done; thereafter consciousness disturbances appeared. Dexamethasone was introduced, followed by significant improvement of nausea and vomiting, which reappeared immediately after its discontinuation. MRI revealed diffuse pachymeningeal thicknening and mild leptomeningeal (especially along folia cerebelli) enhancement. Diagnostic workup did not detect any infectious agents or malignant cells in the CSF. Chemotherapy has continued. Later convulsions with consciousness disturbances appeared on two occasions and responded poorly to anticonvulsants, except dexamethasone. Nausea and vomiting again considerably improved during dexamethasone use, and regression of MRI meningeal findings was observed. Hystological examination of two consequent meningeal biopsies (after considerable regression of MRI findings following dexamethasone and at the time of supratentorial tumor removal) revealed no abnormalities. After second convulsive status, dexamethasone was continuously used for 1.5 years; periodic MRIs have revealed persistent pachymeningeal and slight leptomeningeal thickness, while nausea and vomiting have been considerably controlled. Due to severe side effects, dexamethasone was replaced by methylprednisolone and gradually tapered to very low dose. Nausea and vomiting reappeared, left facial nerve paresis worsened, and progression of MRI findings with diffuse bone thickness of calvaria was found. Immunological workup revealed increased IgM level (1.99 g/liter) and positive ANCA-BPI antibodies. ANA, anti-ENA, s-ACE, LA, and ACA were normal. Elevated erythrocyte sedimentation rate (ESR) and CSF protein concentration were detected on several occasions. A third meningeal biopsy (at the site of a chronic subdural hematoma) did not confirm pachymeningitis. The child received 2 courses of high-dose methylprednisolone, nausea and vomiting diminished again, ESR decreased, and ANCA antibodies were no longer detected. Recently azathioprine was added to methylprednisolone to try to considerably reduce or even omit corticosteroids in the future since they significantly compromise the quality of life of the child, who walks with assistance and has severe osteoporosis, marked cushingoid appearance, and progressive cerebral atrophy. Psychological assessment of his cognitive, physical, language, and psychosocial development show mental and motor developmental delay. Most delayed mental areas are complex language and mathematical concept formation, construction, spatial problem-solving and planning, memory, discrimination, and abstract thinking. The main motor disabilities are balance, coordination of large muscles, finer manipulatory skills of hands and fingers, dynamic movement, and visual-motor integration. His mood is very unstable and varies from depressive to euphoric. We believe we are dealing with idiopathic intracranial pachyleptomeningitis in a child treated for multifocal ATRT, who currently (2 years after treatment conclusion) has no signs of tumor relapse. Persistent clinical and occasional MRI findings of improvements after corticosteroids and persistently elevated ESR favor our diagnosis, despite its not being hystologically confirmed. In our patient idiopathic pachyleptomeningitis might be MPO-ANCA associated and provoked by intravenous or intraventricular chemotherapy in the past.

CR 9. INCREASED NONCLONAL CHROMOSOME ABERRATIONS IN A PATIENT WITH GLIOSARCOMA AND SECONDARY ACUTE MYELOID LEUKEMIA

Zhihong Wang,¹ Kanta Bhambhani,¹ Merlin Hamre,¹ Guo Liu,² Christine Ye,² and Henry Heng²; ¹Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, MI, USA; ²Center for Molecular Medicine and Genetics, Wayne State University, MI, USA.

The theory that genome aberrations rather than gene mutations cause the majority of cancers has gained increasing support from recent experimental data (Heng et al. Genome. 2006;49:195; Heng. BioEssays. 2007;29:783). Genomic instability–generated nonclonal chromosome aberrations (NCCAs) are believed to be a key driving force in cancer progression (Heng et al. J Cell Physiol. 2006;208:461). Both chromosome fragmentation and defective mitotic figures (DFMs) are newly identified forms of chromosome aberrations. In vitro data indicated that frequencies of chromosome fragmentation correlate to levels of genomic instability of cell lines; and many chemotherapeutic agents can generate increased frequency of chromosome fragmentation (Stevens et al. Cancer Res. 2007;67:7686). We report here a patient who developed secondary acute myeloid leukemia (AML) within a year after completion of treatment for gliosarcoma. Significantly increased chromosome fragmentation and NCCAs were detected when he was receiving chemotherapy for gliosarcoma. The patient is a 13-year-old African American male who initially presented with seizures, which led to the evaluation and diagnosis of malignant gliosarcoma of the left frontal region. He had a complete resection of the left frontal mass followed by involved field radiation and chemotherapy with CCNU and temozolamide per COG study ACNS0423. He completed treatment per protocol without major complications. He had an unremarkable past medical history and no family history of brain tumors or other cancers. Nine months after completion of treatment, he developed a forearm hematoma following minor trauma. Workup revealed moderate anemia and severe thrombocytopenia, which prompted bone marrow evaluation and diagnosis of AML with features of extensive myeloid displasia. He had complex cytogenetic abnormalities including 45,XY, del(5)(q13), del(7)(q22q36), r(11)(p15q25), and add(17)(p12). There was no evidence of translocation or deletion of MLL/11q23 by FISH. His AML was refractory to multiple rounds of chemotherapy including arabinoside, daunorubicin, and etoposide (ADE), gemtuzumab, and clofarabine. As part of a pilot study to determine the clinical significance of nonclonal chromosome aberrations, tests for chromosome fragmentation, DMFs, and NCCAs were performed when he was receiving chemotherapy for gliosarcoma. The results showed that he had >20% chromosome fragmentation and 36% NCCAs, both the highest degree among the 15 brain tumor patients we have tested. He also had significantly increased DMFs (>5%) and free chromatin. It is likely that increased chromosome aberrations induced by chemotherapy in this patient contributed to his secondary malignancy. It is uncertain whether he had inherent genome instability, as pretreatment testing was not performed. Nevertheless, we believe that the increased genome instability as demonstrated by increased NCCAs and increased chromosome fragmentation contributed to the rapid onset of secondary malignancy. Although this is only an anecdotal case, it raised some interesting issues. First is whether we can use these tools to monitor the degree of chromosome aberrations and determine the levels of genome instability in patients with high risk of developing cancers. Second is whether the severity of nonclonal chromosome aberration as reflected by NCCAs, chromosome fragmentation, and DNFs could be used as an indicator of increased susceptibity to secondary malignancies.

CR 10. INTRACAVITARY CHEMOTHERAPY WITH LIPOSOMAL CYTARABINE: A CASE REPORT

Benedikt Weiß,¹ Petra Turowski,¹ Gerda Demleitner,¹ Brigitte Wrede,¹ and Ove Peters¹; ¹Children's Hospital, Regensburg, Germany.

Background: We present a white male patient 17 years of age at diagnosis of an alveolar rhabomyosarcoma (T4/pN2c/M0) of the paranasal sinuses with a tumor cyst (6.6 x 5.6 x 4.1 cm; volume 76 cm³) extending per continuity into the left frontal cerebral lobe. This cyst had no contact with the cerebrospinal fluid (CSF) system. In addition to the sarcoma chemotherapy, we locally treated the tumor cyst with repeated intracavitary liposomal cytarabine (DepoCyt) injections. Liposomal cytarabine has been developed as an alternative for the intrathecal chemotherapy of neoplastic meningitis. The drug is composed of the pyrimidine analog cytarabine, encapsulated in multivesicular, lipid-based particles. After a single intrathecal injection, these particles spread throughout the CSF and account for a sustained release of cytarabine, maintaining cytotoxic concentrations up to 14 days.

Procedure: On the third day of the sarcoma chemotherapy (CWS 2002-P: i.v. doxorubicin, ifosfamide/vincristine/dactinomycin, and ITH prednisolone/cytarabine/methotrexate), the intracranial tumor cyst volume increased, resulting in symptoms of elevated intracranial pressure (ICP). We decreased the cyst volume by serial punctures due to an implanted Rickham reservoir. The symptoms of ICP improved rapidly. Each extracted cystic fluid showed a massive amount of rhabdomyosarcoma cells. Because of the recurrent increase in cyst volume, we additionally started an intracavitary chemotherapy by injection of DepoCyt via the implanted Rickham reservoir. The injections were given at days 24 (1 mg), 28 (5 mg), 35 (5 mg), and 49 (5 mg) after initiation of the sarcoma chemotherapy. DepoCyt trough concentrations of the cystic fluid were determined before the injection. Meanwhile, the sarcoma chemotherapy was continued, with interposition of a local irradiation (52.2 Gy) of all primary tumor sites starting 73 days after primary diagnosis.

Results: After four intracavitary DepoCyt injections and concomitant sarcoma chemotherapy within 26 days, a partial shrinkage of the cyst volume and of the primary tumor sites was observed by MRI/sonography before irradiation was started. After the third DepoCyt injection no more rhabdomyosarcoma cells of the cystic fluid were microscopically seen. Since the tumor cyst collapsed completely 53 days after the diagnosis, we terminated the DepoCyt injections. Directly prior to the first DepoCyt injection 24 days after the chemotherapy initiation, the cytarabine concentration of the cystic fluid was zero. Thereafter, the lowest DepoCyt concentration was 2.1 mg/liter 5 days after the first injection (1 mg). The highest concentration was 7.0 mg/liter 8 days after the second injection (5 mg). These DepoCyt trough concentrations are in the range of those of the ventricular CSF after an intraventricularly injected dose of 75 mg DepoCyt in adults. The patient received a total of four DepoCyt injections without consequential side effects or Rickham reservoir complications. Unfortunately, he experienced a multifocal relapse 2 months after termination of the chemotherapy. He died 14 months after the primary diagnosis despite a cytostatic and biologic treatment attempt. At time of relapse, no residual of the tumor cyst was observed.

Conclusion: We conclude that the intracavitary liposomal cytarabine contributed to the disappearance of the intracranial rhabdomyosarcoma cyst and that inoperable or chemotherapy-resistant intracranial malignant cysts could locally be treated with the described strategy.

CR 11. INTRACRANIAL EWING SARCOMA/PERIPHERAL PRIMITIVE NEUROECTODERMAL TUMOR: A REPORT OF TWO CASES

Kazuaki Shimoji,¹ Makoto Hishii,¹ Naoaki Horinaka,¹ Masakazu Miyajima,² and Hajime Arai²; ¹Department of Neurosurgery, Juntendo Nerima Hospital, Nerima City, Tokyo, Japan; ²Department of Neurosurgery, Juntendo University School of Medicine, Bunkyo City, Tokyo, Japan.

Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is a malignant tumor with a pathological appearance of small round cells. It is commonly seen outside the central and sympathetic nervous system such as bone and soft tissue. This tumor may have different characteristics from central PNETs originating in the CNS, which have been described by Hart and Earle. Recently in this tumor, positive immunostaining for MIC2 and characteristic translocation; t(11;22)q(24;q12) related to chromosome 22q12 has been reported. Thus, it has been accepted as a single category. Two cases that had been diagnosed as intracranial ES/pPNET were treated in our institute.

Case 1: An 18-month-old girl was admitted to our hospital suffering from left abducens palsy. The MR image disclosed a cudgel-shaped mass just adjacent to the left pons. She underwent surgery via subtemporal approach. The tumor was partially resected and histological examination showed small round cells with rich chromatin. Homer-Wright rosettes and Flexner-Wintersteiner rosettes were also seen. Immunohistochemical staining revealed positive reaction to MIC2, which led to a diagnosis of intra cranial ES/pPNET. This girl unfortunately died 1 month after surgery despite craniospinal radiation therapy and chemotherapy.

Case 2: A 5-year-old boy was referred to our hospital because his consciousness level had deteriorated to lethargy. The MR image showed a huge mass located at the temporal lobe adjacent to the dura matter. Tumor resection was conducted followed by radiation and chemotherapy. Histological examination was similar to case 1. This boy is still followed up 4 years after surgery without recurrence. In this case expression of EWS/FLI-1 chimeric gene was seen. In the literature, only four reports of intracranial ES/pPNET with histological confirmation are reported. Therefore, this tumor is considered a rare tumor. However, if we focus on the positive reaction of MIC2 in immunohistochemical staining and expression of EWS/FLI-1 chimeric gene, this tumor may not be extremely rare. We would like to discuss the clinical course of our two cases with a review of the literature.

CR 12. MEDULLOBLASTOMA IN A BOY WITH SEPTO-OPTIC DYSPLASIA: CAUSAL RELATIONSHIP?

Antoinette Schouten-Van Meeteren,¹ Jeroen Vermeulen,² Huib Caron,³ Rogier Versteeg,⁴ and Marcel Kool⁴; ¹Pediatric Oncology, Emma Children's Hospital AMC, Amsterdam, Netherlands; ²Pediatric Neurology, VU University Medical Center, Netherlands; ³Pediatric Oncology, Emma Children's Hospital AMC, Netherlands; ⁴Human Genetics, Academic Medical Center, Amsterdam, Netherlands.

Background: The combination of developmental abnormalities of the eye and medulloblastoma is rare and has been described earlier in Gorlin syndrome, Gardner syndrome, and Turcot syndrome. The mutations in these syndromes are detected in the PTCH1 and APC genes, which also have been found in sporadic cases of medullloblastoma. We present a case of a 3.5-year-old boy with medulloblastoma. Staging of the disease showed tumor cells in the cerebrospinal fluid. The treatment consisted of complete resection of the tumor, craniospinal radiotherapy at 54/36 Gy, and chemotherapy with vincristine, lomustine, and cisplatinum for eight courses. Complete remission has been sustained for 5 years now. The medical history of the boy mentioned strabismus at 11 months of age, caused by unilateral optic hypoplasia. Further analysis of this feature revealed optic hypoplasia visible at fundoscopy showing a small pupil with abnormal pigment at the nasal side of the retina, while other sides of the eye were completely normal in development. At physical examination no signs of optic hypoplasia were present, nor were there any doubts about the psychosocial development of the boy, which was normal until the diagnosis of the brain tumor. The endocrine axis showed no abnormalities. MRI revealed absence of the septum pellucidum and the optic nerve was extremely thin, so septo-optic dysplasia was diagnosed. Expression profiling of the tumor tissue of this patient showed that several retina-specific transcription factors such as NRL, CRX, and OTX2 were expressed at high levels. These transcription factors are important for normal eye development, but their role in medulloblastoma pathogenesis is still unclear. Retina-specific gene expression in a medulloblastoma of a patient with optic hypoplasia may suggest that a genetic defect is responsible for both the abnormal eye development and the tumor. In conclusion it is important to examine dysmorphic features in all children with medulloblastoma. Physical examination should be completed with MRI to detect subtle abnormalities of the brain and eye as well. Increased awareness of a possible relationship between neurogenic and oncogenic factors might be helpful.

CR 13. MENINGIOANGIOMATOSIS IN THE BASAL GANGLIA

Takayuki Inagaki,¹ Yasuo Yamanouchi,¹ and Keiji Kawamoto¹; ¹Neurosurgery, Kansai Medical University, Moriguchi, Osaka, Japan.

Introduction: Meningioanigomatosis (MA) is a rare benign disorder. It is a focal lesion involving the leptomeninges and underlying cortex. It was originally described in association with von Recklinghause disease. However, according to Ibrahim Omesis et al., <15% of the cases are associated with neurofibromatosis (NF). MA located at the basal ganglia has not been described previously. We report a case of sporadic MA located in the basal ganglia.

Case presentation: The patient was referred to our hospital after head trauma at the age of one. On computer-assisted tomography, a calcified lesion was found on the left basal ganglion without mass effect or peripheral edema. Because he had no clinical signs he was followed at outpatient clinic. He visited our institute at the age of eight because of slight headache. Follow-up MRI at that time revealed slightly increased size of the mass. The mass was enhanced with Gd. The relationship between the mass and his headache was unclear. Since the tumor was increasing in size, we performed stereotactic biopsies. The first biopsy revealed calcification with normal brain. However, the second biopsy revealed a cluster of meningeal cells which had a whirl formation. The pathological diagnosis of meningioangiomatosis was made. Because of the pathological diagnosis, neither irradiation nor chemotherapy was given. The boy started to have seizures at the age of 10, which were controlled with medication.

Discussion: There are approximately 100 reported cases of MA so far in the English literature. Sixteen cases are associated with NF. The others were thought to be sporadic cases. In both sporadic and NF cases, the lesion was usually located in the leptomeningeal and underlying cortex. There in no report of MA located in the basal ganglia. Total removal of the tumor was recommended in case of leptomeningeal MA. It is not possible in this case because of the location of the mass.

CR 14. RARE EXTRANEURAL METASTASIS FROM A PRIMARY SPNET: DIAGNOSIS AND MANAGEMENT

Jason Fangusaro,¹ Yasmin Gosiengfiao,¹ David Jacobsohn,¹ and Tadanori Tomita²; ¹Pediatric Hematology/Oncology and Stem Cell Transplantation, Children's Memorial Hospital, Northwestern University, Chicago, IL, USA; ²Pediatric Neurosurgery, Children's Memorial Hospital, Chicago, IL, USA.

Although CNS tumors are the most common solid tumor in pediatrics, it is exceedingly rare for a primary brain tumor to spread outside the CNS. When this rare finding does occur, it is often after multiple relapses and is almost universally fatal. We present the case of a 2-year-old female who presented with typical signs of increased intracranial pressure, including emesis and headache. Imaging revealed a large mass in the left occipital region. A near total resection was performed revealing a tumor consistent with a supratentorial primitive neuroectodermal tumor (sPNET). Postoperatively, the patient developed respiratory difficulty, and a chest x-ray and chest CT were performed revealing a paraspinal mass and lung nodules. A biopsy was performed of the paraspinal mass, again revealing a PNET tumor. The tumor samples from both operations were sent for translocation studies and were both negative for the commonly known Ewing sarcoma translocations. Bone scan, abdominal/pelvic CT, spine MRI, lumbar puncture,and bone marrow evaluations were all negative for any other sites of disease. This patient was treated initially using a Ewing's protocol, since the translocation results were yet unknown and Ewing's metastasis to the CNS would be more commonly seen than metastasis outside the CNS. Her lung disease responded completely as seen by imaging, and her CNS disease showed some response of the residual tumor. She subsequently underwent tandem autologous hematopoietic cell rescue after high-dose chemotherapy followed by focal brain radiation. She is currently alive 12 months from diagnosis with a good quality of life. This case demonstrates that extraneural metastasis from primary CNS tumors can occur, and with aggressive treatment, a prolonged event-free survival can be achieved.

CR 15. RETINOBLASTOMA DISPERSION AFTER CHEMOTHERAPY: AN UNREPORTED AND UNDESIRABLE RESPONSE

Reut Parness-Yossifon,¹ Paul Bryar,² Joanna L. Weinstein,³ Janice Lasky Zeid,¹ and Marilyn B. Mets¹; ¹Ophthalmology, Children's Memorial Hospital, Chicago, IL, USA; ²Ophthalmology, Northwestern University Medical School, Chicago, IL, USA; ³Hematology/Oncology, Children's Memorial Hospital, Chicago, IL, USA.

Introduction: Retinoblastoma, the most common intraocular tumor in children, is a highly curable malignancy whose management has changed dramatically over the past decade. Historically, external beam radiation and enucleation were the primary treatment modalities for advanced intraocular retinoblastoma while chemotherapy was reserved for extraocular disease. The morbidity and risk for secondary malignancies associated with external beam radiation prompted further investigation into alternative globe-preserving therapies. In the 1990s promising results with chemoreduction were reported. Chemoreduction refers to the use of chemotherapy to reduce the tumor's volume; the shrunken tumor is then amenable to focal therapy, including cryotherapy, laser photocoagulation, thermotherapy, or plaque radiotherapy. Today, chemoreduction has become the most common approach in moderate to advanced disease. Various uncommon vitreoretinal complications have been reported after treatment. One rare complication reported after focal laser therapy is tumor dispersion, leading to active seeds in the subretinal space and vitreous. This is a devastating complication because vitreous seeds are very difficult to treat and often result in enucleation.

Purpose: To present three cases of retinoblastoma that demonstrated an unusual response to chemotherapy.

Patients: One girl and two boys presented at 8, 28, and 40 months of age, respectively, with large unilateral retinoblastomas. At presentation, cases 1 and 2 had no evidence of vitreous or subretinal seeds; case 3 had a few distant retinal seeds but no vitreous seeds. Shortly after the diagnosis, chemotherapy was started with carboplatin and etoposide in cases 1 and 3, and carboplatin, etoposide, and vincristine in case 2. In all cases examination after one cycle of chemotherapy revealed extensive vitreous seeding. All the eyes were enucleated. Histopathologic exam revealed in all cases either viable tumor cells in the vitreous or significant invasion of the ciliary body, choroid, and inner sclera.

Conclusions: We present three cases of unilateral retinoblastoma that demonstrated an atypical response to chemotherapy with intraocular dissemination. This response has not been previously described in the literature. In all cases the clinical picture changed dramatically in a very brief period of time following chemotherapy, a fact that emphasizes the need for close follow-up. An analysis of Shields et al. found an association between the absence of subretinal fluid, vitreous seeds, or subretinal seeds and the success of chemoreduction. In our cases only one had few subretinal seeds at presentation. We hypothesize that in addition to the clinical characteristics, the histopathologic features, like microscopic seeding, also may have an impact on the tumor's response to chemotherapy. A sudden increase in tumor necrosis in response to chemotherapy may lead to loss of integrity of the tumor mass and dispersion of the tumor debris. However, the close relationship of the initiation of chemotherapy to the tumor dissemination, though suggestive, does not prove a causal relationship. Although these events have not been previously described in the literature, we cannot rule out the possibility that the intraocular dissemination may represent the natural history of these tumors.

CR 16. SPINAL TANYCYTIC EPENDYMOMA PRESENTING AS MENINGITIS

Samuel Blackman,¹ Elizabeth Bundock,² and Mark Kieran¹; ¹Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; ²Office of the Chief Medical Examiner, Commonwealth of Massachusetts, Boston, MA, USA.

Tanycytic ependymoma is a rare variant of ependymoma that most frequently presents in the spine or periventricular area. While distinct from myxopapillary ependymoma, the prognosis for this disease appears favorable. We report a case of tanycytic intramedullary ependymoma of the spinal cord (TE). This case was unusual because the patient presented with findings of meningitis. A 13-year-old boy presented to the hospital for a syncopal episode following several days of back pain radiating to the legs. Physical findings were initially consistent with muscle strain. Over the next several days the patient's pain intensified, accompanied by neck stiffness and headache. Several days later he suffered another syncopal episode and was reevaluated, at which time nuchal rigidity and photophobia were noted. A lumbar puncture yielded purulent fluid containing 90% neutrophils. Cerebrospinal fluid (CSF) cultures were ultimately negative. He was treated with antibiotics and dexamethasone, but several hours later had severe pain, emesis, and paresthesias in both legs. Because of concern for an epidural abscess, an MRI was obtained. This revealed a T1-isointense 3 x 1 cm ovoid lesion at L1–L2 with areas of high T2-signal and enhancement following gadolinium administration. After resolution of the meningitis, the patient underwent extensive resection of the lesion. Two satellite lesions were found on the filum terminale. Pathologic analysis and electron microscopy revealed histologic and ultrastructural features consistent with a TE, characterized by monotonous cells with oval nuclei and fibrillary tanycytic-like processes arranged in loose fascicles and cultures. No areas of hypercellularity or visible mitoses were seen. Of note, the tumor was infiltrated with CD68-positive macrophages and LCA-positive lymphocytes. Based on the extent of resection, histologic analysis, and absence of tumor cells in the CSF, no further treatment was proposed. The patient has been free of disease for >2 years, with no neurologic sequelae. The diagnostic features, pathology, and neuroradiology, and decision making with regard to treatment of TE are discussed. We also review the literature involving presentation of CNS tumors as meningitis.

CR 17. SPONTANEOUS REGRESSION OF A DIFFUSE BRAINSTEM LESION IN AN INFANT

Sofia Nunes,¹ Duarte Salgado,² Gabriela Caldas,³ and Mário Chagas³; ¹Unidade de Neuro-Oncologia Pediátrica, Instituto Português de Oncologia de Lisboa, Lisboa, Portugal; ²Unidade de Neuro-Oncologia Pediátrica, Instituto Português de Oncologia, Portugal; ³Department of Child and Adolescent Oncology, Instituto Português de Oncologia de Lisboa, Portugal.

The authors report a case of a 3-month-old child who presented with a diffuse brainstem lesion that regressed without treatment. He was born after an uneventful full-term pregnancy and his development was considered normal until that time. At 3 months of age, abnormal opsoclonus-like ocular movements were noted for a transient period of about 2 weeks. His physical examination was otherwise normal. MRI showed a large intrinsic expansive lesion centered on the medulla, hypointense on T1, hyperintense on T2 weighted images and without contrast enhancement, consistent with a glioma. Laboratory investigation, including neuroblastoma screening, was inconclusive. Due to age and normal neurological examination, no treatment was considered and surveillance scanning was agreed upon with the parents' consent. Serial MRI taken every 3 months in the first year of follow-up and 6 months apart in the second year showed a clear trend toward regression of the lesion. He is now 2.5 years old, his intellectual and motor development are considered normal for his age, and no neurological deficits have been found so far except for a mild hypertonia of the lower limbs during the first year of follow-up. The spontaneous remission of diffuse brainstem tumors is very rare, although some cases have been reported in patients with neurofibromatosis. To our knowledge only three cases of children <12 months of age have been reported, all diagnosed in the neonatal period and all showing clinical signs of neurological impairment.

CR 18. TRIGEMINAL TROPHIC SYNDROME IN PEDIATRIC BRAIN TUMOR PATIENTS: RECURRENT UNILATERAL FACIAL AND CORNEAL ULCERATIONS

Gesina F. Keating,¹ Francine Kim,² Anthony J. Mancini,³ Zibute G. Zaparackas,⁴ and Tadanori Tomita⁵; ¹Neurology, Children's Memorial Hospital, Chicago, IL, USA; ²Radiology, Children's Memorial Hospital, Chicago, IL, USA; ³Dermatology, Children's Memorial Hospital, Chicago, IL, USA; ⁴Ophthalmology, Children's Memorial Hospital, Chicago, IL, USA; ⁵Neurosurgery, Children's Memorial Hospital, Chicago, IL, USA.

We report three children with brain tumors who developed facial skin ulcerations and corneal irritation during the course of treatment. Trigeminal trophic syndrome (TTS) is a rare condition in which persistent or recurrent skin ulcerations, most typically in the ala nasi region, occur following an insult to the trigeminal nerve, thereby causing altered sensory perception to the face. The resultant anesthesia and paresthesias, combined with self-induced trauma, are believed to lead to the ulcerations, which may become chronic. TTS is typically reported in adults, with the most common etiologies related to stroke or treatment of trigeminal neuralgia. In adults, few reported cases have a history of brain tumor, with reported tumor types including astrocytoma, meningioma, and acoustic neuroma. Aside from the three children with TTS reported here, there are only two others in the literature, neither with brain tumor. All of our patients had benign brain tumors whose location included the cerebellopontine angle, where the trigeminal nerve emerges from the brainstem. They subsequently developed ipsilateral facial and corneal lesions of varying degrees and duration. Although in adults the time to develop TTS ranges from weeks to years, these children all developed lesions within 1 month following surgery. Skin findings in these patients included characteristic crescent-shaped ulcers and crusted plaques at the ala nasi as well as other facial areas, including the chin, cheek, and periorbital region. Treatments included wound care with topical antibiotic ointments and wound dressings, oral antibiotics for secondary superinfection, and the use of elbow-wrist immobilizers while sleeping to minimize manipulation and further trauma to the sites. Although little is discussed elsewhere regarding eye involvement in TTS, neurotrophic keratitis was observed in all three patients here, and ranged from mild and transient to severe and recurrent. In one patient, changes were severe enough to require consultation with a corneal specialist, who recommended using a bandage contact lens to provide protection and promote healing. The child developed a central corneal opacity which decreased his visual acuity to 20/80. Recurrence of lesions has occurred in all three children, though both the frequency and severity have decreased over time. TTS is an important condition for clinicians caring for pediatric brain tumor patients to recognize, so that appropriate diagnosis and intervention can be made in a timely manner. Although TTS may be unpreventable in such patients, prompt recognition may prevent further morbidity, including permanent facial and visual impairment.