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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;