Multidrug resistance proteins (Pgp/MRP1) in human malignantF gliomas: Expression, functional activity and modulation by wild-type and mutant p53
O. Bähr, W. Wick, M. Weller
Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tuebingen,
Tuebingen, Germany

The molecular mechanisms underlying resistance to chemotherapy of human malignant gliomas are poorly understood. Understanding and overcoming multidrug resistance may be a promising strategy to develop more effective pharmacotherapies for malignant gliomas.
RT-PCR, immunoblot analysis and flow cytometry showed that human malignant glioma cell lines (n=12) exhibit heterogeneous mRNA expression, protein levels and functional activity of the mdr-1 gene-encoded P-glycoprotein (Pgp) and the MDR-associated protein (MRP1). Inhibition of Pgp (Verapamil, PSC833) or MRP (Indomethacin, Probenecid) resulted in enhanced cytotoxic effects of vincristine, doxorubicin and teniposide. Cytotoxicity of Taxol was only affected by Pgp inhibition, whereas cytotoxicity of cisplatin, topotecan, gemcitabine and lomustine was unaffected. Functional activity was the best predictive marker for the ability of MDR inhibitors to sensitize glioma cell lines for chemotherapeutic drugs. To consider the possible role of the blood-brain-barrier for chemotherapy, we examined the human cerebral endothelial cell line SV-HCEC. These cells exhibited the strongest Pgp activity of all cell lines while MRP expression and activity were comparable with the glioma cell lines.
Since loss of wild-type p53 activity and acquisition of a multidrug resistance phenotype may be interrelated, we examined the influence of p53 on expression and functional activity of Pgp and MRP1. Therefore we introduced the temperaturesensitive p53V135A mutant in 5 human malignant glioma cell lines with different p53 status. This mutant assumes wild-type conformation at 32.5°C and acts as a dominant-negative mutant at 38.5°C. There was a rather heterogeneous modulation of the MDR phenotype by both mutant and wild-type p53 that could not be predicted by the p53 background.
In conclusion, our data provide new insights on the MDR phenotype and the regulation of Pgp/MRP1 by p53 in human malignant glioma cell lines.

Teratoma of head and neck (H&N) in neonates (MAKEI 83-96)
B. Bernbeck¹, G. Calaminus¹, D. Harms¹ for the MAKEI study group
¹Dept. of Pediatric Oncology, Hematology and Immunology, University Hospital, Düsseldorf,
²Inst. for Paidopathology, University Kiel, Germany

Objectives: Mature and immature teratoma are diagnosed most often in neonates at the sacrococcygeal region and seldom at H&N. Despite benign histology they grow very quickly and have the potential of transformation into highly malignant tumors. Whereas the tumor biology of sacrococcygeal teratoma has been studied intensively, information about H&N teratoma is limited.
Methods: The prospectively registered H&N teratoma have been analyzed in respect to clinical signs, therapy and prognosis.
Results: From 1983 until 2000 230 neonatal teratomas were registered in MAKEI 83, 86, 89 and 96, of whom 32 (14%) were located in the H&N. In 18 cases diagnosis was done by prenatal ultrasound. Twelve babies were preterm and the median birth weight was 3.050 g (range 1.700-4500 g). Because of mechanical airway obstruction by the tumor 12 newborns needed postpartal intubation; only 6 newborns showed normal respiration. The tumor sites were the neck 19x, face 7x and intracranial 6x. One of the newborns with intracranial tumor died within a few hours and two others needed an extracranial liquor device by emergency. Tumor resection was performed between day 1 (n=3) and day 89 (n=1) and was complete in 19 cases and incomplete in 7 cases. Five pts were biopsied only. The tumor weight ranged between 2 and 673 g, median 168 g. Histology: mature Teratoma 5x, immature Teratoma grade 1 7x, grade 2 15x, grade 3 1x, microfoci of yolk sac tumor were present in 2 pts., Hamartoma 1x. Relapses occurred only in 5 infants. All children with neck and face teratoma survived and 3 out of the 6 with intracranial site.
Conclusions: Irrespective of tumor size teratoma of the neck and face have an excellent prognosis whereas prognosis is impaired in intracranial teratoma. Complete tumor resection is not a prerequisite for survival.

Supported by Deutsche Krebshilfe

Carboxypeptidase rescue in patients with methotrexate intoxication and renal failure
U. Bode¹, S. Buchen¹, G. Fleischhack¹, C. Hasan¹, R. Melton^2
1Department of Pediatric Hematology/Oncology, University of Bonn, Germany, ²ENACT Pharma, United Kingdom

High-dose methotrexate (MTX) bears a considerable risk of acute and delayed toxicity, for example, mucositis, CNS, liver and renal toxicity. Even high doses of posttreatment leucovorin (LV) are not likely to completely reverse the toxicity from MTX, when plasma MTX concentrations are persistently greater than 10µM.
Hemodialysis and hemoperfusion have only variable efficacy. Thus, alternative methods of rescue are needed. MTX and other folates are inactivated by the enzyme carboxypeptidase G1 (CPDG2), which hydrolyze MTX to the inactive DAMPA and glutamate.
In a phase I/II study between May 1997 and March 2002 CPGD2 was administrated in a median dose of 50 U/kg (range, 33-60 U/kg) intravenously in 82 patients with renal failure and MTX intoxication. Eligible were patients with plasma concentrations of >10µM 36 hrs. or >5µM 42 hrs. or >3µM 48 hrs. after start of a MTX infusion and with documented renal failure (i.e. >50% increase in serum creatinine level and decreased diuresis). LV had to be omitted four hours previously and reintroduced one hour following the injection.
Immediately before CPDG2 administration a median MTX serum level of 11,9 µM (range, 1.5-901 µM) were documented. CPDG2 was given in median 52 hrs. (range, 25-178 hrs.) following start of a MTX infusion of 1.5-12 g/m_. One hour after CPGD2 injection the MTX levels (estimated by HPLC) decreased in median to less than 2 % (range, 1% to 22%) of the original values. CPDG2 related toxicity, i.e. like allergic reactions were not observed. Related to the MTX intoxication gastrointestinal, liver and skin toxicity was documented in about one half of patients. Four patients died due to severe myelosuppression and septic complications 7 to 22 days following the MTX infusion.
CPDG2 provides a well tolerated, safe and very effective pathway of MTX elimination by converting it to inactive and non-toxic metabolites.

Intercranial Germinoma: Prognosis after Irradiation (CSI) or combined Chemo-Radiotherapy (CT-RT): Results of SIOP GCT 96
G. Calaminus¹, M.L. Garre, C. Alapetite, D. Frappaz, R. Kortmann, J. Nicholson, U. Ricardi, F. Saran, C. Patte, U. Göbel¹. for the study group: Universities and Children’s Hospitals Düsseldorf¹, Genova, Paris, Lyon, Tübingen, Cambridge, Sutton, Villejuif, Germany, Italy, France, UK

Objectives: Within SIOP CNS GCT 96, after histological diagnosis (Dx) and negative markers (serum/CSF) pts received a CSI, or a combined CT-RT, including a focal RT in localized disease. (negative CSF-cytology, no metastases in imaging). In view of further protocol development we analyzed factors influencing outcome.
Patients/Methods: Until1/2001, 142 protocol pts with germinoma were accrued, of whom 114 showed localized disease and 28 pts were metastatic. 33 children offered a bifocal tumor. In 90 pts Dx was made by biopsy, whereas 40 children received a tumor resection. One pt was diagnosed through tumor cells in CSF. In 6 pts no information were available. Diagnostic work-up was incomplete in 64 pts. 93 pts received CSI (24 Gy/16 Gy), whereas 45 children were treated with CT-RT (2x Carbo-PEI, 40 Gy), in 4 children no information were available. We analyzed: age, sex, localisation, histologic component (+/- teratoma), markers, surgery, complete/incomplete work-up, tumor response, residual disease after treatment in respect to outcome.
Results: The EFS in pts treated with CSI is 0.92+/- 0.05, compared to 0.84+/-0.06 in pts with combined CT-RT. 10 events occurred. After combined CT-RT, 4 of 6 events were ventricular failures, whereas after CSI only local relapses were observed. 7/64 pts with incomplete diagnostic work-up developed recurrence, compared to 3 out of 78 completely staged pts. 3/10 children with events had marker elevation at time of recurrence, who had not been staged for tumor markers in CSF at Dx. All other investigated factors were not of prognostic value.
Conclusions: A complete diagnostic work-up is of major importance for outcome in germinoma. In localized disease and combined CT-RT the ventricular system must be included in the irradiation field.
Supported in part by Deutsche Krebshilfe

Secreting CNS germ cell tumors (GCTs): Prognostic factors for outcome: Results of SIOP CNS GCT 96
G. Calaminus¹, M.L. Garre, C. Alapetite, D. Frappaz, R. Kortmann, J. Nicholson, U. Ricardi, F. Saran, C. Patte, U. Göbel¹. for the study group: Universities and Children’s Hospitals Düsseldorf¹,
Genova, Paris, Lyon, Tübingen, Cambridge, Sutton, Villejuif, Germany, Italy, France, UK

Objectives: Within SIOP CNS GCT 96, after diagnosis (Dx) by markers (serum/CSF) pts received a combined chemotherapy/radiotherapy (CT-RT) including a focal RT in localized disease (negative CSF-cytology, no metastases in imaging) or a craniospinal RT (CSI) in case of metastases. In view of the future protocol we analysed factors influencing outcome.
Patients/Methods: Until1/2001, 105 protocol pts with secreting CNS GCT were accrued, of whom 81 showed localized disease and 24 pts were metastatic. 36 pts was diagnosed by markers and imaging, whereas 44 children were primarily operated (total resection: 16 pts, subtotal: 17 pts, partial: 11 pts.) 25 pts receive a stereotactic biopsy. 37 pts were treated with 4 x PEI+ CSI (30/24 Gy). 61 pts received 4xPEI and focal RT (54 Gy). 5 pts did not receive RT (3pts < 4yrs, 2pts DOC). Incomplete information are available in two children. 28/105 children showed residual tumor after treatment. We analysed: age, sex, localisation, histology, dissemination, markers, surgery, complete/incomplete work-up, tumor response, residual disease after treatment in respect to outcome.
Results: The EFS in pts treated with chemo and CSI is 0.72+/- 0.08, compared to 0.64+/-0.08 in pts with CT and focal RT. Of prognostic value are elevation of AFP (> 1000 IU/L), age and residual tumor were detected.
Conclusions: A complete diagnostic work-up and documentation is of major importance. A clinical diagnosis is encouraged in all pts to reduce morbidity. In the forthcoming protocol resection of residual disease and treatment intensification in high risk pts is emphasized.
Supported in part by Deutsche Krebshilfe

Quality of life in children with brain tumors (BT): Results obtained from the German Brain Tumor Studies within a retrospective setting
G. Calaminus¹, J.Kühl², H. Müller³, A. Wiener¹, M. Neubauer¹, U. Göbel1
Universities/Child. Hospitals Düsseldorf¹, Würzburg², Oldenburg³, Germany

Objectives: Although within recent years the prognosis of pediatric brain tumors (BT) has dramatically improved these group of patients still have to face a high burden of long term sequelae. To offer adequate rehabilitation it is necessary to know about the objective and subjective quality of survival of these patients. QoL evaluation with validated questionnaires offers the possibility to obtain easily information from affected patients about their well-being. The aim was to get a first overview about QoL of pediatric BT patients which could be used to optimize care processes.
Patients/Methods: We evaluated 263 children, adolescents and young adults, treated for brain tumors. Median age at Dx had been 8 years (2-16 yrs). The median age at evaluation was 12 yrs (8-31 yrs). Diagnosis were: Craniopharyngeoma: 148 pts, malignant germ cell tumors: 92 pts and PNET 23 pts. All pts were in 1.CR at time of evaluation. Three different QoL measures were used: 8-18yrs: KINDL, PEDQOL, > 18yrs: EORTC-QLQ-30.
Results: The overall QoL of the BT patients was satisfiable (65% positive ratings) although it was significantly lower than their healthy controls (80% positive ratings) (p<0.05). Specifically affected are the domain cognition and autonomy. In respect to disease pts with PNET expressed the lowest overall QoL scores, affected domains differ between the disease groups. F.e. craniopharyngeoma patients rate body image lower than other disease groups.
Conclusion: QoL evaluation offers an easy possibility to get subjective information about the quality of survival of BT patients. This could be used additionally to further optimize care of these patients.

Preliminary report (radiotherapy) on the prospective, cooperative trial of GPOH/APRO „HIT 2000“ for children and young adults with medulloblastoma, stPNET and ependymoma
Elke Dannenmann-Stern¹, Eva Rex¹, Jutta Scheiderbauer1, Silke Metzger¹, Joachim Kühl², Michael Bamberg¹, Rolf-Dieter Kortmann^1
1Department of Radiotherapy, University of Tübingen, ²Children`s Hospital, University of Wuerzburg

Aims: The multicenter, prospective trial „HIT 2000“ for children and young adults with medulloblastoma (MB), stPNET and ependymoma (EP) consists of several treatment protocols including a randomized phase III trial for standard-risk MB aged over 4 yrs. This is the current status of the data available with special regard to radiotherapy.
Methods: Data of all patients who entered the study between 01.08.2000 and 31.12.2002 have been analyzed for histological diagnosis, recruiting rate per protocol, feasibility and acute toxicity with respect to treatment arms and for early relapses.
Results: 244 patients have been evaluated: 164 pat. with MB, 26 pat. with stPNET and 54 pat. with EP (n=14 WHO°II, n=40 WHO°III). 91/164 pat. with MB showed standard-risk disease over 4 yrs.; 70 of them have been randomized in either hyperfractionated (n=34) or conventional fractionated (n=36) postoperative radiotherapy. 182/244 pat. underwent irradiation. Dose prescriptions according to the protocol guidelines have been followed in 137/144 documented cases (95,1%). Time interval between surgery and XRT was 33 d median (range 11-188 d). In 45 cases (35,4%) this was within the protocol recommendations (14-28d). CTC Grade IV toxicity during XRT was registered in 20/100 documented cases (n=15 with myelotoxicity), particularly when high dose chemotherapy preceded XRT. The total number of SAE-reports was 10; 4 of them were related to XRT. There are 22/80 follow-up cases with early relapse (9 MB, 7 stPNET, 6EP).
Conclusions: 1. The recruiting rate in all protocols of „HIT 2000“ was as high as expected, although the number of patients enrolled in the randomized trial for standard risk MB was yet lower than expected. 2. All irradiation schedules show good feasibility and low acute toxicity even in combination with intensive chemotherapy. 3. There are no unexpected early relapses.
Sponsored by the Deutsche Kinderkrebsstiftung

Alkylglycerol-mediated improvement of drug delivery to the normal brain and to C6 gliomas in rats: regulation of the effect and comparison with other means
Erdlenbruch B¹, Kugler W¹, Eibl H², Lakomek M^1
1Universitätskinderklinik Göttingen, D-37075 Göttingen, ²Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany

Response of malignant brain tumors to chemotherapy is often poor or transient. Unsatisfactory penetration of most of anticancer agents across the blood-brain barrier (BBB) into the brain tissue emphasizes the need for new strategies in neurooncology to overcome the BBB in order to achieve effective tissue concentrations. We have investigated the increase in the transfer of cytotoxic drugs to the normal brain and to C6 gliomas in rats using intraarterial injection of short chain alkylglycerols. Results were compared with those obtained using hypertonic mannitol or bradykinin. Intracarotid infusion of bradykinin (10 µg/kg/min) was only effective in tumor tissue resulting in 2.4 fold higher MTX concentrations as compared to controls with intraarterial MTX. In contrast, a very strong increase in the transfer of MTX was observed in the ipsilateral brain of normal animals after osmotic blood-brain barrier disruption with 1.4 M mannitol (99-fold). In C6 gliomas, mannitol increased MTX concentrations 8.3-fold in the tumor and 15-fold in surrounding brain. Using 1-Opentylglycerol (120 to 300 mM), delivery of MTX was enhanced in a concentrationdependent manner. In the ipsilateral brain of tumor-free rats, MTX concentrations were 5.6- to 84-fold higher as compared to controls. MTX accumulation in C6 tumors amounted to 1.5 to 18 in tumor tissue and to 1.8 to 28 in surrounding normal brain. Apart from variations in the concentration and chemical structure of the alkylglycerols, time schedule of drug administration and combinations of different alkylglycerols were identified as instruments to adjust the amount of chemotherapeutic drugs delivered to the brain.
There is evidence from these data that intraarterial chemotherapy in conjunction with alkylglycerols might be superior to the use of bradykinin or mannitol.
Supported by Deutsche Krebshilfe (10-1554-Er 2).

Outcome for Newly-Diagnosed Young Children with Central Nervous System Primitive Neuro-ectodermal Tumors (PNET) Treated on the “Head Start I and II” Treatment Regimens, 1991-2002.
Jonathan Finlay
New York University School of Medicine, New York, U.S.A.

Between 1991 and 2002, over 150 young children newly-diagnosed with malignant brain tumors were enrolled on two serial trials, known as “Head Start I” (1991- 1997) and “Head Start II” (1997-2002). Treatment on “Head Start I” was uniform for all patients, consisting of an induction chemotherapy regimen (five cycles of vincristine, cisplatin, cyclophosphamide and etoposide, administered over a three day period and repeated at 3 to 4 week intervals), followed by consolidation with marrow ablative chemotherapy (carboplatin, thiotepa and etoposide) with autologous hemopoietic stem cell rescue. No irradiation was administered, provided patients were without evidence of any residual disease, either radiographically or cytologically, at the conclusion of induction chemotherapy. On “Head Start II”, the treatment strategy was the same, except that children with evidence of leptomeningeal dissemination at diagnosis were enrolled on an induction regimen intensified by the addition of high dose (400mg/Kg) methotrexate (MTX) with each cycle. Eligibility on both studies was restricted to children aged < 3 years at diagnosis for those with non-disseminated medulloblastoma. Children with non-cerebellar PNET or with medulloblastoma/PNET with leptomeningeal dissemination, were eligible <6 years of age in “Head Start I” and <10 years of age in “Head Start II”.
For 20 children <3 years old with non-metastatic cerebellar medulloblastoma/PNET, the 1,2 and 5 year Kaplan Meier (K-M) analyses of overall survival (OS) are 79%, 67% and 61%, while the event-free survival (EFS) are 73%, 43% and 37%. Age < 18 months versus 18 to 36 months appeared to confer no adverse impact upon outcome. A benefit from gross total resection is suggested but is not statistically significant. Over half of those surviving children have avoided irradiation. For 21 children (all but one < 6 years old) with disseminated medulloblastoma, enrolled on the “Head Start II” regimen with MTX intensification, the complete response rate to induction chemotherapy exceeds 80%, and the early K-M analysis of EFS exceeds 60% at 2.5 years. – superior to the EFS at 2 years for non-metastatic medulloblastoma treated with the non-intensified “Head Start I” regimen.
For 43 children with non-cerebellar PNET enrolled on both “Head Start I and II” protocols, the 1,2 and 5 year K-M analyses of OS are 71%, 49.6% and 43.4%, while the EFS are 66.2%, 42.8% and 40%. A significantly poorer outcome is noted for children <36 months of age versus those >36 months: one and 5yr OS for children <36 months of age are 67% and 28%, while for >36 months of age OS are 74% and 57%. A trend is noted for benefit from radical surgical resection. A difference is noted between location of pineal tumors: for pineal PNET, the 1,2 and 5 year OS are 57%, 43% and 43%, while for non-pineal PNET, the OS are 77%, 58% and 50%. All 4 children with brainstem PNET died rapidly of tumor progression. Of 22 surviving children, 14 (64%) have never been irradiated.
The toxic mortality for all patients, irrespective of pathology and extent of disease, enrolled on the ‘standard” induction regimen, was 5.4% on “Head Start I” and 2.6% (n=38) on “Head Start II”. The toxic mortality for the MTX intensified induction chemotherapy regimen is 6.3% (n=32). The toxic mortality from the marrow ablative chemotherapy regimen was 8.5% (n=47) during “Head Start I” and 2.2%(n=46) on “Head Start II”.
This treatment strategy appears to offer the prospect of durable remission with avoidance of irradiation for the majority of young children with CNS PNET.

Interdisciplinary therapy of relapsed primitive neuroectodermal brain tumors in childhood and adolescents: Results of the HIT-REZ- 97 Studyz
G. Fleischhack¹, M. Zimmermann¹, C. Hasan¹, J. Kuehl², U. Bode¹
Departments of Pediatric Hematology/Oncology, University of ¹Bonn and ²Würzburg, Germany

In the HIT-REZ-97 study a curative treatment arm was designed for patients with a relapsed primitive neuroectodermal brain tumor (PNET) who are able to get again an intensive therapy. In this arm patients achieving a CR or PR following chemotherapy with continuous infusion of carboplatin and VP16 (800 and 400 mg/m_ over 96 hrs., 3 to 4 courses) were qualified for high dose chemotherapy (HDCT, thiotepa 600 mg/m_, carboplatin 2000 mg/m_, VP16 1000 mg/m_) with autologous stem cell support. For all patients the opportunities of local therapy should be exploited. Between 1997 and 2003 72 of 108 (66.7%) study patients with relapsed medulloblastomas/PNET's entered the intensive therapy arm (median age: 12.6 yrs.). 84.7% and 15.3 % of them, respectively, were treated for the first or a later relapse. 29 (40.3%) of these patients got in CR/PR (28) or SD (1) HDCT with autologous stem cell support. Local therapy was applied in 33.3% of patients (tumor resection in 15.3 %, radiotherapy in 22.2%, both in 4.2%). 17 (23.6%) patients with poor response received further palliative chemotherapy.
Following the therapy with carboplatin/VP16 59.7 % of evaluable patients achieved a CR or PR. Unfortunately, only 24.2 % of all patients were free of progression at a median follow-up time of 13 months (range, 3-65 months). The overall survival time was longer in patients who have got a local therapy too. Immediately after HDCT 21 of 25 (84%) of evaluable patients were in CR/PR. 7 of them (28%) are free of progression at a median follow up time of 18 months (range, 5.5-65 months). The main toxicities of the HDCT were severe myelosuppression and severe oral and intestinal mucositis associated with severe infections (WHO scale III/IV) in about two third of patients. Severe ototoxicity was observed in about one half of patients. Two (6.9%) patients died therapy-related due to septic complications. This intensive relapse regimen induced a high response rate in poor prognosis medulloblastomas/PNETs with tolerable toxicity. However, the duration of response is short in the majority of patients. HDCT prolong the overall survival only in a few patients for only several months. Further investigations are necessary to explore the efficacy of this regimen in combination with other and new therapy modalities.
Supported by the German Research Foundation for Cancer in Children

A Novel Approach for the Imaging of Embryonal CNS Tumors: Somatostatin Receptor Scintigraphy (Octreoscan^®)
M.C. Frühwald, C.H. Rickert, M.S. O'Dorisio, R. Sträter, H. Jürgens, J. Kühl, H.L. Müller

Introduction: The timely diagnosis of residual or recurrent CNS tumors is often hampered by inaccuracies of imaging techniques such as conventional MRI and CT. This is due to unspecific tissue changes, eliciting false positive signals. These are mainly caused by radiotherapy and to a lesser extent by surgery and chemotherapy. We have previously demonstrated the utility of scintigraphic imaging of somatostatin receptor 2 (sst2) in the diagnosis of recurrent and residual medulloblastomas. The determination whether it is worthwhile to study a tumor using sst2-receptorscintigraphy (SSRS) has been rather laborious due to the lack of a rapid and inexpensive test assessing the sst2 status of a given tissue specimen. Until recently no sst2 antibodies have been available.
Methods: In the current study we used a novel antibody to test expression patterns of sst2 in malignant brain tumors of childhood other than medulloblastoma. We screened nine high-grade gliomas (five glioblastomas, three anaplastic astrocytomas), three atypical teratoid/rhabdoid tumors (AT/RT), five stPNETs and nine ependymomas. Tumors with a positive expression pattern were further studied by somatostatin receptor scintigraphy (Octreoscan^®).
Results: Our findings of an abundant expression of the sst2 receptor in all studied stPNETs and some ependymomas warranted our first in vivo imaging analyses. We thus evaluated five patients with stPNETs, one with an intracerebral rhabdomyosarcoma, two with ependymomas, one with a cerebral neuroblastoma, one with a medulloepithelioma and one with a glioblastoma. In all cases scintigraphic visualization of the tumors was possible. I.e. non-specific disturbances of the bloodbrain- barrier could be excluded using DTP-Tc scanning. In cases where scanning was possible after complete resection the imaging showed a lack of enhancement.
Conclusion: The scintigraphic imaging of sst2 receptors should be considered when in doubt about the presence of residual or recurrent tumor in embryonal CNS tumors such as stPNETS, medulloepithelioma and cerebral neuroblastoma. Rapid testing for sst2 protein expression should be performed before scintigraphy. Furthermore, tagging of the sst2-antibodywith higher energy nuclides might be a therapeutic option for patients with recurrent high-grade embryonal CNS malignancies. Supported by the Karl Bröcker Stiftung, Geseke

Smac peptides as new cancer therapeutics: synergy with TRAIL to eradicate malignant glioma in vivo without toxicity to the normal brain
Fulda S.¹, Wick W.², Weller W.², Debatin K.M.^1
1University Children’s Hospital, Ulm, ²Department of Neurology, University of Tübingen, Tübingen, Germany

Due to resistance of tumors to established therapies, current attempts to improve cancer survival largely depend on strategies to target tumor resistance. “Inhibitor of Apoptosis Proteins” (IAPs) contribute to the resistance of cancers, since they are expressed at high levels in many tumors. Recently, Smac was identified as a mitochondrial protein that promotes apoptosis by antagonizing IAPs. We developed cell-permeable Smac peptides using the TAT protein transduction domain that sensitized various tumor cell lines in vitro and malignant glioma cells in vivo for apoptosis induced by death receptor ligation or cytotoxic drugs. Cell-permeable Smac peptides even bypassed the Bcl-2 block in tumor cells with high expression Bcl-2, which prevented the release of Smac from mitochondria into the cytosol. Also, Smac peptides sensitized resistant neuroblastoma cells lacking caspase-8, melanoma cells lacking Apaf-1 or even resistant patient-derived primary tumor cells ex vivo. Most importantly, Smac peptides strongly enhanced the antitumor activity of TRAIL in vivo in an intracranial malignant glioma xenograft model. Maximal synergy of the combined treatment with Smac peptides and TRAIL was found at suboptimal concentrations of TRAIL that on their own only temporarily delayed tumor growth. Complete eradication of established tumors and survival of mice was only achieved upon combined treatment with Smac peptides and TRAIL, whereas mice treated with TRAIL or Smac peptides alone all eventually died. Importantly, Smac peptides do not reverse the lack of toxicity of TRAIL on nontransformed, primary human cells of different lineages including human astrocytes. Also, co-injection of TRAIL and Smac peptides into normal mouse brain in vivo showed no cytotoxic effects and caused no acute or delayed neurotoxicity as assessed by neurological scores. In sharp contrast, injection of Smac peptides into human malignant glioma xenografts grown intracranially in mice strongly enhanced TRAILinduced apoptosis in glioma cells. This indicates the specificity, and thus the potential safety, of the sensitization effect of Smac peptides for tumor cells, but not for normal cells. Interestingly, injection of FITC-labelled Smac peptides into the normal mouse brain and into human malignant glioma grown intracranially in mice revealed no difference in the distribution of Smac peptides in the normal brain tissue compared with tumor tissue showing that Smac peptides did not preferentially localize to tumor cells. Taken together, by demonstrating that even resistant tumor cells can be sensitized by Smac agonists for TRAIL- or anticancer drug-induced apoptosis, our studies performed in cell lines, primary tumor cells and in a malignant glioma tumor model in vivo have important implications for cancer therapy. Thus, Smac agonists represent novel promising cancer therapeutics to enhance the efficacy of cytotoxic therapies even in resistant tumors.

Low Grade Glioma in Children with Neurofibromatosis NF I: Natural History and Treatment Results from the Trial HIT-LGG 1996.
A.K. Gnekow¹, R. Kortmann², O.D. Wiestler³, T. Pietsch³, M. Warmuth-Metz⁴, S. Söllner^1
1I. Hospital for Children and Adolescents, Augsburg, University Hospital: ²Radiotherapy Department, Tuebingen, ³Reference center for Neuropathology, Bonn, ⁴Reference center for Neuroradiology, Wuerzburg, for the Study Committee of the Low-grade-Glioma-Study HIT-LGG 1996 of the Brain Tumor Group, GPOH.

The occurrence of low grade glioma within the visual pathways is one of the major diagnostic criteria of Neurofibromatosis NF I. There are no prospective studies, so data concerning incidence and natural history have been derived mainly retrospectively from larger centers or case reports. The tendency for progression and necessity of therapy are considered to be low.
Between October 1st., 1996, and March 31st., 2002, 572 protocol patients were entered into the trial HIT-LGG 1996, 58 ( 10,1 %, 32 f, 26 m ) of these had NF I according to clinical criteria. At diagnosis there was no child under 1 year of age, the age ranged from 1,5 to 15,2 years. 50 Tumors were located in the supratentorial midline (42/50 in the visual pathways), 5 in the cerebral hemispheres and 3 in the caudal brain stem. Diagnosis was made on radiologic grounds in 34 cases. Histology following 24 surgical interventions was pilocytic Astrocytoma in 20, Astrocytoma II° in 3 and pleomorphic Xanthoastrocytoma in 1. At last follow-up 21 children were observed, 29 had received chemotherapy and 8 had received external radiotherapy.
radiotherapy. 1. Observation group: Following an observation time of median 27,8 (1,9-52,7) months 4 patients are in CR, 14 SD, 1 in PD and 2 children died.
2. Chemotherapy group: Vincristin/Carboplatin-chemotherapy was given in 13 children at diagnosis, in 16 following an observation period of 5,1 to 83,3 months, age at start of therapy 1,6 to 16,5 years. „Best“ response was achieved after median 3,15 months: 12 PR/OR, 14 SD, 2 tumors showed primary progression (1 too early, 1 unknown). 7 children suffered from progression 1,6 to 56,6 months after start of chemotherapy, 2 were irradiated, 2 received alternative chemotherapy and 2 had no further therapy. 55,1 (8,8 to 162,9) months following diagnosis all children are alive, 1 with regressive and 25 with stable residual tumor, progression in 1 ( 2 too early ).
3. Radiotherapy group: 2 children were irradiated at diagnosis, 6 following an observation period of 3,1-88,5 months at an age between 5,7 and 18,7 years. „Best“ response after median 3,33 months were 2 PR/OR and 4 SD, 1 tumor showed primary progression ( 1 too early ). 2 children suffered from progression 7,5 and 27,5 months after start of therapy. 1 Tumor stabilized without therapy, 1 tumor further progressed despite chemotherapy. 53,6 (5,7-171,5) months following diagnosis all children are alive, 1 with regressive and 5 with stable residual tumor, progression in 1 ( 1 too early ).
3-Year progression free survival in the chemotherapy group is favorable with 0,74. It is 0,72 for children aged 1-4 years, 0,71 at age 5-10 years and 1,0 for those above 10 years. Progression beyond 3 years seems rare, but observation time is still short. Since comparably favorable results following chemotherapy have been reported from other low grade glioma studies, and since the risk for developing late effects after radiotherapy is high, children and adolescents with NF I shall be treated with Vincristin/Carboplatin-chemotherapy irrespective of age and tumor location in the next LGG trial, if there is an indication for non-surgical therapy. Following progression further chemotherapy is recommended.

Cooperative Multi-center Study for Children and Adolescents with a Glioma of low-grade Malignancy–Concept of the Study SIOP-LGG 2003
A.K. Gnekow¹, R. Kortmann², O.D. Wiestler³, T. Pietsch³, M. Warmuth-Metz⁴, S. Söllner¹, A. Faldum⁵, A. Emser^1
1I. Hospital for Children and Adolescents, Augsburg, University Hospital: ²Radiotherapy Department, Tuebingen, ³Reference center for Neuropathology, Bonn, ⁴Reference center for Neuroradiology, Wuerzburg, ⁵IMBEI, Mainz, for the Study Committee of the Low-grade-Glioma- Study HIT-LGG 1996 of the Brain Tumor Group, GPOH.

The study HIT-LGG 1996 was the first study for children and adolescents with a low grade glioma in the German speaking countries demonstrating that a uniform therapeutic strategy is useful and sensible. The significant risk of progression for incompletely resected tumors independent of their location requires stringent guidelines for the use of non-surgical therapy, although the results of deferring radiotherapy by applying chemotherapy in younger children are not satisfying in various international study groups and for children following radiotherapy the risks of late effects have to be reduced. Since patient numbers are small, randomized studies to improve current therapeutic approaches are only possible in cooperative European trials.
The study SIOP-LGG 2003 offers a common therapy strategy for all children and adolescents with a histologically ( WHO criteria ) or radiologically confirmed low grade glioma. Following complete resection patients will only be observed, as will be patients without symptoms or progression after incomplete resection or clinical diagnosis. Non-surgical therapy will be instituted at the presence of defined indications following incomplete resection, non-resectable relapse or progression of an unresectable tumor.
Older children (³ 8 years) receive primary radiotherapy. Modern planning and treatment techniques shall reduce long term side effects upon surrounding tissues and organs at risk. At the presence of specific conditions these children may receive chemotherapy as well. The indication for interstitial radiotherapy is not age restricted. Younger children (< 8 years) receive primary chemotherapy. Children affected by Neurofibromatosis NF I shall be treated with chemotherapy at all ages. The duration of chemotherapy is 18 months. Children without NF I (stratified for age and tumor localization) will be randomized to receive standard induction with Vincristin and Carboplatin or intensified induction with Vincristin, Carboplatin and Etoposide, to test, if there is a difference in progression free survival. Additionally the distribution of tumor response at week 24 shall be investigated. Consolidation therapy will consist of 10 6-week cycles of single dose Carboplatin and Vincristin.
For all children overall survival, progression free and event free survival will be calculated. The influence of clinical and histologic findings upon these parameters will be investigated. The extent of late effects of primary tumor and therapy shall be documented prospectively.

Erucylphosphocholine induces different modes of cell death in A172 and U373 glioma cell lines
Dagmar E.H. Heinemann¹, Bernhard Erdlenbruch², Wilfried Kugler², Hansjörg Eibl¹und Max Lakomek^2
1Max-Planck Institut für Biophysikalische Chemie, ²Universitäts-Kinderklinik Göttingen

Erucylphosphocholine (ErPC) is an alkylphosphocholine which exerts cytostatic and cytotoxic effects on glioma cell lines. It can be given intravenously and crosses the blood-brain barrier. Therefore, ErPC is a promising anti-neoplastic drug for treating malignant brain tumors.
We compared the mode of ErPC-induced cell death in the human glioma cell lines A172 and U373. Both are highly sensitive to ErPC as measured by the WST-1 test. Morphologically, they showed different features of cell death. U373 cells showed mainly typical apoptotic appearence and stained positively for active caspase-3 after 24 h incubation with 25 to 50 µM ErPC. In contrast, in ErPC-treated A172 cultures only a small percentage of cells exhibited apoptotic morphology and active caspase- 3 was hardly detected. Using fluorescence microscopy, propidium iodide was shown to be incorporated mainly in intact nuclei of unfixed A172 cells, an indication of the oncotic pathway.
Butylated hydroxyanisole (BHA), an intracellular scavenger for reactive oxygen species (ROS), was used as an indirect indicator of oxidative stress. In both cells lines ErPC-induced cell death was reduced by BHA at concentrations between 333 and 555 µM. In spite of an increased viability, a higher number of apoptotic cells was observed. In addition, A172 cells were positive to caspase-3 staining. Dichlorofluoresceindiacetate (DC-FDA), a non-fluorescent agent which penetrates into the cell, was used for detection of ROS. In ErPC-treated A172 and U373 cultures the oxidized green fluorescent dye was observed by fluorescence microscopy whereas untreated control cells showed no signs of ROS production.
From our results we conclude that ErPC can induce different pathways leading to cell death depending on the cell lines studied. In U373 cells, the preferred pathway is apoptosis, in A172 cells oncosis.

Dexamethasone tissue levels in glioma patients and effects of analogous concentrations in vitro
Lücke, M., Nestler, U., Boeker, D.-K., Winking, M.
Department of Neurosurgery, Justus-Liebig-Universitaet, Giessen

Background: Focal brain edema in Glioma patients can be successfully treated by administration of dexamethasone (DEX). However, glucocorticoids exert far more effects, some of them potentially beneficial, others rather harmful. Only few data have been published on intratumoral levels and effective concentrations of DEX. Some authors suggested a proliferative, others an antiproliferative effect of DEX on glioma growth, which might be partially mediated by modulation of vascular endothelial growth factor (VEGF) expression. Gliomas express VEGF of different amounts. The aim of this study was the evaluation of DEX tissue levels, the cytotoxic concentration in vitro and the modulation of in vitro expression of VEGF and the two VEGF receptors KDR and FLT-1.
Methods: For cytotoxicity assays 104 cells of 3 established cell lines (U87, A172 and U73) and 15 primary cell cultures obtained from glioma specimens were exposed to 18 different concentrations of DEX ranging from 1 ng to 1 mg ml-1. After one week, cell viability was determined by the Alamar blue assay and the CD50 was calculated.
For determination of DEX tissue levels specimens of 51 neurosurgical tumor resections were collected (26 glioblastomas, WHO grade IV, 12 anaplastic gliomas, WHO grade III, 2 gliomas, WHO grade II and 9 metastasis). 25 of the patients received 0.6 mg kg-1, 26 were on a stable dose of 12 to 32 mg. Concentrations of DEX were evaluated by HPLC assay.
The three cell lines U87, A172 and U73 and 7 primary glioma cultures were exposed to 200 ng/ml of DEX for 7 days. The expression of VEGF, KDR and FLT-1 was detected by Westernblot analysis.
Results: The CD50 of DEX to the exposed cell lines ranged between 62.5 and 500 µg/ml (median 212.5). In 22 of the glioma specimens DEX was detectable in concentrations between 12 and 2093 ng/g (median 117). VEGF expression was detectable in only three of the cell lines, in two of these a slight suppression of VEGF expression was observed in the DEX group compared to control. KDR was detected in 8, FLT-1 in only 2 of the 10 cell lines. Expression of KDR and FLT-1 was not influenced by DEX at a concentration comparable to tissue levels in vivo.
Discussion: Cytotoxic doses of DEX exceeded 1000 times those detected in vivo. No proliferative effect of DEX on Gliomas was observed in vivo. In conclusion, direct effects of DEX on glioma growth appears to be irrelevant. Moreover, according to our preliminary results, an influence of DEX on proliferation via a modulation of expression of VEGF and its receptors seems to play no major role.

Kraniopharyngeom 2000 – Prospective multicenter surveillance study of childhood craniopharyngioma - Interim Report
Müller HL¹, Etavard-Gorris N¹, Gebhardt U¹, Sörensen N².
¹Children’s Hospital, Klinikum Oldenburg gGmbH, ²Dep. of Pediatric Neurosurgery, University Hospital, Würzburg.
(E-Mail: mueller.hermann@klinikum-oldenburg.de)

From 100 participating clinics in Germany, Austria and Switzerland 30 children and adolescents with craniopharyngioma aged 3,3 to 17,9 years at diagnosis were recruited since May 2001, with a peak at a median age between 8 and 9 years. In 18 patients the follow-up is more than one year. Symptoms leading to diagnosis of childhood craniopharyngioma were growth retardation (37%), impairment of view (47%), headache (52%), pubertas precox / tarda (7%). In primary surgery complete resection was achieved in 10 cases. In 14 children a subtotal resection was performed. Two children were treated repetitively by cystic drainage. Three patients received radiotherapy following subtotal resection.17 patients (57 %) were treated in pediatric departments of Universities, 47 % by oncologists, 18% by endocrinologists and 35% by neuro pediatricians.
We received first reports after diagnosis in 27 of 30 patients. Surgical reports from 22 patients and histopathological reports from 19 patients were analyzed. In 25 cases the brain tumor reference center (Dep. of Neuropathology, University of Bonn) provided reference pathology services. Imaging of 21 patients was reevaluated by our reference panel (Dep. of Neuroradiology, University of Würzburg). The postoperative follow-up visits were reported in 13 patients after three months, in 9 patients after six months, in 6 patients after nine months and in 10 patients after twelve months. The instruments for Quality of Life evaluation (FMH, Epworth Sleepiness Scale, Eating disorders, CBCL/4-18, PEDQOL (children´s and parents´version), CHQ-PF28 (parents´ version) LKJ-J), which were sent directly to the families, were completed and sent back in 75%. Supported by the Deutsche Kinderkrebsstiftung, Bonn, Germany

Growth in patients with childhood craniopharyngioma – Study on weight and height development before diagnosis
Hermann L Müller¹, Gina Bruhnken¹, Rudolf Oeverink¹, Nicole Etavard-Gorris¹, Ursel Gebhardt¹, Reinhard Kolb¹ and Niels Sörensen².
¹Department of Pediatrics, Zentrum fur Kinder und Jugendmedizin, Klinikum Oldenburg gGmbH, 26133 Oldenburg, ¹Department of Pediatric Neurosurgery, University Hospital, 97080 Würzburg, Germany

Patients with childhood craniopharyngioma frequently suffer from weight gain and short stature. Patients at risk for severe obesity presented with higher body mass index (BMI) SDS and lower height SDS already at the time of diagnosis (1). We asked whether pathogenic factors such as hypothalamic tumor involvement (2) have influence on weight development and growth already before diagnosis. Therefore, we analyzed growth and weight in 60 patients at standardized age-related time points before diagnosis and in yearly intervals after diagnosis. Weight and height before diagnosis were evaluated based on patients records of a standardized prospective health care survey (Vorsorgeuntersuchungen). Furthermore, BMI and height SDS at the time of diagnosis, at yearly intervals (year 1 to 8) after diagnosis and at the latest visit were evaluated. Auxiological parameters were compared between the groups of patients with obesity (BMI >2SD) (n=36) and normal weight at latest visit (BMI We conclude that hypothalamic involvement is a risk factor for obesity in patients with craniopharyngioma leading to increased weight already at the time of diagnosis. Low birth weight could be a risk factor indicating a genetic disposition for obesity. However, significant increases in weight were not found during long-term history before diagnosis but seemed to occur shortly before diagnosis. The results of our retrospective analysis are currently tested in a prospective study Kraniopharyngeom 2000 (www.kraniopharyngeom.com).
References: (1) Müller HL et al.: Obesity after childhood craniopharyngioma. Klin Padiatr 213: 244-249, 2001.
(2) Müller HL et al.: Melatonin secretion and increased daytime sleepiness in childhood craniopharyngioma patients. J Clin Endocrinol Metab 87: 3993-3996, 2002.
Financial Support: Deutsche Kinderkrebsstiftung, Bonn, Germany

Quality of life and body composition in patients with sellar masses
– Cross-sectional study on 403 children and adolescents

Hermann L Müller¹, Gina Bruhnken¹, Andreas Faldum², Rudolf Oeverink¹, Nicole Etavard- Gorris¹, Ursel Gebhardt¹, Reinhard Kolb¹ and Niels Sörensen³.
¹Department of Pediatrics, Zentrum fur Kinder und Jugendmedizin, Klinikum Oldenburg gGmbH, 26133 Oldenburg, ²Institute for Medical Biometry, Epidemiology and Statistics (IMBEI), University of Mainz, and ³ Department of Pediatric Neurosurgery, University Hospital, 97080 Würzburg,Germany

Object: Patients with childhood craniopharyngioma frequently suffer from reduced quality of life (QoL) due to obesity resulting from hypothalamic lesions. Similar sequelae is found in patients with sellar tumors of other histology. We evaluated Qol and body composition in children and adolescents with sellar masses. Aim of the study was to analyze the specific impact of tumor localization and histology on QoL and the degree of obesity during follow-up.
Methods: In 403 children and adolescents with sellar masses (276 craniopharyngioma, 14 germinoma, 21 optic/chiasma glioma, 40 hypothalamic glioma, 13 cysts of Rathke`s cleft and 39 other sellar masses) QoL was evaluated using a normalized questionnaire for self-assessment of QoL (Fertigkeitenskala Münster-Heidelberg [FMH]). Obesity was quantified at the time of diagnosis and at the time of latest evaluation by body mass index SDS [BMI]. Besides diagnosis, the influence of gender, age at diagnosis, age at evaluation, time from diagnosis to evaluation, irradiation, tumor localization and hypothalamic tumor involvement on QoL and BMI were analyzed. Using a stepwise variable selection General Linear Models with explanatory influential variables were built.
Results: In multivariate analysis, only age at diagnosis (p<0.001) and hypothalamic involvement (p=0.005) had relevant impact on QoL. R2 for the model was 14.2%. The second General Linear Model showed BMI at the time of diagnosis (p<0,001), hypothalamic involvement (p<0.001) and craniopharyngioma (p=0,004) to be influential on BMI at the latest evaluation. R2 for the second model was 43.7%.
Conclusions: Hypothalamic tumor involvement and young age at diagnosis had major impact on QoL. Therefore, treatment especially of young infants with sellar masses should be confined to specialized centers. Obesity mainly occurred in patients with hypothalamic involvement of craniopharyngioma who presented with increased BMI already at the time of diagnosis. Accordingly, risk factors for obesity could be easily identified already at the time of diagnosis. The results of our retrospective analysis are currently tested in a prospective study Kraniopharyngeom 2000 (www.kraniopharyngeom.com).
Financial Support: Deutsche Kinderkrebsstiftung, Bonn, Germany

HIT 2000: Infants and young children with medulloblastoma, PNET and ependymoma (SKK)
Stefan Rutkowski, Children’s Hospital, University of Wuerzburg

The prospective multicenter trial HIT 2000-SKK of German-speaking countries is designed for treatment of children younger than 4 years of age with medulloblastoma, supratentorial PNET and ependymoma. According to the previous SKK’92 trial, children younger than 3 years received 3x4 cycles of polychemotherapy (cyclophosphamide, vincristine, methotrexate, carboplatin, etoposide). Additional administration of 36 x 2 mg intraventricular methotrexate via Rickham-reservoir was introduced to avoid or delay radiotherapy with possible subsequent neuropsychological late effects. Results of different treatment groups are explained. By SKK’92 strategy, 5-year-PFS of infants with medulloblastoma without postoperative residual tumor was 74% without any radiotherapy. Toxicity of systemic and intraventricular chemotherapy was acceptable. 2 of 6 children with VP-shunt developed abdominal ascites after intraventricular methotrexate. Neuropsychological outcome of children receiving intraventricular methotrexate in SKK’92 was better than of children receiving radiotherapy without intraventricular methotrexate in the former SKK’87 study.
Therapeutic strategies of the current HIT 2000 trial with prolonged chemotherapy will be shown. In the first 22 months, 41 infants were included, among them 20 ependymoma, 16 medulloblastoma and 5 supratentorial PNET. First interim analysis of HIT 2000 will be available until summer 2003.

Tumor vaccination for patients with relapse of malignant brain tumors: Results of a pilot study
Stefan Rutkowski, Steven De Vleeschouwer, Eckhart Kaempgen, Johannes Wolff, Frank Van Calenbergh, Christian Plets, Stefaan W. Van Gool
Catholic University of Leuven (SDV, FVC, CP, SWVG), Würzburg (SR, EK) and Regensburg (JW)

Introduction: Most malignant brain tumors have a bad prognosis in spite of intensive oncological treatment. Therefore, new treatment modalities are warranted. Based on basic science data, an immune therapeutical strategy was developed using autologous mature dendritic cells loaded with autologous tumor homogenate.
Patients: In the pilot phase of the phase I/II trial HGG-IMMUNO-2003, 14 patients with a median age of 32 years (range: 11 - 78 y) were treated. All patients had a high grade histology. Eight patients were vaccinated at second relapse, while 6 patients had more than one malignant event prior to vaccination. Vaccination was given at week 1, 3, and further each 4 weeks.
Results: A median of 5 (range: 2 - 7) vaccines was given. In 6/14 patients, a response was observed. A response was considered as induction of SD (n = 2), partial remission (n = 1) or maintenance of CCR (n = 3: 23, 22, 10 months). Seven patients died after a postoperative survival of 7 months (median, range: 4 - 12 m). Seven other patients are still alive with a postoperative follow up of 10 months (median, range = 5 - 23 m). There was no difference between the overall survival period of the first group of patients versus the follow up period of the latter group of patients. The age of the patients in both groups was also not different. There were no major side effects unless the patient had gross tumoral disease prior to vaccination.
Conclusion: Immune therapy for patients with relapsed malignant brain tumors is feasible without major side effects. Although the assessment of efficacy is far too early, some interesting case histories are promising and support the continuation of dendritic cell immune therapy for malignant brain tumors.

Prospective, longitudinal study on quality of life after diagnosis of childhood craniopharyngioma

Hermann L Müller¹, Gina Bruhnken¹, Rudolf Oeverink¹, Nicole Etavard-Gorris¹, Ursel Gebhardt¹, Reinhard Kolb¹ and Niels Sörensen^2
1Department of Pediatrics, Zentrum fur Kinder und Jugendmedizin, Klinikum Oldenburg gGmbH, 26133 Oldenburg, ²Department of Pediatric Neurosurgery, University Hospital, 97080 Würzburg, Germany

Craniopharyngiomas are rare embryogenic malformations. As the survival rate after childhood craniopharyngioma is high (92%), prognosis and quality of life (QoL) in survivors mainly depend on adverse late effects. In a cross sectional study, patients with severe obesity after childhood craniopharyngioma had lower self-estimation of their QoL (1). Information on longitudinal development of QoL and risk factors for a lower self-estimation of QoL in patients with childhood craniopharyngioma is rare.

We longitudinally analyzed QoL by Fertigkeitenskala Münster Heidelberg (FMH) ability scale in 50 patients treated at the Department of Pediatric Neurosurgery, University of Würzburg between 1980 and 2000. Craniopharyngioma was diagnosed at a median age of 8.2 years ranging from 0.05 to 18 years. The first evaluation of QoL was performed at an age of 13.7 years ranging from 2.3 to 30.7 years, the second evaluation was prospectively performed after a median follow-up interval of 3.3 years (range: 1.7 to 4.3 years) at a median age of 17.3 years (range: 4.0 to 34.2 years). Furthermore, body mass index (BMI SDS), hypothalamic tumor involvement, the degree of surgical resection and irradiation were evaluated.
QoL did not change in patients (n=32) with obesity (BMI >2SD) and patients (n=30) with hypothalamic tumor involvement during longitudinal follow-up evaluation. QoL in normal weight (BMI<2SD) patients (n=18) and patients without hypothalamic tumor involvement (n=20) significantly (p<0.05; p<0.05) improved during followup and when compared with QoL in obese patients (p<0.001) and patients with hypothalamic involvement (p<0.01) during follow-up. No differences or changes in QoL were detectable in relation to irradiation, gender or the degree of surgical resection.
We conclude that obesity and hypothalamic tumor involvement in patients with childhood craniopharyngioma have significant impact on self-estimation of QoL. In normal weight patients and patients without hypothalamic involvement an improvement of QoL is detectable during longitudinal follow-up. Accordingly, rehabilitative efforts should be increased especially in obese patients and patients with hypothalamic involvement in order to improve their QoL. The results of our analysis are further tested in a prospective surveillance study Kraniopharyngeom 2000 (www.kraniopharyngeom.com).
References: (1) Müller HL et al. Obesity after childhood craniopharyngioma. Klin Pädiatr 2001;213:244-9.
Financial Support: Deutsche Kinderkrebsstiftung, Bonn, Germany

Magnet Resonance Spectroscopy in children with diffuse pontine glioma
Warmuth-Metz M, Kühl J*, Haddad D^#, Neuberg Th^#, Bartsch A, Bendszus M, Solymosi L
Department of Neuroradiology, Institute of Experimental Physics^# and Department of Pediatric Oncology*, University of Würzburg, Germany

Objective: Prognostic imaging features in diffuse pontine gliomas have not yet been defined. Prognosis has remained extremely poor. As novel treatment strategies might be different in prognostically different varieties of this tumor, such information is expected to be very valuable.
Materials and Methods: In a prospective study proton-MR-Spectroscopy (1HMRS) was performed in 8 children with typical diffuse pontine glioma diagnosed on Magnetic Resonance Imaging (MRI). All children deceased and survival time was calculated to the individual time points of MRS. Ratios between Choline containing compounds (Cho), Creatine (Cr) and N-Acetylaspartate (NAA) were calculated using AMARES on MRUI.
Results: Correlation of the ratio between Cho and Cr and survival time revealed a significant correlation. All others ratios did not correlated significantly. Dichotomization between long and short term survivors showed significant differences in mean values for the Cho/Cr ratio.
Conclusion: 1H-MRS is a valuable tool for the definition of prognosis in children with diffuse pontine gliomas. 1H-MRS might be useful for the evaluation of response in future treatment trials as has already been demonstrated for adults with malignant gliomas.

Atypical teratoid/rhabdoid tumor of the central nervous system: Case report
O Peters¹, N Graf ⁴, P Stadler², M Herbst², M Friedrich³, UW Ullrich³, H Hauch¹, S Wagner¹, JEA Wolff¹ Departments of Paediatric Oncology and Haematology of ¹Regensburg and ⁴Homburg, Germany, ²Department of Radiotherapy, Regensburg, Germany, ³Department of Neurosurgery, Regensburg, Germany

Background: The atypical teratoid/ rhabdoid tumor (AT/RT) is a very rare tumor occurring all over the body but mostly in the kidneys and the central nervous system (CNS). Newborns and infants are usually affected. Especially in CNS tumors the long term survival is exceptionally poor. No effective chemotherapy is established and a successful multimodal therapy does not exist.
Patient and history: 18 months old white boy with a 4 week history of balance disturbance, vomiting and headache. On admission he presented a right hemiparesis and a right central seventh nerve palsy. Diagnosis: Magnetic resonance imaging (MRI) revealed a contrast enhancing tumor of the left basal ganglia (max. diameter 4,0 cm) accompanied by a severe midline shift to the right. After subtotal resection the diagnosis was confirmed by two German Tumor Reference Centers (University of Bonn and Kiel). No leptomeningeal seeding was noticed (M0) on MRI of the spinal axis or in the cerebrospinal fluid (CSF).
Therapy and outcome: Only 20 days after a subtotal resection of the tumor a local tumor progression (max. diameter 2,0 cm) was observed. Without any further tumor resection a Rickham reservoir was implanted in the left ventricle and a newly developed chemotherapy strategy was started immediately. After the first chemotherapy course (doxorubicin: 25 mg/m2/d, 12 h i.v., d 1, 2, 3; actinomycin D: 45 µg/m2/d, i.v. push d 1; cisplatin: 70 mg/m2/d, 6 h i.v., d 4; vincristine 1,5 mg/m2/d, i.v. push d 8, 15; methotrexate: 2,0 mg single dose, intrathecal d 1, 2, 3, 4) a partial response was seen on MRI. Therefore a second chemotherapy course (same as 1. course) was applied within 3 weeks As a result a complete response was diagnosed prior to conventional local radiotherapy (tumor region +1cm safety margin: 54 Gy, 5 x 1.8 Gy/week, 6 MeV photons plus adjuvant carboplatin 80 mg/m_/d, i.v. 6 h, 4d). After radiotherapy as well as after a third chemotherapy course (same as 1. course) a continuous complete remission was achieved until 6 months after diagnosis when a continuous chemotherapy (CCNU, cisplatin, vincristine) was started. Within this period of time no leptomeningeal seeding was observed by MRI of the spinal axis or by microscopy of the CSF. The toxicities following the 1.-3. chemotherapy course were severe: Haemetological grade 4, stomatitis grade 4, general condition grade 3, infection grade 2, skin grade 2. No toxicity was observed after or within the time of radiotherapy. The initial neurological deficits resolved almost complete and the boy is doing well.
Conclusion: The current response data to a newly developed chemotherapy regimen are encouraging. They show in this case of a AT/RT a marked and rapid sensitivity to the applied chemotherapy.

Low-grade Oligodendroglioma in Childhood
Peters O¹, Gnekow AK², Rating D³, Hauch H¹, Wagner S¹, Wolff JEA¹
Departments of Paediatric Oncology and Haematology, of: ¹Regensburg, ²Augsburg,³Heidelberg, and 4Würzburg

Contact address: St. Hedwig Hospital, Steinmetzstrasse 1-3, 93049 Regensburg, Germany, email: Ove.Peters@barmherzige-regensburg.de
Background: In childhood oligodendrogliomas represent only 0.3-4 % of all intracranial gliomas. Most of them are of low-grade histology (91%) and supratentorial location (94%). Treatment recommendations follow guidelines of childhood lowgrade astrocytomas or adult oligodendrogliomas: Complete tumour resection is felt to provide the best opportunity for long-term survival, while irradiation and chemotherapy is still discussed controversial in children. Therefore we retrospectively analysed pediatric patients exclusively suffering from primary low-grade oligodendroglioma which have been referred to the German Childhood Cancer Registry
Methods: We pooled data from two prospective multicentre studies (HIT-DOK/- LGG) performed in the German-speaking group (GPOH). Eligibility criteria were: 1) primary malignant disease, 2) histology of pure oligodendroglioma WHO grade II (excluding mixed oligoastrocytoma), 3) intracranial location (excluding spinal tumours), 4) age: <18 years, 5) date of diagnosis: 1990 to 2000, 6) observation time: >6 months. All data were revised and patients were updated by time of submission. Statistical analysis were calculated by using the SPSS-software.
Results: 13 girls and 19 boys met the above criteria (median age: 10.3 years, range 0.6 to 16.4 years). Tumour locations included: 23 cerebral hemisphere/ 3 cerebellum (i.e. peripheral tumours) and 4 thalamus/ 1 frontal diencephalon / 1 basal ganglia (i.e. central tumours). Surgery was classified as complete resection in 19 (15 cerebral hemispheres, 3 cerebellum, 1 thalamus), and less than complete in 13 patients (3 subtotal resection, 7 partial resection, 3 biopsy). The event free survival (EFS) of all patients was 81,3%, the overall survival (OS) 84,4% (median observation time: 3,8 years, range 0,8 to 10,9 years). All of the 26 children with peripheral tumours (hemisphere, cerebellum) were alive and progression-free. In contrast all children with central tumours (thalamus, diencephalon, basal ganglia) experienced progressive disease (one to three times) regardless if adjuvant treatment was applied (median time to first progression: 6 months). Five of six patients of this group died of disease (median time to death: 1,6 years, range 0.9 to 3.7 years). The survival difference between the two groups of tumour locations (i.e. peripheral versus central) was statistically significant for EFS as well as for OS (P <0.0001, log-rank test). There was no significant survival difference between complete resection versus incomplete resection (P > 0,06, log-rank test).
Conclusion: We conclude that the outcome of children with central low-grade oligodendrogliomas is particularly poor, while tumours of the cerebral hemisphere and cerebellum offer an excellent prognosis, even with incomplete tumour resection.

Studies on loading peripheral blood monocyte-derived dendritic cells with proteins from glioblastoma tumor homogenates
De Vleeschouwer S, Spencer Lopes I, Prud'Homme L, Arredouani M, Cadot P (1), Adé M, Ceuppens JL, Van Gool SW
Laboratory of Experimental Immunology, and Department of Neurosurgery (DVS) and Pediatric Hemato-oncology (VGSW), University Hospital Gasthuisberg, Leuven, Belgium.

Background: Dendritic cells (DC) are professional antigen-capturing and-presenting cells (APC). Their fundamental role in initiating and directing a primary immune response is well established, and is used to design tumor vaccination strategies. Successful pulsing of DC with tumor antigens has been reported using different sources: RNA, peptides, proteins from tumor lysate or homogenate, or apoptotic or necrotic cell bodies. We studied some further aspects on the process of loading and maturation of DC with glioblastoma tumor homogenate. Materials and methods: Freshly resected tumor tissue from patients with glioblastoma multiforme was homogenised mechanically. The tumor protein content in the homogenate was measured with Coomassi Blue technique. DC were differentiated out of freshly isolated human PBMC from volunteers (MoDC) in the presence of rIL-4 and rGMCSF. After 7 days, the homogenate was added to the DC culture. To study whether tumor proteins were taken up and handled appropriately by DC, proteins were labelled with FITC and kept frozen. The uptake and handling of the FITC-labelled tumor proteins was measured using FACS and confocal microscopy. In other experiments, DC were loaded with different amounts of tumor homogenate for 1 or 3 days, and the Antigen-Presenting Cell quality of DC were analysed by FACS and by their capacity to induce allogeneic responses.
Results: With the use of FITC-labelled tumor proteins, we showed a time-dependent uptake and re-appearance on the cell surface of the FITC-proteins by the MoDC. Uptake and processing of the FITC-proteins was dependent on cell metabolic activity, and was not present when the cells were kept at 4°C. The re-appearance on the membrane was polarised. The uptake and re-appearance on the membrane surface of the MoDC was extremely strong in case of overnight loading. The appearance of FITC-proteins on the cell surface remained stable, even when loaded DC were kept frozen and were thawed afterwards. Addition of the DC maturation cytokines rTNF-a, rIL-1ß and PGE2 at time of loading did not interfere with the uptake and processing of FITC-proteins. Loading with different amounts of tumor proteins revealed that viability and APC quality of DC diminished above 200 µg million DC. Loading for three days instead of one day did not improve the APC phenotype of the DC.
Conclusion: We clearly show that the protein fraction of a crude glioblastoma tumor homogenate is taken up and processed by MoDC, resulting in a polarised reappearance on the cell surface. DC could be loaded with up to 200 µg protein per million DC. These data support the use of MoDC, which are loaded with tumor homogenate proteins, as tumor vaccine to treat patients with glioblastoma multiforme according to our phase I/II trial HGG-IMMUNO-2003.

Searching for a genetic program in neuroblastoma cells that controls differentiation and spontaneous regression
Olaf Witt¹,² and Arnulf Pekrun^2
1Functional Genome Analysis Group, DKFZ, Heidelberg, Germany, ²Department of Pediatrics I, University of Göttingen, Germany

Spontaneous regression of malignant tumors is a clinically well documented phenomenon occurring in a wide variety of cancer diseases in children and adults. In early infancy even disseminated neuroblastoma often shows spontaneous regression. Histopathologically, many of these tumors show features of differentiation into a benign ganglioneuroma.
However, the underlying molecular mechanisms controlling these events are far from being understood. In vitro, neuroblastoma cells can be differentiated into a neuron-like phenotype by histone-deacetylase (HDAC) inhibitors. The morphological differentiation is accompanied by upregulation of cell cycle inhibitors, tumor suppressor-genes and differentiation markers and down-regulation of the oncogene n-myc. Based on genome-wide DNA-microarray analysis we identified 20 candidate ESTs which may play a pivotal role in controlling this differentiation process. Using chromatin-immunoprecipitation assays we demonstrate that gene activation occurs by histone-hyperacetylation in-vivo.
We propose a model, in which inhibition of HDACs activate a genetic program that eventually leads to loss of the malignant properties and acquisition of a benign phenotype. Our data may give insights into the mechanism of spontaneous tumor regression and may provide molecular targets for differentiation therapy of neuroblastoma.

Pilot study with high-dose MTX followed by simultaneous radiochemotherapy in children with high-grade glioma
Sabine Wagner¹, Hansjörg Schmid², Anne-Kathrin Liebeskind³, Norbert Graf⁴, Ove Peters¹, Johannes Wolff¹
Depts of Pediatric Oncology/Haematology of ¹Regensburg, ²Hannover, ³Berlin-Buch, ⁴Homburg

Background: In children with high-grade glioma, a simultaneous radiochemotherapy was the frontline therapy of two protocols of the HIT-GBM-study with encouraging results regarding response data and toxicity (Anticancer Res 22:3569-3572, 2002). Before starting the new HIT-GBM protocol (HIT-GBM D), a pilot study was conducted to evaluate toxicity and response of the induction therapy with additional high-dose methotrexate (HD-MTX) prior to radiochemotherapy.
Patients and Methods: 7 boys and 2 girls were enrolled in this evaluation (median age: 11 years, range 4-15 years). 5 anaplastic astrocytoma and 3 glioblastoma (1 low grade glioma=pontine tumor) were found in the cortex (n=3), brainstem (n=3), spinal cord (n=1), IVth ventricle (n=1) and as gliomatosis cerebri (n=1). Four tumors were biopsied, one partially, three subtotally and one completely resected. The patients received two cycle of high-dose MTX (5 g/m_, over 24h i.v.) within a 14 days interval. Two weeks after MTX, a standard fractionated radiotherapy (54-60 Gy total dose) started simultaneously with two cycles of chemotherapy (first cycle: cisplatin 20 mg/m_/d x 5d, etoposide 100 mg/m_ x 3d, vincristine 1,5 mg/m_ x 1d, second cycle: cisplatin, etoposide, ifosfamide 1,5 g/m_/x 5d) and a weekly vincristine between the two cycles. The toxicity was recorded by means of the NCI-common toxicity criteria.
Results: Two cycles of HD-MTX were tolerable concerning toxicity. The toxicity of the simultaneous radiochemotherapy (SRCT) after HD-MTX was: toxic death 0/7, grade IV toxicity 7/7 (haematotoxicity: n=7, uncertain neurotoxicity: n=1), grade III toxicity 6/7 (infection n=6, nephrotoxicity n=1, ileus n=1). As grade I and II toxicity haematotoxicity, gastrointestinal toxicity, infection, reduction of general condition, skin toxicity and nephrotoxicity were found. Two weeks after SRCT, the response data were: CCR: 1/7, PR: 2/7, SD: 4/7. The tumor of one patient with PR showed complete response after 5 months of further therapy. Children were progression free after a follow-up time of four months (n=1), five months (n=2), six months (n=2), seven months (n=1) and eight months (n=1, unknown: n=1). There was one progression after 5 months.
Conclusion: The toxicity results of the study were tolerable. The response data were promising for the new HIT-GBM D protocol.

Phase III Study of children and adults with choroid plexus tumors: preliminary results
S Wagner¹, T Hassall², A Moghrabi³, T Kutluk⁴, JEA Wolff¹
Depts. of Pediatric Oncology/Haematology of ¹Regensburg, Germany, ²Victoria, Australia, ³Montreal, Canada, ⁴4Ankara, Turkey

Background: Choroid plexus tumors are epithelial brain tumors, most frequently diagnosed in infants. The tumor has an incidence of 0.4-0.8% of all brain tumors. Limited information is available regarding the tumor biology and the best current treatment.
Patients and Methods: The world wide CPT-SIOP 2000 study enrolls children and adults with choroid plexus tumors. After surgery, randomization into either a carboplatin-etoposide-vincristine arm (350 mg/m_/d day 2-3 – 100 mg/m_/d day 1-5 – 1.5 mg/m_/d day 5) or a cyclophoshamide-etoposide-vincristine arm (1g/m_/d day2-3 – 100 mg/m_/d day 1-5 – 1.5 mg/m_/d day 5 ) is open for patients with choroid plexus carcinomas (CPC), anaplastic plexus papillomas (APP) with residual tumor or metastases and choroid plexus papillomas (CPP) with metastases. For patients with CPP without metastases, and for patients with APP without residual tumor and without metastases a wait and see approach followed surgery. In both randomization groups, a total number of six chemotherapy cycles is given, two of them are given before consideration of irradiation (54 Gy, patients >= 3 years) or further surgery.
Results: 24 patients (14 males) were enrolled from October 2000 to February 2003. The median age at diagnosis was 2.1 years (range 02.2-22.4 years). Of these patients 13 were younger than three years, five patients older than 12 years. 10 CPP, 5 APP and 9 CPC were localized in the left lateral ventricle (n=6), the right lateral ventricle (n=7), the third ventricle (n=3) and the fourth ventricle (n=4, unknown: n=4). MRI scan after surgery shows no residual tumor in 13, residual tumor in 7 patients (unknown: n=4). Response data of 5 patients after the first two cycles of chemotherapy were: CCR in 2/5 patients, PR 2/3 patients and SD in 1/2 patients. One patient died after surgery, all other patients are still alive.
Conclusion: The preliminary treatment results are encouraging. Further patients need to be enrolled.

Toxicity of HD-MTX followed by simultaneous radiochemotherapy in nine children with high-grade glioma
Sabine Wagner¹, Ulrich Leuthold², Karl-Friedrich Classen³, Ove Peters¹, Johannes Wolff¹
Pediatric Oncology Depts of ¹Regensburg, ² Siegen, ³Ulm

Background: Simultaneous radiochemotherapy was feasible in the frontline therapy in two protocols of the HIT-GBM-study (Anticancer Res 22:3569-3572, 2002). In preparation of the new HIT-GBM-protocol (HIT-GBM-D), the toxicity of a treatment with high-dose methotrexate (HD-MTX) prior to simultaneous radiochemotherapy (SRCT) was studied.
Patients and Methods: Two cycle of high-dose MTX (5g/m_, 24h-infusion) were given within an 14 days interval. Two weeks after MTX, a standard fractionated radiotherapy (single dose of 1.8 Gy up to 54 Gy total dose) started simultaneously with two cycle of chemotherapy (first cycle: cisplatin 20mg/m_/d x5, etoposide 100 mg x3d, vincristin 1,5 mg/m_/x1d, second cycle: cisplatin, etoposide, ifosfamide 1,5 g/m_/x5d) and weekly vincristin between the two cycles. 7 boys and 2 girls were enrolled in this pilot study (median age: 11 years, range 4-15 years). The toxicity was recorded using the NCI-common toxicity criteria.
Results: The protocol was followed with minor modifications: two patients received MTX additional intrathecally, 1 patient received only one block of MTX, in one patient the interval of the two MTX-cycles was only one week, in one patient the interval of MTX-therapy and SRCT was five weeks. The toxicity after HD-MTX was: no toxic death, IV° toxicity: 1/9 (infection; interval of seven days between the two MTX cycles), III° toxicity: 1/9 (infection), II° toxicity: 2/9 (stomatitis, vomiting, haematotoxicity), I° toxicity: 3/9 (stomatitis, haematotoxicity, exanthem,. transaminase). The toxicity after the SRCT was: no toxic death, IV° toxicity: 7/7 (haematotoxicity 7/7, uncertain neurotoxicity 1/7), III° toxicity: 6/7 (haematotoxicity 1/7, infection 6/7, ileus 1/7), II° toxicity: 7/7 (vomiting, nausea, fatigue, infection, stomatitis, nephrotoxicity, haematotoxicity, I° toxicity: 6/7 (fever, nausea, vomiting, stomatitis, nephrotoxicity, infection, exanthem, fatigue).
Conclusion: The toxicity of HD-MTX prior to simultaneous radiochemotherapy was tolerable. We conclude that the new HIT-GBM-D protocol is feasible regarding the toxicity.

Immunochemotherapy with Interferon Gamma and low dose cyclophosphamide in children with high-grade malignant glioma
Wolff JEA¹, Wagner S¹, Reinert C¹, Gnekow AK ², van Gool S³, Kühl J⁴
Depts of Pediatrics of ¹Regensburg, Germany, ²Augsburg, Germany, ³Leuven, Belgium, ⁴Würzburg, Germany

Background: Clinical evidence shows that low dose cyclophosphamide may increase immune response. Treatment with interferon gamma results in a immune stimulation by regulating MAC 1 and MAC 2 class molecules and by stimulating antigen processing and antigen presenting cells. Patients and Methods: Interferon gamma and cyclophosphamide were given to children after an induction treatment with simultaneous radiation and chemotherapy (HIT-GBM-B, Wolff, Anticancer Res 2002). Children were treated with individually increasing interferon gamma doses starting from 25 mg/m² daily up to a maximum of 175 mg/m² within 7 weeks. Cyclophosphamide was given at 300 mg/m² as a one hour infusion every 21 days. Control group: patients treated belong to HITGBM- A protocol (Wolff, Cancer 2000).
Results: 40 patients (22 males) were available for evaluation with a median age of 8.5 years (range: 3-18 years). 14 glioblastomas, 14 anaplastic astrocytomas and 2 low grade gliomas (diffuse pontine glioma) were found in the cerebral cortex (n=8), basal ganglia (n=4), brainstem (n=24), cerebellum (n=3) and spinal cord (n=1). With a maximum dose of 175 mg/m² per day interferon gamma, no toxic death was found. NCI-CTC Grade IV toxicity for thrombocytopenia was 10% and for leucopenia 2.5%. There was 2.5% grade III central nervous toxicity and 5% grade III hepatic toxicity 5%. The observation time of the 6 surviving patients ranged from 1.1 to 4.3 years. The median overall-survival at 1 year (46%) has no significant difference from a historical control group (HIT-GBM A: 43%) For patients excluding pontine glioma, the one year survival was 69 % (HIT-GBM A: 47%), the two year survival was 38% (HIT-GBM A: 27%). For the subgroup of pontine gliomas, the tendency was even worse. In the subgroup of patients with WHO Grade IV tumors the one year survival was 66% (HIT-GBM A: 46%), the two year survival was 33% (HIT-GBM A: 26%).
Conclusion: There was no survival advantage of patients who were treated with interferon gamma according to the HIT-GBM-B protocol compared to the control group treated according to the HIT-GBM-A protocol. In further protocols, interferon gamma would not be a treatment choice.