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HIGH RISK NEUROBLASTOMA STUDY 1 OF SIOP-EUROPE
WRITING COMMITTEE
STUDY CHAIR
Ruth Ladenstein AUSTRIA
SIOP EUROPE NEUROBLASTOMA BOARD

Jean Michon Bruno De Bernardi Victoria Castel Ruth Ladenstein Andy Pearson FRANCE / President ITALY / Vice President SPAIN / Vice President AUSTRIA / Secretary UK / Advisory former President
PAEDIATRIC ONCOLOGY NATIONAL CO-ORDINATORS

Ruth Ladenstein Genevive Laureys Henrik Schroeder Dominique Valteau-Couanet Isaac Yaniv Roberto Luksch Ingebjorg Storm-Mathisen Ana Forjaz de Lacerda Victoria Castel Per Kogner Maya Nenadov-Beck Penelope Brock AUSTRIA BELGIUM DENMARK FRANCE ISRAEL ITALY NORWAY PORTUGAL SPAIN SWEDEN SWITZERLAND UNITED KINGDOM
FURTHER CANDIDATES
CZECH REPUBLIK, GREECE, HUNGARY, POLAND
STUDY TO BE ACTIVATED IN NOVEMBER 2001 R0 to be opened in November 2001 R1 to be activated in December 2001 R2 expected to be activated in July 2002
SIOP EUROPE NEUROBLASTOMA SUB-COMMITTEES

for the HR-NBL-1/ESIOP Study and Sub-Committee Chairs
BIOLOGY BONE MARROW STUDIES IMMUNOTHERAPY NUCLEAR MEDICINE PATHOLOGY RADIOLOGY RADIOTHERAPY SURGERY STEM CELL COMMITTEE STATISTICAL COMMITTEE Peter Ambros Lawrence Faulkner Holger Lode Simon Meller Klaus Beiske Dominique Couanet Sylvie Helfre Keith Holmes Sandro Dallorso David Machin Ulrike Poetschger Vronique Mosseri Paolo Bruzzi AUSTRIA ITALY GERMANY UK NORWAY FRANCE FRANCE UK ITALY UK AUSTRIA FRANCE ITALY
THIS PROTOCOL WAS PRODUCED FOLLOWING DISCUSSIONS FROM THE FOLLOWING COUNTRIES AND GROUPS
AGPHO AIEOP BSPHO ISPHO GPOH NOPHO SEOP SFOP UKCCSG SIAK
Austrian Group of Paediatric HaematoOncology Associazione Italiana Ematologia Oncologia Pediatrica Belgian Society of Peadiatric HaematoOncology Israeli Society of Paediatric Haematology Oncology German Group of Paediatric HaematoOncology Nordic Society for Paediatric Haematology and Oncology (Norway, Sweden, Denmark) Spanish Society of Paediatric Oncology Socit Franaise d Oncologie Pdiatrique United Kingdom Childrens Cancer Study Group Switzerland Portugal
Final version of HR-NBL 1/ESIOP 14.03.06
ABBREVIATIONS USED IN THIS PROTOCOL IN ALPHABETICAL ORDER ABMT ANC ARDS AUC BM BUMEL CADO CBDCA CCRI CCSG CCNU CDDP CEM CGH CI CMV COG COJEC CYC CR CRP CT CTV DLT DNA EBMT EFS ENSG ENQUA G-CSF GD2 GFR GI GTV Gy HSV HVA ICRU IL2 INCR INSS LDH LMCE LI MAT mIBG MLC MNC MRI MSKCC MR MTD NB (L) NSE OS PBSCH PBSCR PCP PCR PD autologous bone marrow transplantation absolute neutrophil count acute respiratory distress syndrome area under the concentration versus time curve bone marrow busulphan and melphalan MAT regimen French induction regimen consisting of cyclophosphamide, adriamycin, vincristine carboplatin Childrens Cancer Research Institut Childrens Cancer Study Group CCNitrosourea cisplatin carboplatin, etoposide and melphalan MAT regimen comparative genomic hybridisation confidence interval cytomegalyvirus Childrens Oncology Group rapid, platinum-containing induction schedule (CBDCA, CDDP, CYC, VCR, VP16) cyclophosphamide complete response C-reactive protein computer tomography clinical target volume dose-limiting toxicity deoxyribonucleic acid European Group for Bone Marrow (Stem Cell) Transplantation event-free survival European Neuroblastoma Study Group European Neuroblastoma Group for Quality Assessment of Biological Markers granulocyte stimulating growth factor (filgrastim, Neupogen ) ganglioside glomerular filtration rate gastro-intestinal tract gross tumour volume Gray herpes simplex virus homovanillic acid international commission of radiation units interleukin-2 International Neuroblastoma Response Criteria International Neuroblastoma Staging System lactate dehydrogenase French Paediatric Neuroblastoma Group: Lyon-Marseille- I. Curie(Paris)- East of France local irradiation myeloablative therapy meta-iodobenzylguanidine multi-leaf collimator mononuclear cell magnetic resonance imaging Memorial Sloan-Kettering Cancer Centre mixed response maximum tolerated dose neuroblastoma neuron-specific enolase overall survival peripheral blood stem cell harvest peripheral blood stem cell rescue pneumocystis carinii pneumonia polymerase chain reaction progressive disease 3
PEM PR PTV RA RNA R0 R1 R2 SD SIOP SFOP SMZ TBI TMP VAMP VCR VGPR VMA VOD VP16 VZU W WBC
MAT regimen using cisplatin, etoposide, melphalan, partial remission planning target volume 13-cis retinoic acid ribonucleic acid randomisation on the use of G-CSF (filgrastim) during induction randomisation of MAT regimen randomisation of ch14.18 antibody GD2 stable disease Socit Internationale dOncologie Pdiatrique Socit Franaise dOncologie Pdiatrique sulfamethoxazol total body irradiation trimethoprim MAT regimen including teniposide, adriamycin, melphalan, cisplatin, vincristine very good partial remission vanillyl mandelic acid veno-occlusive disease etoposide varicella-zoster virus weeks white blood cells
INTERNATIONAL DATA CENTRE

Study Chair / Contact Address:
Ass. Prof. Dr. Ruth Ladenstein St. Anna Childrens Hospital / CCRI Unit for Applied Clinical Research and Statistics (UARCS) A-1090 Vienna, AUSTRIA Kinderspitalg. 6 Phone: 0043-1-40170 Fax: 0043-1- 40170- 430 Email address: ladenstein@ccri.univie.ac.at, HRNBL1@ ccri.univie.ac.at
IMPORTANT NOTE
This document is intended to describe a collaborative study in High-Risk Neuroblastoma over the age of one at diagnosis and to provide information for entering patients. The trial committee does not intend it to be used as an aide-memoire or guide for treatment of NON-registered patients. Amendments may be necessary; these will be circulated to known participants of the trial, but institutions entering patients are advised to contact the appropriate study centres to confirm the correctness of the protocol in their possession. Although this protocol was written and checked with care, typing errors may have occurred and the physician in charge therefore has the final responsibility of controlling the drug dosages given to individual patients. Before entering patients into one of the studies clinicians must ensure that the study protocol has been cleared by their ethical committee. SERIOUS ADVERSE EVENTS All serious adverse events have to be notified to the National Data Centre within 24 hours following onset. The Data Centre will then inform the Study Co-ordinator at the International Data Centre also within 24 hours.
CONTENTS
SPECIFIC AIMS........................................................................................................................................................ 10 1.1 1.2 Primary Aims ....................................................................................................................................................... 10 Secondary Aims ................................................................................................................................................... 10 Induction Chemotherapy (Supportive Care Randomisation -Ro)....................................................................... 11 PBSC Harvest....................................................................................................................................................... 12 Surgery ................................................................................................................................................................. 12 Myeloablative Therapy and Peripheral Blood Stem Cell Rescue (First Randomisation -R1)........................... 12 Radiotherapy ........................................................................................................................................................ 13 Differentiation Therapy ....................................................................................................................................... 13 Novel Immunotherapy Approach (Second Randomisation - R2)....................................................................... 13 Background .......................................................................................................................................................... 14 Rationale for the Various Therapeutic Elements of the Proposed Study ........................................................... 15 Eligibility Criteria for the Study .......................................................................................................................... 26 Eligibility Criteria for the R0 Randomisation ..................................................................................................... 26 Eligibility Criteria for the R1 Randomisation ..................................................................................................... 27 Eligibility Criteria for the R2 Randomisation .................................................................................................... 27 Pre-treatment Investigations ................................................................................................................................ 28 Necessary Interactions to Secure Tumour Material Flow................................................................................... 31 Investigations at Diagnosis, during Induction and pre-MAT Phase ................................................................... 32 Investigations after the Induction Phase until the End of Treatment.................................................................. 33 Follow Up Investigations..................................................................................................................................... 33 Introduction .......................................................................................................................................................... 35 Aims ..................................................................................................................................................................... 35 Timing .................................................................................................................................................................. 35 Definition of Procedures ...................................................................................................................................... 36 Definition of Major Surgical Complications....................................................................................................... 36 Aspects of Surgical Procedures ........................................................................................................................... 37 Risk Factors Related to Localisation ................................................................................................................... 37 General Remarks.................................................................................................................................................. 39 Recommendations for Handling of Tumour Material......................................................................................... 39 Role of Pathologist............................................................................................................................................... 39 Further Important Aspects ................................................................................................................................... 40 Type of Biological Studies................................................................................................................................... 41 Material for Biological Studies of Neuroblastoma ............................................................................................. 41 Important Remarks............................................................................................................................................... 42 Introduction .......................................................................................................................................................... 43 Timing of Peripheral Stem Cell Harvest ............................................................................................................. 43 Recommended Procedure for PBSC Mobilisation and Day of Collection......................................................... 43 Vascular Access ................................................................................................................................................... 44 Standard Procedure .............................................................................................................................................. 44 Freezing Procedure and Storage .......................................................................................................................... 44 Purging of PBSC Harvests................................................................................................................................... 44
TRIAL DESIGN......................................................................................................................................................... 11 2.1 2.2 2.3 2.4 2.5 2.6 2.7
BACKGROUND AND RATIONALE ..................................................................................................................... 14 3.1 3.2
ELIGIBILITY AND PATIENT ENTRY CRITERIA........................................................................................... 26 4.1 4.2 4.3 4.4
ASSESSMENT OF EXTENT OF DISEASE, RESPONSE AND TOXICITY .................................................. 28 5.1 5.2 5.3 1.4 1.5
SURGERY .................................................................................................................................................................. 35 6.1 6.2 6.3 6.4 6.5 6.6 6.7
PATHOLOGY............................................................................................................................................................ 39 7.1 7.2 7.3 7.4
BIOLOGICAL STUDIES ......................................................................................................................................... 41 8.1 8.2 8.3
PERIPHERAL STEM CELL HARVEST .............................................................................................................. 43 9.1 9.2 9.3 9.4 9.5 9.6 9.7
10 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11 11.1 11.2 11.3 11.4 11.5 12 12.1 12.2 12.3 12.4 13 13.1 13.2 13.3 13.4 13.5 13.6 14 14.1 14.2 14.3 15
CHEMOTHERAPY REGIMEN DETAILS....................................................................................................... 45 COJEC Induction ............................................................................................................................................. 45 Check List prior to MAT ..................................................................................................................................... 51 Monitoring for Ototoxicity .................................................................................................................................. 52 Monitoring of Renal Function ............................................................................................................................. 53 BUMEL MAT Regimen ...................................................................................................................................... 63 CEM MAT Regimen......................................................................................................................................... 64 Patients with Normal Renal Function.................................................................................................................. 65 Patients with Abnormal Renal Function.............................................................................................................. 67 Stem Cell Reinfusion ........................................................................................................................................... 69 RADIOTHERAPY................................................................................................................................................. 70 Indication.............................................................................................................................................................. 70 Timing of Radiotherapy ....................................................................................................................................... 70 Fields and Dose of Radiotherapy......................................................................................................................... 70 Normal Tissue Tolerance..................................................................................................................................... 71 Quality control ..................................................................................................................................................... 71 13-CIS RETINOIC ACID THERAPY ................................................................................................................ 72 Treatment Schedule for 13-cis RA ...................................................................................................................... 72 Suggested Supportive Care .................................................................................................................................. 72 Criteria prior to Each Cycle of 13-cis RA ........................................................................................................... 72 Dose Modifications .............................................................................................................................................. 72 IMMUNOTHERAPY WITH CH14.18 ANTI-GD2 MAB ................................................................................. 74 Indication.............................................................................................................................................................. 74 Ch14.18 Anti-GD2 mAb Treatment Schedule ..................................................................................................... 74 Mode of Action .................................................................................................................................................... 74 Mode of Administration of ch14.18 anti-GD2 mAb Treatment.......................................................................... 74 Toxicity ................................................................................................................................................................ 75 Special Supportive Care during Ch14.18 Anti-GD2 mAb Treatment................................................................. 75 USE OF G-CSF....................................................................................................................................................... 77 G-CSF (filgrastim) during Induction Treatment (Supportive Care Randomisation R0)................................... 77 G-CSF for PBSC Mobilisation ............................................................................................................................ 79 G-CSF after MAT ................................................................................................................................................ 80 SUPPORTIVE CARE GUIDELINES ................................................................................................................. 82 Anti-Emetics......................................................................................................................................................... 82 Hydration.............................................................................................................................................................. 82 Blood Component Therapy.................................................................................................................................. 82 Central Lines ........................................................................................................................................................ 82 Treatment of Infections........................................................................................................................................ 82 Pneumocystis Pneumonitis Prophylaxis.............................................................................................................. 82 Nutrition ............................................................................................................................................................... 83 Additional Supportive Care during the Acute Phase of MAT ............................................................................ 83 Prophylaxis, Diagnosis and Management of Hepatic Veno Occlusive Disease............................................... 83 Renal Function Monitoring.............................................................................................................................. 85 End Points ............................................................................................................................................................ 86 Randomisation...................................................................................................................................................... 87 Patient Number Estimates.................................................................................................................................... 88 Power Considerations .......................................................................................................................................... 89 Analysis ................................................................................................................................................................ 89 Monitoring Guidelines for Severe Toxicities...................................................................................................... 91 APPENDIX STAGING CRITERIA ACCORDING TO INSS....................................................................... 92 Diagnosis of Neuroblastoma................................................................................................................................ 92 Assessment of Extent of Disease......................................................................................................................... 92 INSS Staging ........................................................................................................................................................ 93 INRC Definition of Response.............................................................................................................................. 93 7
15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 15.10 16 16.1 16.2 16.3 16.4 16.5 16.6 17 17.1 17.2 17.3 17.4
STATISTICS AND BIOMETRICAL METHODOLOGY ............................................................................... 86
18 APPENDIX PATHOLOGY AND BIOLOGY GUIDELINES FOR RESECTABLE AND UNRESECTABLE NEUROBLASTIC TUMOURS AND BONE MARROW EXAMINATION GUIDELINES. 94 18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8 18.9 18.10 18.11 18.12 18.13 18.14 19 19.1 19.2 19.3 19.4 20 20.1 20.2 21 21.1 21.2 21.3 21.4 21.5 21.6 21.7 21.8 21.9 21.10 21.11 22 22.1 22.2 23 23.1 23.2 23.3 24 24.1 24.2 25 26 26.1 26.2 27 28 Remarks and Recommendations for Pathology .................................................................................................. 95 Sectioning and Securing Tumour Material in Case of Resected Tumours......................................................... 95 Securing Tumour Material in Case of Unresectable Tumours ........................................................................... 96 Histology/Cytology Report................................................................................................................................ 100 Tumour Material Obtained after Cytotoxic Therapy ........................................................................................ 101 Regional Lymph Node Examination ................................................................................................................. 101 Immunohistology/-cytology............................................................................................................................... 101 Exact Determination of the Tumour Cell Content ............................................................................................ 102 Pathology Review .............................................................................................................................................. 102 General Remarks and Recommendations for Biology.................................................................................. 102 Procedures for the Determination of the Tumour Cell Content.................................................................... 102 Genetic Parameters to be analysed ................................................................................................................ 103 General Remarks on Bone Marrow Aspirations ........................................................................................... 107 Handling of the bone marrow cells in the laboratory.................................................................................... 110 Objectives........................................................................................................................................................... 113 Patient Preparation ............................................................................................................................................. 113 Tracer Activity ................................................................................................................................................... 113 Scanning Protocol .............................................................................................................................................. 114 APPENDIX MONITORING OF RENAL TOXICITIES .............................................................................. 117 Recommended Procedure for GFR Evaluation ................................................................................................. 117 Prescription sheet for GFR estimation............................................................................................................... 119 APPENDIX DRUG INFORMATION ............................................................................................................. 121 Carboplatin ......................................................................................................................................................... 121 Cisplatin.............................................................................................................................................................. 121 Etoposide ............................................................................................................................................................ 122 Cyclophosphamide............................................................................................................................................. 123 Mesna (sodium 2-mercaptoethanesulfonate (MESNA) .................................................................................... 124 Vincristine .......................................................................................................................................................... 125 Busulphan........................................................................................................................................................... 126 Melphalan........................................................................................................................................................... 126 Granulocyte Colony Stimulating Factor............................................................................................................ 127 13-Cis-Retinoic Acid (ISOTRETINOIN , ROACCUTANE)........................................................................... 128 Monoclonal Anti-GD2-Antibody (ch 14.18)................................................................................................. 130 Pharmacodynamics and pharmacogenetics of high-dose busulphan and melphalan ....................................... 131 Pharmacodynamics and pharmacogenetics of CEM MAT............................................................................. 133 LEUCAPHERESIS GUIDELINES ................................................................................................................... 138 Paediatric apheresis procedure........................................................................................................................... 138 Cryopreservation of PBSC Products ................................................................................................................. 139 PBSC Infusion.................................................................................................................................................... 140 APPENDIX PERFORMANCE SCALES ......................................................................................................... 141 Lansky Play-Performance Scale ........................................................................................................................ 141 Karnofsky Performance Scale ........................................................................................................................... 141 APPENDIX GUIDELINES FOR REPORTING TOXICITY/SERIOUS ADVERSE EVENTS ............... 142 APPENDIX: TOXICITY GRADING................................................................................................................ 143 TOXICITY AFTER HIGH DOSE CHEMOTHERAPY.................................................................................. 143 CTC-NCIC CRITERIA ..................................................................................................................................... 144 APPENDIX PYSCHO-SOCIAL SUPPORT ................................................................................................... 146 APPENDIX INTERNATIONAL TRIAL CO-ORDINATION ...................................................................... 147
APPENDIX SCINTIGRAPHY PROTOCOL FOR MIBG SCANNING..................................................... 113
APPENDIX: PHARMACOLOGICAL GUIDELINES FOR MAT............................................................... 131
28.1 28.2 28.3 28.4 28.5 28.6 28.7 28.8 28.9 28.10 28.11 28.12 28.13 28.14 28.15 28.16 28.17 28.18 28.19 28.20 28.21 28.22 29 29.1 29.2 29.3 29.4 29.5 29.6 29.7 29.8 29.9 29.10 29.11 29.12 29.13 30 30.1 30.2 30.3 31 31.1 31.2 31.3 31.4 31.5 31.6 31.7 31.8 32
Status of Study ................................................................................................................................................... 147 The Protocol ....................................................................................................................................................... 147 Study Forms ....................................................................................................................................................... 147 Inclusion Procedure for New Patients ............................................................................................................... 147 Data Collection................................................................................................................................................... 147 Randomisation Procedure .................................................................................................................................. 147 Confidentiality of Patient Data .......................................................................................................................... 148 Data Quality Control.......................................................................................................................................... 148 Data Analysis and Monitoring........................................................................................................................... 148 Adverse Events............................................................................................................................................... 148 Chemotherapy Review................................................................................................................................... 148 Pathology Review .......................................................................................................................................... 148 Biological Review.......................................................................................................................................... 149 BM /PBSC Harvest Review........................................................................................................................... 149 MIBG Review ................................................................................................................................................ 149 Radiological Review...................................................................................................................................... 149 Follow Up Data .............................................................................................................................................. 149 Study Approval .............................................................................................................................................. 149 Institutional/Local Ethical Approval and Patient Consent............................................................................ 149 Data Monitoring and Safety Committee (DMSC) ........................................................................................ 149 Toxicity Monitoring....................................................................................................................................... 150 Treatment Stopping Rules for Individual Patients ........................................................................................ 150 SIOP EUROPE NEUROBLASTOMA BOARD .......................................................................................................... 151 DATA MONITORING COMMMITTEE .................................................................................................................... 152 NATIONAL CO-ORDINATORS ............................................................................................................................... 152 BIOLOGY SC ....................................................................................................................................................... 155 BONE MARROW STUDIES SC.............................................................................................................................. 158 IMMUNOTHERAPY SC ......................................................................................................................................... 160 NUCLEAR MEDICINE AND PHYSICS .................................................................................................................... 161 PATHOLOGY SC .................................................................................................................................................. 164 RADIOLOGY SC................................................................................................................................................... 167 RADIOTHERAPY .............................................................................................................................................. 168 STATISTICAL SC ............................................................................................................................................. 170 STEM CELL SC................................................................................................................................................ 171 SURGERY......................................................................................................................................................... 172 Introduction ........................................................................................................................................................ 175 Basic Principles for All Medical Research........................................................................................................ 176 Additional Principles for Medical Research Combined with Medical Care .................................................... 178
APPENDIX - ADDRESS LIST....................................................................................................................... 151
APPENDIX: DECLARATION OF HELSINKI............................................................................................... 175
APPENDIX: SAMPLE INFORMATION SHEETS / CONSENT FORMS ................................................ 179 Sample Information Sheet/ Consent Form ........................................................................................................ 179 Trial consent form.............................................................................................................................................. 189 Supportive Care Randomisation on the Use of G-CSF: R0.............................................................................. 190 Consent Form for Supportive Care Randomisation R0 .................................................................................... 193 Randomisation R1 concerning the choice of MAT regimen ............................................................................ 194 Consent Form for Randomisation R1................................................................................................................ 197 Immunotherapy Randomisation R2................................................................................................................... 198 Consent Form for Randomisation R2................................................................................................................ 201 REFERENCE LIST............................................................................................................................................ 202
Specific Aims
1.1 Primary Aims
To test the hypothesis that myeloablative therapy (MAT) with busulphan and melphalan (BUMEL) in patients with high risk neuroblastoma (stage 4 disease older than one year, stages 2 and 3 disease with MycN amplification) will result in a superior, 3-year, event free survival (EFS) than MAT with continuous infusion carboplatin, etoposide and melphalan (CEM). To test the hypothesis that immunotherapy with chimeric 14.18 anti-GD2 antibody, following MAT, in addition to differentiation therapy with 13-cis retinoic acid, will improve 3-year EFS in patients with high-risk neuroblastoma.
Slettet: Slettet: Sideskift
1.2
Secondary Aims
Related to diagnosis and induction phase To evaluate the response at metastatic sites following induction chemotherapy with a high dose, rapid, platinum-containing schedule (COJEC). To determine the effect of response of metastatic disease following induction therapy, on EFS, progression free survival (PFS) and overall survival (OS). To determine the effect of complete surgical resection of the primary tumour on local control, EFS, PFS and OS. To collect data on selected, validated biological features and to determine the effect of these on EFS, PFS and OS. To compare the toxicity and, in particular, episodes of febrile neutropenia associated with induction therapy with a rapid schedule platinum regimen (COJEC) when administered with and without G-CSF (filgrastim). To determine the effect of elective haemopoietic support with G-CSF (filgrastim) during induction therapy on the success of peripheral blood stem cell harvesting (PBSCH) after induction therapy. Related to MAT phase To determine the effect of MAT with BUMEL or CEM on 5-year EFS, 3- and 5-year PFS and 3- and 5-year OS. To determine and compare the acute and long term toxicities of BUMEL and CEM used as MAT. To monitor drug levels of MAT (in particular of busulphan and melphalan) and to relate them to patients outcome and toxicity. Related to post MAT phase To determine the effect of radiotherapy on the pre-surgical tumour volume at the primary site on local control, EFS, PFS and overall survival. To determine the tolerance of 13-cis retinoic acid (RA) following MAT with and without the addition of immunotherapy with chimeric anti-GD2 antibody.
10
Trial Design
This will be a randomised study of the European SIOP Neuroblastoma group in high-risk neuroblastoma (stage 4 disease over the age of one and stages 2 and 3 MycN-amplified neuroblastoma). The protocol consists of a rapid, dose intensive induction chemotherapy (COJEC) with the randomised use of G-CSF (filgrastim), peripheral blood stem cell harvest (PBSCH), attempted complete excision of the primary tumour, randomisation of myeloablative therapy (BUMEL compared to CEM) followed by peripheral blood stem cell rescue (PBSCR), radiotherapy to the site of the primary tumour and randomisation, either to differentiation therapy with 13-cis RA alone, or differentiation therapy and immunotherapy with chimeric 14.18 anti-GD2 antibody.
2.1 Induction Chemotherapy (Supportive Care Randomisation R0)

The COJEC Schedule lasts ten weeks and proceeds regardless of the neutrophil or platelet count and controlled infections. COJEC consists of three different courses of chemotherapy: Course A, Course B and Course C. One of these courses is given every 10 days. Drug doses used in each of these are summarised here as cumulative dose per course. Course A consists of carboplatin (CBDCA) 750mg/m/course, etoposide (VP16) 350mg/m/course in two divided doses and vincristine 1.5mg/m/course (maximum dose of 2 mg). The dose of drugs used in Course B are: cisplatin (CDDP) 80mg/m/course as continuous intravenous infusion and vincristine (VCR) 1.5mg/m/course (maximum dose of 2mg). Course C consists of etoposide 350mg/m/course, cyclophosphamide (CYC) 2.1g/m/course and vincristine 1.5mg/m/course (maximum dose 2 mg). Course A starts on days 0 and 40, B on days 10, 30, 50 and 70 and C on days 20 and 60. Daily doses and detailed infusion schedules are given in detail in Section 12. The study will address a supportive care question (R0) on the randomised use of G-CSF (filgrastim) during the induction period (Details in Section 14). Dose /day R0G-CSF 5g/kg CBDCA Vp16 VCR CDDP CYC DAY COURSE CYCLE HARVEST SURGERY STAGING
Primary site MRI or CT, ultrasound, mIBG, Bone Marrow: 2 aspirates/ 2 biopsies Bone Marrow: 2 aspirates, primary site ultrasound Primary site: control of surgical success (method optional ultrasound, MRI, CT: 2 weeks after surgery ; if positive repeat it but not prior to day 60 post MAT) Disease assessment according to the International Neuroblastoma Staging and Response Criteria.
days days days days days days days days

3-8 12-18 23-28 32- 38 43- 48 52-58 63-68 72 till harvest
750mg/m 175mg/m 1.5mg/m 80mg/m 1050mg/m 0
ctn
ctn
ctn
ctn
10
20
30
40
50
60
70
80
90
100 110
A
1
B
2
C
3
B
4
A
5
B
6
C
7
B
8
H H Sx H
R1 MAT
11
2.2 PBSC Harvest

Patients achieving a complete response (CR) or partial response (PR) at metastatic sites on mIBG scanning, with no evidence of disease on two bone marrow aspirates on cytomorphological evaluation, will undergo harvest of peripheral blood stem cells (PBSC). Harvest is scheduled to be performed after a full re-staging of disease status ONLY following stimulation with GCSF(filgrastim). Harvest may be scheduled either after the last chemotherapy cycle or out of steady state mobilisation prior or after surgery.
2.3 Surgery
Complete surgical excision of the primary tumour should be attempted in all patients at day 95 with the major aim of the study to achieve complete resection and to improve local control.
2.4 Myeloablative Therapy and Peripheral Blood Stem Cell Rescue (First Randomisation -R1)
First randomisation should be sought with the results of the metastatic response evaluation by day 90. Irrespective of the amount of post-operative residual primary tumour, patients in CR, VGPR or PR, but without evidence of neuroblastoma on cytomorphological examination of two bone marrow aspirates will be randomised to receive either BUMEL or CEM as MAT regimen followed by autologous PBSCR by day 110. Patients not fulfilling these response criteria will not be eligible for randomisation and are off protocol. However, these patients may receive MAT/ SCT if partial response is obtained secondarily with either a classical conventional chemotherapy regimen for example with adriamycin-containing courses or a phase-I or phase-II study. The BUMEL MAT regimen consists of the administration of busulphan (600mg/m/course) divided in 16 oral doses and melphalan (140mg/m/day). Melphalan is administered as a 15 minute short infusion at least 24h after the last busulphan dose. Peripheral blood stem cells are reinfused 24 hours after melphalan administration. The CEM MAT-regimen uses three drugs: the dose of carboplatin must be based on renal function with a target area under the concentration versus time curve [AUC] of 16.4mg/ml.min/course, etoposide 350mg/m/course and melphalan 210mg/m/course. Patients with a GFR >100ml/min/1.73m will receive full dose of etoposide and melphalan. CBDCA and VP16 are given in parallel as a continuous infusion over 4 days. Melphalan is given in split doses on three consecutive days as a 15 minute intravenous infusion. Patients with a GFR below 100 ml/min/1.73m need reduced CEM-MAT doses, otherwise they may experience major toxicities. Reinfusion of peripheral stem cells 72 hours after the end of the infusion of CBDCA and VP16. Infusion plans, in particular calculation of CBDCA dose, as well as dose reduction guidelines must be followed and are outlined in detail in section 12.
12
2.5 Radiotherapy
Radiation treatment will be given to all patients to the pre-operative extension of the primary tumour after MAT prior to differentiation and minimal residual disease therapy. A total volume of 21 Gy will be delivered to the target volume at the primary tumour site in daily fractions of 1.5 Gy starting as close as possible to day 60 after PBSCR and no later than day 90, if a longer interval is needed due to prolonged transplant related toxicities (i.e. VOD, ARDS, renal impairment, uncontrolled infection).
2.6 Differentiation Therapy

All patients (including those refusing second randomisation) will receive 6 cycles of 13-cis RA. Six cycles of 13-cis RA given by mouth should be administered at a dose of 160mg/m/day over 14 days every 4 weeks and should be started after completion of local irradiation on day 90 post PBSCR, and certainly no later than by day 120 post MAT. Treatment Schedule for 13-cis Retinoic Acid (RA): W1 RA W13 RA W2 RA W14 RA W15 rest W3 rest W4 rest W5 RA W16 rest RA RA W6 RA W17 W18 W19 rest W7 rest W8 rest W9 RA W10 RA W21 RA W22 RA W11 W12
W20 rest
W: weeks related to start of 13-cis RA treatment
2.7 Novel Immunotherapy Approach (Second Randomisation - R2)

Patients will be randomised for additional immunotherapy with the chimeric 14.18 anti-GD2 monoclonal antibody (mAb) at a dose of 20mg/m/day over 5 days every 4 weeks for 5 courses. The first course will start three weeks after initiation of 13-cis RA . Treatment schedule for 13-cis RA and the chimeric 14.18 anti-GD2 mAb for patients elected by R2 W1 RA W13 RA W2 RA W14 RA W3 rest W4 W5 W6 RA W17 RA W18 RA W7 rest W8 W9 W10 RA W21 RA W22 RA W11 rest W12 GD2
GD2 RA W15 rest GD2 W16
13
3 Background and Rationale

In this protocol the term high-risk neuroblastoma refers to children above one year of age at diagnosis with either disseminated disease (INSS stage 4: about 40 to 50% of all neuroblastomas), or INSS stage 2 and 3 disease with amplification of the MycN proto-oncogene (about 3% of all neuroblastomas). Between 10% and 20% of children with stage 3 and occasional patients with stage 2 disease are characterised by amplification of the MycN gene in their tumours. This biological characteristic has clearly been shown to be associated with a greater risk of relapse and death from disease progression. These patients may benefit from very aggressive treatment and, based on this hypothesis, they are included in this protocol. Since the literature related to high-risk neuroblastoma mainly focuses on disseminated disease most of the background will refer to stage 4.
3.1 Background
Children with this type of presentation and age represent the largest neuroblastoma subgroup. Their prognosis remains poor in most cases and our ability to predict the clinical course and the outcome of the individual patient is modest. With conventional (i.e. non-intensive) chemotherapy long-term survival of these children used to be sporadic (< 5% of the cases).1-12 The great improvement in supportive care, which occurred in the early 80s, led to the widespread use of dose-intensive regimens.13-21 Treatment intensification usually included an intensified induction regimen followed by the resection of the primary tumour and a final cycle of MAT combined with stem cell transplantation (SCT).22-28 As a result of this change a remarkable progress in therapeutic results was achieved in terms of (a) response rate, (b) 3- and 5- years EFS and OS, and (c) number of potential cures. Early results from UK and USA29-33 were confirmed by several reports from France, Italy, Germany, Japan and other national groups.23;34-42 However, the expectation that the refinement of these strategies would progressively improve patients outcome was frustrated by a number of trials that failed to increase the 5-year event-free survival above 30%.18;42-45 In addition, even 5-year survivors showed only an 80% chance of continued remission.46-48 A few reports on smaller patient series claiming superior results appeared most likely to be related to limited statistical power due to small numbers and, possibly, patient selection.49 Few clinical factors seem to have prognostic significance in children treated with modern high-dose protocols. The most consistent one is the disappearance of all detectable metastatic disease.48;50;51 However, the lack of uniform criteria to document the regression of skeletal and bone marrow disease (the most common sites of metastatic disease) have made it difficult to compare results.52-56 The advantage of having a few large co-operative groups investigating major therapeutic questions related to high-risk neuroblastoma appears obvious. Biological studies 57-68 as well as some serologic factors, i.e. LDH, ferritin and NSE 69-71, which are of great prognostic value for localised disease, were reported to be less indicative for high-risk patients. However, this may be related to the inconsistent evaluation and data sets of these factors in most studies reporting on their prognostic value. In summary, even the currently used highly intensive chemotherapy regimens, although superior to the conventional ones, are ineffective in more than half of the children with stage 4 neuroblastoma older than one year of age at diagnosis. Therefore, new additional approaches are needed.
14
3.2 Rationale for the Various Therapeutic Elements of the Proposed Study
The protocol proposed in the following sections will have the largest European patient subset of any previous European Neuroblastoma protocol. This current study is expected to make an important contribution towards the understanding of the many issues which remain unresolved in disseminated aggressive neuroblastoma due to its recruitment of a large number of patients in a relatively short period of time. Based on the above considerations the present protocol has been designed with the following goals: a) To employ an induction regimen capable of rapidly and effectively reducing the bulky disease. b) To find the most effective MAT. c) To reduce the incidence of local relapse. d) To promote the eradication of the minimal residual disease. To realise these goals six major therapeutic choices were included in this protocol: a) To administer a rapid, intensive induction therapy (COJEC) adapted from the ENSG 5 protocol (A. Pearson, oral communication) and to investigate in a randomised way the role of G-CSF (filgrastim) during this induction. b) To compare the therapeutic benefit and toxicity of two myeloablative therapy regimens (CEM v BuMel). 18;50;72;73 c) To encourage extensive surgical removal of the primary tumour at the end of induction. d) To administer local irradiation to all patients after MAT. e) To administer differentiation therapy (13-cis RA) after MAT and irradiation. 74-77 f) To investigate in a randomised fashion the potential of the anti-GD2 ch14.18 antibody (an anti-neuroblastoma monoclonal antibody78;79) to eradicate minimal residual disease in addition to 13-cis RA. The motivations and rationale of the above choices are summarised in the next sections.
3.2.1
FIRST TREATMENT MODALITY: COJEC AS INDUCTION REGIMEN
Many different induction regimens for high-risk neuroblastoma have been described in the literature. Most regimens utilise cisplatin and/or carboplatin, cyclophosphamide, etoposide and vincristine, with some including doxorubicin. No regimen has been proven to be conclusively superior, as there has been no randomised study. It is difficult to compare event free survival (EFS), progression free survival (PFS) and overall survival (OS) rates between different published induction regimens as the influence of MAT, local treatment and differentiation therapy have to be considered. In addition, variable definitions and the inconsistent use of mIBG scanning rates following induction therapy hamper the comparison of response data. Established regimens utilised in North America, e.g. CCG 389118, have not been demonstrated to be superior. Preliminary evidence suggested that the N6 regimen 80 (N7 regimen: no longer using a continuous infusion of VCR) developed by Kushner and colleagues at Memorial Sloan Kettering, New York resulted in superior response rates and EFS. However the effect of the N7 regimen was confounded by the influence of surgical resection of the primary tumour and the use of immunotherapy with an anti-GD2 antibody. Recent results from two European groups (SFOP and Austria) have failed to confirm the high response rates reported for this regimen.81 In Europe no clearly superior induction regimen is evident from the literature (Table 1). The NB 87 regimen of SFOP, comprising high dose cisplatin and etoposide, alternating with cyclophosphamide, doxorubicin and vincristine (CADO) has been most extensively used for over
Final version of HR-NBL 1/ESIOP 14.03.06 15
10 years.15 Again, the exact effect of this regimen on EFS and OS is unclear as post-induction therapy varied according to the degree of initial response - further chemotherapy with carboplatin and etoposide; single MAT with vincristine, total body radiation, melphalan or two MAT procedures with the second utilising CCNU and etoposide (LMCE1, LMCE3).38;82 Table 1: Comparison of European Neuroblastoma Studies National Groups Austria France Protocol A-NB94 No. of Event-free Overall Cases Survival Survival 28 43% (3yrs) 72 99 47 135 206 106 76 170 60 72 130 125 8% 29% 20% 32% 27% 26% 28% 24% 30% (4yrs) 18.6% 39.6% Journal Med.Ped.Onc. 1997 J. Clin. Oncol. 1991 J.Clin.Oncol. 1997 Med.Ped.Onc. 2000 Klin.Pdiatrie 1990 Oral communications (B. DeBernardi) Eur. J. Cancer 1995 Oral communication (A. Pearson)
LMCE1 LMCE3 F-NB97 Germany NB85 NB90 NB-85 Italy NB-89 NB-92 N-I-87 Spain N-II-92 ENSG5 UK -OPEC/OJEC -COJEC
18% 17% 16%
17.7% 31.3%
A retrospective review of the therapy of over 700 patients with stage 4 neuroblastoma throughout Europe between 1990 and 1994 by Dr Valteau-Couanet, on behalf of the SIOP Europe Neuroblastoma group indicated a high (40%) local relapse rate, but no conclusive superiority of any induction regimen used during this period.83 Between 1990 and 1999 the European Neuroblastoma Study Group (ENSG) conducted a randomised study to investigate the effect of dose intensity of induction therapy on EFS for stage 4 neuroblastoma over the age of one. Two hundred and fifty-five patients were randomised to receive one of two induction regimens - COJEC or OPEC/OJEC. Each regimen utilised the same drugs cisplatin, carboplatin, etoposide, cylophosphamide and vincristine - in the same dose, but the dose intensity (in mg/m2 per week) of COJEC was 1.8 fold higher (Table 2). 57-61 Table 2: Comparison of the total Doses and Dose Intensity in OPEC/OJEC and COJEC . DRUG Cisplatin Carboplatin Cyclo Etoposide Vincristine TOTAL DOSE OPEC/OJEC 320 1500 4200 1400 10.5 DOSEINTENSITY COJEC OPEC /OJEC 320 17.8 1500 83.3 4200 233 1400 77.8 12.0 0.58 FACTOR COJEC 32 150 420 140 1.2 1.8 1.8 1.8 1.8 2.03
Therapy in the COJEC arm was administered every 10 days, regardless of neutrophil and platelet counts and controlled infection, whilst it was delivered every 21 days in the OPEC/OJEC arm if there was haematological recovery (absolute neutrophil count greater than 1 x109/L and platelet count 100 x 10 9/L). In those patients who were responding after induction therapy and had achieved a bone marrow complete response (no evidence of detectable disease after
cytomorphological evaluation of two bone marrow aspirates and two trephines), attempted surgical excision of the primary tumour was undertaken, followed by MAT with single agent melphalan at a dose of 180 mg/m2, and haemopoietic stem cell rescue in the form of autologous bone marrow. Median time elapsing between the last course of chemotherapy and MAT was about 3 months. No radiation therapy was administered. Those patients who were within six months of myeloablative therapy in 1999 received six months of 13-cis RA, as a result of the superior EFS published by the CCG.18 With a minimum of 23 months follow-up, the 4-year EFS was higher for those patients treated with COJEC (31.3%) than with OPEC/OJEC (17.7%) p = 0.02. In particular the COJEC regimen resulted in significantly higher response rates (Table 3) as assessed at 14.3 weeks (100 days) after commencing therapy. Table 3: Comparison of Response and Survival Rates OPEC/OJEC and COJEC No. Cases COMPLETE RESPONSE RATES BM response (%) mIBG response (%) Catechol normal (%) Primary resection (%) OVERALL RESPONSE RATE CR VGPR PR SD PD SURVIVAL RATE ESTIMATE 6-y OS (%) OPEC/OJEC 130 56 49 80 56 58 7 19 10 6 19.8 COJEC 125 79 71 86 79 78 6 10 3 3 34.7 P-value <0.001 <0.017 NS 0.02 <0.001
0.01
ENSG 5 OVERALL SURVIVAL BY TREATMENT GROUPS

100 90 80 70 60 50 40 30 20 10 0 0 1 2 3 4 5 Time in years Rapid COJEC (n=125) Std OPEC/OJEC (n=130) 6 7 8 9 10
There was no significant difference in the toxicity observed with the two regimens.
17
Table 4: Comparison of the toxicity of OPEC/OJEC and COJEC TOXICITIES HOSPITAL DAYS Median treatment duration in days (range) Mean hospital days for treatment Mean hospital days for toxicity HAEMATOLOGICAL TOXICITY Days with ANC < 0.5 x 109 /l Days with WBC <1.0x 109 / l Time to ANC >1.0x 109 / l Time to WBC>1.0x 109 / l Time to platelets >100x109 / l after last CHT course (prior to Sx) Median number of red packed cell transfusions Median number of platelet transfusions INFECTIONS Episodes of febrile neutropenia Episodes of septicaemia Toxic deaths ORGAN TOXICITY GFR: median ml/min /1.73m2 Weight loss > 10% OPEC/OJEC 140 15 8.5 38 45 COJEC 70 16 9.8 69 75 82 78 87 2.9 2.7 4.1 0.9 5 97 23 0.8 0.9 p-Value
0.9 0.9
2.1 1.2 3.6 1.1 3 89 12
0.8
A recent analysis has been undertaken, comparing the EFS and OS of NB 87 (used as induction chemotherapy in the LMCE 3 regimen), OPEC/OJEC and COJEC (Pearson ADJ, Frappaz D, Phillip T, Imeson J). There was no significant difference in EFS or OS between LMCE 3 and COJEC; however, LMCE 3 had a superior EFS and OS compared to OJEC/OPEC.
COMPARISON OF EVENT FREE SURVIVAL: LMCE3 AND RAPID COJEC

100 90 80 70 % 60 50 40 30 20 10 0 0 1 2 3 4 5 T im e in y e a r s 6 7 8 9 10
E N S G 5 - C O J E C (n = 1 2 5 ) L M C E 3 (n = 9 9 )
18
It is noteworthy to state that the ENSG5 study used only melphalan as post-induction consolidation either after OPEC/OJEC or COJEC, while a more intense MAT approach was used within LMCE 3. It is further important to note the LMCE 3 (Lyon-Marseille-Curie East of France) strategy: After induction with the French Society of Pediatric Oncology NB87 regimen and surgery patients having achieved complete remission immediately proceeded to consolidation therapy with vincristine, melphalan, and fractionated total-body irradiation (VMT). All other patients underwent a postinduction strategy before VMT, either an additional megatherapy regimen or further chemotherapy with etoposide/carboplatin. The progression-free survival (PFS) is 29% at 7 years from diagnosis. Based upon the above data, COJEC will be used as the induction therapy for the current study as: 1. There is no significant difference in EFS and OS between different European induction regimens.83 2. The EFS and OS are very similar following NB 87 (LMCE 3) and COJEC. MAT intensity was greater after LMCE3 induction than after COJEC induction, i.e. melphalan alone in the latter. 3. The response rates with COJEC are similar to those obtained with NB 87/LMCE 3. However, induction therapy is completed within 10 weeks with COJEC, in contrast to 18 weeks with NB 87. No doxorubicin is utilised in the COJEC regimen. 4. In over 95% of patients there is no significant impairment of glomerular infiltration rate (GFR) (GFR < 80 ml/min/1.7m2) with the COJEC regimen). The use of COJEC as the induction regimen will produce a rapid response and allow myeloablative therapy to be delivered 110 days after diagnosis, in contrast to 200 days with NB 87. In addition, the COJEC regimen will not cause mucositis, cardiac damage or significant renal impairment.
3.2.2
SECOND TREATMENT MODALITY: SURGERY
This study aims to achieve complete primary tumour excision prior to MAT to improve local control. Surgical issues are discussed in detail in section 6.
3.2.3
THIRD TREATMENT MODALITY: COMPARISON OF TWO MAT REGIMENS BUMEL VS CEM
A very large number of MAT regimens have been employed since this therapeutic modality was introduced in the early 80s. Their large variety, and consequently the limited number of patients treated with each individual regimen has hindered the ability to reach firm conclusions on their respective antitumour effect and toxicity. Most investigators however, currently agree on the following statements: Children should be spared total body irradiation, given its severe toxicity on organ development and risk of secondary cancer.48 Peripheral blood progenitor cells are preferable to bone marrow progenitors, particularly because of the rapid recovery of the peripheral blood count they induce which lowers the procedure related morbidity. 84. Data comes from a number of sources to support the use of myeloablative therapy. 72;72;72;85 The most encouraging one is the CCG 3891 protocol (although limited to the 3-year EFS) randomising MAT followed by autologous purged bone marrow against further intensive chemotherapy without stem cell support.18
In the current protocol HR-NBL-1/ESIOP two MAT regimens will be applied in a randomised fashion: CEM and BUMEL. CEM is considered as the US standard MAT regimen and BuMel the European regimen. The hypothesis is that BUMEL may provide an improvement in the 3-year EFS (see Statistical Section of the protocol). MAT toxicity related risks are in particular gut and pulmonary toxicity for the CEM regimen while in the BUMEL regimen anticipated risks are particularly related to VOD, pulmonary (although very unusual without the use of alkalyting agents prior to the BUMEL regimen), cutaneous acute toxicities and the additional disadvantage of infertility, in particular ovarian failure in females.
A.
CEM BACKGROUND
The CCG mainly focused on TBI based regimens (VAMP-TBI in the CCG-321P3 study 48) working on drug adaptation to improve efficacy and toxicity profiles (PEM-TBI, CEM-TBI). CEM was selected for further dose-escalation, in an attempt to find an optimal regimen substituting for the TBI-containing one employed in the CCG 3891 protocol.18 The 91LA6 protocol (CEM-LI) treated 106 evaluable patients from July 1991 to January 1999 to determine the MTD of continuous infusion carboplatin and etoposide, with a fixed dose of melphalan at 210 mg/m2 (given as 70 mg/m2/day for 3 days).72 All patients received infusion of purged autologous bone marrow. Irradiation (1500-2100 cGy) was given to all patients to the primary tumour site (and other sites of residual tumour). Doses of carboplatin and etoposide were escalated in two separate cohorts: patients with a GFR < 100 ml/min/1.73 and patients with a GFR > 100 ml/min/1.73 m2. Patients with GFR 100 ml/min/1.73m The tolerable doses of carboplatin and etoposide were found to be significantly higher after the elimination of TBI, which may improve EFS rates. In the new A3973 COG study for high risk neuroblastoma defined doses are 1700 mg/m2 for carboplatin, 1350 mg/m2 for etoposide and 210 mg/m2 for melphalan. At this dose level no toxic deaths occurred in the US. Patients with a GFR 100 ml/min/1.73m The determined MTD for carboplatin was an AUC of 16mg/ml.min/course (dose calculated by the paediatric Calvert formula), 800 mg/m for etoposide and 180 mg/m for melphalan. The current A3973 COG Study for high risk neuroblastoma is using these dose levels for low GFR patients. The European HR-NBL1/ESIOP Study will use identical CEM MAT dose adaptations as in the ongoing US-A 3973 Neuroblastoma High Risk Study. This will allow investigation of the efficacy of these different MAT regimens in a comparable way. The US and Europe have decided to exchange toxicity data on a three-monthly basis. If further DLT occurs, the same dose modifications shall be applied in both protocols.
Results of CEM (LI) As of January 1999, the 3-year EFS from the time of transplant is 62% [CI: 20%] for all 77 patients transplanted after CEM prior to progression so far. The only significant prognostic factor is MycN amplification, with age, stage and pre-BMT response status not being significant. The 3-year EFS for all stage 4 patients > 1 year of age is 59% [CI: 32%]. It is 73% [CI: 38%] for MycN non-amplified and 51% [CI: 32%] for MycN amplified. In patients with normal GFR who received ABMT in 1st and 2nd response, respectively, no toxic deaths occurred. The overall toxic death rate was 4%. The 3-year EFS for patients having received CEM-LI in 1st response (76 pts) or 2nd response (29 pts) was 65% and 22%, respectively [Judy Villablanca for the CCG Group, oral communication].
20
CCG 3891 as compared with CEM-LI Comparing the regimens of CCG 3891 with the CEM-LI regimen, is the same as comparing TBI-containing versus TBI-non containing regimens: In the CCG 3891 protocol the dose of carboplatin was 1000mg/m, of etoposide 800mg/m and of melphalan 210mg/m. Local irradiation at the primary tumour site was given to a 1000cGy in addition to a 1000cGy of TBI. The CEM-LI Regimen used carboplatin at a dose of 1700mg/m, of etoposide 1350mg/m, and of melphalan 210mg/m. Local irradiation to the primary tumour site was within the range of 1500 to 2100cGy. The CEM-LI Regimen based on 77 patients in first response resulted in a 3-year EFS of 65%, whereas the CEM-TBI regimen, including 43 patients, had a 3-year EFS rate of 40% only.
B.
BUMEL BACKGROUND
Experience with BuMel is largely European and derives from studies performed in neuroblastoma and Ewings sarcoma.
Institut Gustave Roussy Data (Olivier Hartmann et al)
A multivariate analysis on prognostic factors was performed in data sets of 218 metastatic neuroblastoma patients over one year of age 50. Skeletal disease was present in 79% of cases and bone marrow involvement in 93%. MycN oncogene amplification was found in 27% of the patients studied. The probability of EFS at 5 years post-diagnosis was 29% in this series. Three major favourable prognostic factors were significant and independent in the multivariate analysis: age under 2 years at diagnosis (P<0.01), absence of bone marrow metastases at diagnosis (p<0.04) and the MAT regimen containing the busulphan/melphalan combination (p = 0.001). The quality of response to conventional primary chemotherapy was close to significance (p = 0.053). Thus factors related to the patient (age) and extent of disease were predictive of outcome in neuroblastoma patients treated with conventional chemotherapy, surgical excision of the primary and consolidation with MAT/SCT. The type of conditioning regimen appeared to be the most important prognostic factor.
EBMT Data
Equally the EBMT registry was able to depict in the year 2000 analysis a significant advantage for the BUMEL/MAT regimen in high-risk neuroblastoma patients. (Ladenstein et al, oral communication, BMT 2000). Patients after the BUMEL/ MAT regimen had an overall survival of 51% at 5 years (p=0.033) in comparison with other MAT regimens used Europe-wide.
21
E B M T 2 0 0 0 a n a ly s is : 1 0 1 1 N B L p a tie n ts w it h s in g le M A T
1 0 .8 0 .6 0 .4 0 .2 0 0 5 y e a rs 10 15 p = 0 .0 3 3
pSU
B u s u lfa n /M e lp h a la n w ith o u t C Y C -T T P : n = 1 3 3 , p S U a t 5 y e a rs = 0 .5 1 0 .0 6 B u s u lfa n /M e lp h a la n + C Y C -T T P : n = 1 2 1 , p S U a t 5 y e a rs = 0 .3 4 0 .0 5 M e lp h a la n a lo n e : n = 1 8 6 , p S U a t 5 y e a rs = 0 .3 7 0 .0 4 M e lp h a la n + : n = 2 0 6 , p S U a t 5 y e a rs = 0 .4 3 0 .0 4 o th e rs : n = 4 0 , p S U a t 5 y e a rs = 0 .3 7 0 .1 0 T B I: n = 3 2 5 , p S U a t 5 y e a rs = 0 .3 2 0 .0 3
3.2.4
FOURTH TREATMENT MODALITY: RADIOTHERAPY
All patients will receive local irradiation following MAT/PSCR according to the presurgical tumour volume at the primary site even if complete excision was achieved. According to the CCG data, consistent local radiation should be well tolerated without the use of TBI and may improve the local control rate at the primary site. This radiation has been piloted in a MSKCC protocol and in a limited institution study.72 Seeger et al, reported relapse at the primary site in 32 of 68 (47%) patients with disease progression following BMT on the CCG-321P3 study, in which 1000-2000 cGy were given (as 150 cGy BID for seven days) ONLY if disease was present at these sites immediately prior to BMT at primary and metastatic sites.44 Also in the retrospective European analysis of stage 4 patients over the age of one treated in Europe between 1990 and 1994, undertaken by Valteau et al on behalf of the SIOP Europe Neuroblastoma Group, a 40% local failure rate was detected (oral communication). In the non-TBI regimen utilised by Kushner et al, only one in ten (10%) relapses involved the primary site.31 These patients were irradiated with 21 Gy on a fractionated schedule at the site of primary disease and regional lymph nodes, as defined at diagnosis. Kremens et al, also utilised 21Gy (given as 150 cGy BID for 7 days) to the primary site and BMT in 26 children with neuroblastoma, and observed 4/14 (29%) relapses involving the primary site.40 Mugishima et al (1995) transplanted 36 children with advanced neuroblastoma. No relapses occurred at the primary site if intraoperative radiation was given.37 A trend for improved five year PFS and for less failure in sites of disease irradiated prior to BMT (doses of 8-24 Gy) was found in a review of 26 patients with advanced neuroblastoma transplanted with a variety of chemotherapy/TBI regimens.86 In the pilot study using CEM-LI (91LA6), 56 patients were treated prior to disease progression. Of 16 reviewed relapses only 3 involved the primary site. 72 This is considerably better than the local control rate seen with single daily fractions and lower dose irradiation used in CCG321P3, where the primary tumour was the site of relapse in more than 50% of the patients.87 In summary, the above data suggests that improved local control may be achieved with local radiation given to the primary tumour site. Radiotherapy guidelines are given in detail in section 11.
22
3.2.5
FIFTH THERAPEUTIC CHOICE: DIFFERENTIATION THERAPY WITH 13-CIS-RETINOIC ACID
3.2.5.1 Rationale for using 13-cis RA in Neuroblastoma The rationale for using 13-cis RA as adjuvant treatment is based on numerous reports that 13-cis RA is effective in inducing neuroblastoma differentiation and apoptosis in vitro. This results in a marked decrease in neuroblastoma cell proliferation which has also been shown to be effective in cell lines that are multi-drug resistant to conventional chemotherapeutic drugs. 74;75 Based on these pre-clinical evaluations, a Phase I study was initiated to evaluate the maximum tolerated dose and toxicity profile of 13-cis RA in children with stage 4 neuroblastoma.77 3.2.5.2 Phase I Study with 13-cis RA This trial revealed minimal toxicity of 13-cis RA following BMT. Interestingly, the treatment induced clearing of bone marrow from neuroblastoma cells in 3 of 10 treated patients as determined by histology. Treatment was well tolerated with mild toxicity primarily consisting of cheilitis, dry skin and hypertriglyceridemia.88 Hypercalcaemia has also been reported, albeit only at higher doses.89 3.2.4.3 Prospective randomised Multi-Centre Trial with 13-cis RA These promising results led to a prospective randomised multi-centre trial to address the question of whether there was a benefit for patients receiving adjuvant treatment with 13-cis RA. 18;76 In this trial, all patients who completed cytotoxic therapy (either chemotherapy or ABMT consolidation) without disease progression were randomly assigned at week 34 of therapy to receive no further treatment or treatment with 13-cis RA for 6 months. Since toxicity tends to increase with repetitive daily dosage, the schedule was 2 weeks of treatment, followed by 2 weeks of rest. This regimen was well tolerated in both the phase I trial 75 and this study. Specifically, patients in the 13-cis RA arm received six cycles of 13-cis RA (160 mg/m2 per day) administered orally in two doses for 14 consecutive days in a 28 day cycle. The result of this trial revealed a clear increase in the survival rate three years after the randomisation among the 130 patients who were assigned to receive 13-cis RA in contrast to the 129 patients receiving no further therapy (46 6 % versus 29 5 %, p=0.027). Looking at MycN-amplified patients only, the 13-cis RA group had a survival of 40%, and those not having received 13-cis RA had a survival of only 20%. This result also was significant. This advantage for EFS was also demonstrated in subgroups. The highest overall EFS was observed for patients treated with MAT/ABMT and 13-cis RA (55 10 %), followed by patients with MAT/ABMT but no 13-cis RA (EFS 39%), then patients with chemotherapy consolidation and 13cis RA (EFS 32%), and finally the cohort of patients with chemotherapy and no 13-cis RA (EFS18% 6 %). Based on these findings, the current protocol will include 13-cis RA as differentiation therapy following local radiotherapy after MAT/PBSCR. 3.2.6 SIXTH THERAPEUTIC CHOICE: RANDOMISED IMMUNOTHERAPY WITH MONOCLONAL ANTI-GD2 ANTIBODY CH14.18
3.2.6.1 Background of Immunotherapy with ch14.18 antiGD2 mAb One concept in the treatment of neuroblastoma is the application of monoclonal antibodies directed against neuroblastoma cells.90 The chimeric monoclonal antibody (mAb) ch 14.18, which recognises the ganglioside GD2 on neuroblastoma cells, is expressed by virtually all neuroblastoma cells. This antibody could therefore have an important role in the treatment of neuroblastoma. This antibody is called chimeric, because it was genetically engineered to consist of 30% mouse-protein (variable domain of the protein) and of 70% human protein (constant domains of human IgG1). Furthermore, it has been shown that this antibody induces killing of tumour cells in vitro by both antibody dependent cellular cytotoxicity (ADCC) and complement dependent cellular cytotoxocity (CDC).91;92
3.2.6.2 Previous Experience with the ch 14.18 anti-GD2 mAb First experiences using ch14.18 antiGD2 mAb antibody in children with neuroblastoma were generated at the University of California at San Diego by Dr. Alice Yu in a phase I trial.79 Ten patients were treated with increasing doses. Side effects consisted primarily of pain, tachycardia, hypertension, fever, and urticaria. Objective responses were observed in 5 patients: One partial (PR), four mixed responses (MRs) and one stable disease (SD). A second phase I trial was conducted at the Childrens Hospital in Tbingen.78 Nine patients with stage 4 neuroblastoma were treated with increasing doses of 30, 40 and 50 mg/m2/day for 5 days. The maximum tolerated dose (MTD) per injection was 50 mg/m2/day. Seven patients were given more than one course per treatment. Side effects in this trial were pain in the joints and abdomen, pruritus and urticaria. One patient showed temporary pupil atonia, and two patients suffered from unilateral atrophy of the optic nerve. It was not clear whether these side-effects were also attributed to preceding therapies. Clinical responses observed were: two patients with complete remission, two patients with partial remission, one patient with minor response and one patient with stable disease. In 3 patients the tumour progressed. 3.2.6.3 The recent use of ch14.18 anti-GD2 mAb in Germany. In the current German stage 4 study the ch 14.18 anti- GD2 mAb is given intravenously at a dose of 20mg/m/day in an eight hour infusion over 5 days in a hospital setting with adequate supportive care, six times two monthly. At the time when 70 patients had received a total of 240 courses toxicity data was available on 44 patients for a total of 170 courses. Overall, 151 sideeffects were noted. Fever occurred in 57%, pain in 34%, CRP elevation in 32%, treatment-resistant cough in 31%, hypotonia in 12%, GOT- and GPT- elevations in 8%, reduced general condition in 5%, oedema in 5%, and serum sickness in 1%. Less common side-effects were: unilateral iridoplegia in 3 patients, bilateral accomodation iridoplegia in 1 patient, stridor with allergic cough in 3 patients, without cough in 3 patients, seizures with fever in 2 patients, oxygen requirements in four patients and capillary leak syndrome in 1 patient. Conclusions from the study were not to administer the antibody during an infectious condition. The infusion time was prolonged from 8 to 12 hours. Patients with evidence of a damaged blood brain barrier, particularly after irradiation to the brain, are now excluded from ch 14.18 anti-GD2 mAb treatment (F.Berthold oral communication). 3.2.6.4 Current COG Study Based on results from a pilot study (CCG-0935A), patients currently participating in the COG study are randomised to receive cycles of Interleukin 2 alternating with GM-CSF, and the ch14.18 anti-GD2 mAb after MAT/SCT. 3.2.6.5 Chimeric antiGD2 mAb in this European Trial Based on the above experience, it is clear that the question of whether adjuvant immunotherapy with ch 14.18 anti-GD2 provides a survival benefit for treated patients needs to be addressed clearly in a large cohort. The HR-NBL1/ESIOP Study provides an ideal patient population with which to answer this question. Based on the reported survival benefit using 13-cis RA after MAT/SCT, it has been agreed that 13-cis RA should be given to all patients eligible for randomisation.76 3.2.6.6 Aspects of combining 13-cis RA and ch14.18 antiGD2 mAb The aim is to combine two independent mechanisms of anti-neuroblastoma activity realised by these two adjuvant therapies. Immunotherapy targeting GD2 using the ch14.18 anti-GD2 mAb is aimed at directing the hosts Fc-receptor positive immune cells (natural killer cells, granulocytes, macrophages) against GD2 positive neuroblastoma cells and thereby eliciting an anti-tumour effect. Treatment with 13-cis
RA is aimed at the neuroblastoma cell directly either by inducing neuroblastoma cell death via apoptotic mechanisms or via induction of neuroblastoma cell differentiation. The combination of these approaches is likely to be synergistic, since the tumour is targeted from two different angles. One is indirect via activation of the immune system, the other is direct by induction of neuroblastoma cell death. Antagonism is unlikely, since cis retinoic acid has no reported immunosuppressive activity. Furthermore the change of expression of the target antigen GD2 on neuroblastoma cells upon exposure to retinoic acid has been investigated with more reports of increases 93-95 than decreases 96 of the target antigen GD2. However, the only report on a decrease of GD2 following 13-cis RA showed that there was significant residual GD2 expression sufficient for antibody-targeting. There is no report of a complete loss of GD2 expression following 13-cis RA treatment. Based on these observations, the aim of this protocol is to address the question of whether there is a survival benefit for patients receiving ch14.18 antiGD2 mAb in addition to 13-cis RA therapy.
25
Eligibility and Patient Entry Criteria
4.1 Eligibility Criteria for the Study

Established diagnosis of neuroblastoma according to the International Neuroblastoma Staging System (INSS). Age over one year at diagnosis and below 21 years. Stage 4 neuroblastoma and all stage 2 and 3 diseases with MycN amplification Patients who have received no previous chemotherapy except the first chemotherapy cycle for localised unresectable patients. In this situation the first COJEC cycle may be replaced by the first cycle of the unresectable protocol (etoposide / carboplatin). Written informed consent, including agreement of parents or legal guardian for minors, to enter a randomised study if the criteria for randomisation are met. Tumour cell material available for determination of biological prognostic factors. Registration of all eligibility criteria with the data centre within 6 weeks from diagnosis. Provisional follow up of 5 years. National and local ethical committee approval. Any negative answer will render the patient ineligible. The date of diagnosis is defined as the date when the pathological diagnosis is obtained. It is essential that this is prior to any treatment other than surgery. The date of eligibility is defined as the date at which all criteria for entry into the study have been checked by the co-ordinating centre along with the referring physician. The date of diagnosis will be the starting point for subsequent follow up.
4.2 Eligibility Criteria for the R0 Randomisation

Participation in R0 is optional and non-participation in R0 does not exclude a patient from study entry. Study eligibility criteria fulfilled. Written informed consent to participate in R0. Randomisation prior to starting COJEC induction treatment Any negative answer will render the patient ineligible.
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Study entry criteria fulfilled. COJEC induction treatment given (strictly no anthracyclines, no investigational anti-tumour chemotherapy) Complete re-staging of disease after completion of induction therapy. CR or PR at metastatic sites after induction treatment , i.e. at least 50% reduction in skeletal mIBG positivity and not more than 3 positive, but improved spots on mIBG as well as cytomorphological CR in 2 BM aspirates. Organ functions (liver, kidney, heart, lungs, ears) fulfilling criteria prior to MAT. ALT, bilirubin < 3 x normal Creatinine clearance and/or GFR 60 ml/min/1.73m and serum creatinine < 1.5mg/dl Call study co-ordinator for MAT dose modifications if GFR < 60ml/min/1.73m and serum creatinine 1.5mg/dl. Shortening fraction 28%, or ejection fraction 55%, no clinical congestive heart failure. Normal chest X-ray and oxygen saturation. Sufficient stem cells available. Minimum required: 3 x10 6 CD34 cells /kg body weight, if a BM harvest was unavoidable at least 3 x 10 8 MNC /kg body weight. Written informed consent for R1, and for minors an agreement by parents or legal guardian.
Any negative answer will render the patient ineligible. Note: Once the patient has fulfilled the eligibility criteria, the patient should be randomised. The result of surgery will not influence randomisation. R1 is not allowed later than 120 days from the first day of chemotherapy.

Participation in R1 Complete re-staging after MAT following recovery from major transplant related toxicities No signs of disease progression at post MAT evaluation. Local radiotherapy completed. Stable WBC above 2 x 109/l (or stable neutrophil count greater than 0.5 x 109/l) in 2 counts taken 48h apart after cessation of G-CSF (filgrastim). Written informed consent to participate in R2, and for minors an agreement by parents or legal guardian. Any negative answer will render the patient ineligible. Note.
The patient should be randomised as soon as possible once restaging after MAT is completed aiming to start 13-cis RA treatment by day 80 but not later than day 120 after MAT and completed radiotherapy to the primary tumour site.
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5 Assessment of Extent of Disease, Response and Toxicity

Disease assessment will be according to the Revised International Neuroblastoma Criteria for Diagnosis, Staging and Response to Treatment published in 1993. 97(see chapter 17)
5.1 Pre-treatment Investigations

It is essential that disease evaluation is carried out prior to any therapy. If treatment is required as an emergency, pre-treatment investigations must be completed within seven days of the start of therapy. 5.1.1 FULL HISTORY WITH ATTENTION TO presence and duration of symptoms, such as pallor, sweating, weight loss, diarrhoea, irritability
CLINICAL EXAMINATION
5.1.2
Measurements of weight, height and blood pressure Note signs of spinal cord compression
5.1.3
HAEMATOLOGY Full blood count including haemoglobin, white blood cell, neutrophil, lymphocyte and platelet counts. Coagulation profile Important: 10ml EDTA (ACD) peripheral blood sample for constitutional DNA to the national biological reference laboratory (a prerequisite for any PCR studies!) SERUM Renal and liver function (Na, K, Ca, Mg, PO4, urea, creatinine, glucose, total protein, bilirubin, transaminases) Serum lactate dehydrogenase (LDH) Serum ferritin, serum NSE Pre-transfusion serum tests URINE ANALYSIS Catecholamine metabolites: Determination of vanillomandelic acid (VMA), homovanillic acid (HVA) and dopamine, expressed in relation to creatinine excretion Strip test for albumin, glucose, ketones, blood, pH prior to platinum derivatives to exclude underlying renal disease IMAGING Isotope scintigraphy preferably I-mIBG123: mIBG scan assessing the uptake on the primary tumour, the number and the location of bone metastases If negative mIBG, bone scintigraphy (bone scan) with 99mTc-hydroxy-methylenediphosphanate (MDP) scintigraphy AP chest x-ray CT or MRI scan of primary tumour (with 3D measurements) including search for dumbbell extension in relevant regions Radiological visualisation of any other evaluable disease
5.1.4
5.1.5
5.1.6
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5.1.7
BONE MARROW EVALUATION
Detailed information on handling of material is given in the Appendix on Guidelines for Bone Marrow Evaluation (chapter 18). Evaluation of the bone marrow (BM) is mandatory. Bone marrow aspirates and trephines should be obtained from right and left posterior iliac crests, i.e. a total of four samples, two aspirates and two trephines according to INSS guidelines.
BONE MARROW ASPIRATES (2 sides - left and right posterior iliac crests) On each side use 3 syringes for aspiration as given: first aspiration (native) of 0.2 to 0.5 ml BM for 10 smears per side air dried for cytology (Pappenheim stained, keep at least 5 slides unstained!) second aspiration of 5 ml BM plus 200l heparin (5000IE/ml) per side to produce from 4ml BM the mononuclear fraction for cytospins (6 to 10 wide diameter Hetich centrifuge cytospins) for immunocytology and FISH techniques (allowing investigation for MycN amplification but also other biological markers (for details see the Appendix on Biological Guidelines, chapter 18) and studies of chromosomal losses and gains by CGH [Comparative Genomic Hybridization]) and 1 ml BM processed according to a cytogenetic protocol allowing classical cytogenetic analysis third aspiration of 2-3 ml per side for RT-PCR techniques plus EDTA, ACD (1:5) or according to the requirements of the local laboratory (i.e. guanidinum thiocyanate containing tubes: for supply contact Sue Burchill (ICFR Cancer Medicine Research Unit, St. James University Hospital, Leeds) mix tubes contents and store at 80C). (for PCR based MRD studies and for the analyses of genetic markers e.g. MycN, 1p del, gene products; remember also that the established standards of the ENQUA Group include quality controls by a second method for biological markers in clinical trials). Please do not pool bone marrow aspirates! Since PB is a prerequisite for any PCR based LOH technique, there is a need to supply in addition 5 to 10ml of peripheral blood (PB) plus EDTA, ACD (1:5), or according to the requirements of the local laboratory (i.e. guanidinum thiocyanate containing tubes: for supply contact Sue Burchill (ICFR Cancer Medicine Research Unit, St. James University Hospital, Leeds), mix tubes contents and store at 80C). (Be reminded that detection of tumour typical genes in the PB at diagnosis, detected by RT-PCR, as published by S. Burchill was shown to be an important early prognostic indicator in stage 4 disease over the age of one year. This observation has potential future impact on treatment strategies if confirmed.98) 2 BONE MARROW TREPHINES (2 sides-as above) Trephine biopsies should contain at least 0.5cm of marrow (better 1cm); 1 cm of cortical bone/cartilage and 2 mm of BM is inadequate for assessment. Trephine biopsies must be obtained by an experienced operator!
* BM will be centrally reviewed Cytospins at diagnosis to establish Europe-wide standardised immunocytology including controls based on tumour cell specific biological markers and development of standardised BM response criteria. BM trephines (Europe-wide retrospectively)
5.1.8
HISTOLOGY OF PRIMARY TUMOUR
Detailed information on handling and storage of material is given in the Appendix on Pathology Guidelines (chapter 18). It is recommended that in all children material from the primary tumour, even in the presence of metastatic disease (providing this can be obtained with minimal trauma to the child) is obtained. A biopsy of the primary tumour is desirable, allowing histopathological assessment. In cases where gain of primary tumour material is considered too hazardous by virtue of the site of the tumour or the condition of the child, cytological diagnosis is accepted, provided adequate and sufficient material from heavily invaded bone marrows is obtained to enable identification of tumour cells and MycN status and other biological and genetic markers. The ENQUA group stresses the importance of collecting both tissues, BM and primary tumour. Primary tumour material may be gained with fine needle aspirates and /or true cut biopsies under imaging control (CT or sonography as appropriate).
5.1.9
BIOLOGICAL STUDIES
Rationale and handling of material is given in detail in the Appendix on Biological Guidelines (chapter 18). The strong need to study primary tumour samples, samples from second look surgery and bone marrow aspirates is stressed and advised by the ENQUA group. Only the analysis of all these samples will enable the study of metastatic processes and the study of clonal evolution during cytotoxic treatment. Besides the direct work-up of the tissue samples and BM (see Appendix, chapter 18), it is strongly advised by the ENQUA group that tumour material is snap frozen in the laboratory as soon as possible after the operation (primary tumour: -80) and that isolated BM cells are frozen also in liquid nitrogen with DMSO. In addition, laboratories undertaking PCR studies will store material in accordance with PCR techniques [molecular biology: DNA (-80), RNA(-80)]. The following studies have been agreed upon by the ENQUA group within the scope of the High-Risk NBL 1/ESIOP Study. Material has to be adequately handled and prepared in local centres and is to be transferred to the national reference laboratory for specific studies.
(National Co-ordinators: Add the address of your national reference laboratory here for clinicians in individual national centres) 5.1.9.1 First priority investigations within this protocol MycN copy number Ploidy Cytogenetics 5.1.9.2 Second priority investigations within this protocol 1p36 gain of 17q CGH (Comparative Genomic Hybridisation) to study of other chromosomal gains and losses Biology of disseminated tumour cells
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5.2 Necessary Interactions to Secure Tumour Material Flow

Clinician
Information on study, therapy, tissue
Surgeon Tumour material - every time

sterile, native, urgent
Pathologist
diagnosis splitting
tu cell cont. within 1 w.
Natl. Biology RC
native, fresh, touch slides, frozen tissue MYCN 1p36 gain 17q ploidy
Bone marrow trephine - at dx. Peripheral blood - at dx.

2-10 ml EDTA or similar send at r.t. 1 ml into GT, stored at -80C, send to RC.
Natl. Natl. Tu. bank
Bone marrow aspirates - every time

0.5 ml ---- BM smears 5 ml in Heparin 0.5 ml into GT buffer, stored at -80C, send to RC. - every time except for day 40
Cytologist
~ % tu cells 10-20x106 MNCs immediately at r.t. if no tumour: 50x106 MNCs! 2 cytospins at dx. & d 40, r.t.. CCRI Cytogenetics, N. Bown, New Castle
Co-ordinating clinician: Major co-ordinating function by phone to secure the necessary workup of tumour material by specialists, performs bone marrow aspirates and trephines, and sends out peripheral blood. Pathologist: Sectioning and securing of tumour material (touch slides, native tumour material (RPMI), freezing), establishment of histological diagnosis (on tumour and BM trephines) and of tumour cell content on material further investigated for biological profile [ENQUA guidelines] Biologist: [National Biology Reference Center] Securing, culturing, biological diagnosis [MycN copy number, ploidy, 1p36, 17q, cytogenetics, CGH, biology of disseminated tumour cells] Cytologist: BM analysis of tumour cells
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5.3
Investigations at Diagnosis, during Induction and pre-MAT Phase

Early timing of investigations is an important issue! Register the patient immediately at diagnosis and ask for R0 randomisation! (registration sheets in the Appendix section, chapter 30) At diagnosis adequate material from the primary tumour and BM for biology including 10ml peripheral blood (EDTA or ACE) is mandatory! Informed consent signature! Centres and/or departments performing imaging, PBSC harvest, surgery and MAT should be informed early! On study documentation within first 40 days! Ask for R1 Randomisation with the results of post induction investigations and after harvest of sufficient stem cells!
INVESTIGATIONS
FLOW SHEET 1
DIAGNOSIS AND INDUCTION PHASE

A 1 0 B 2 10 C 3 20 B 4 30 A 5 40 B 6 50 C 7 60 B 8 70
PRE-MAT PHASE
Prior PBSC
Restaging
Course Cycle Number Days

Height Weight Blood pressure Full blood counts
Prior Sx 90
Prior MAT 100
80
Biochemistry
renal/liver function, serum protein, Mg, Ca, Phosphate)
Serum markers
LDH, NSE Ferritin only at Dx Urine check + prox. tubular function Creatinine Clearance
GFR EDTA Urine Catecholamines 2 BM aspirates 2 BM trephines* Primary Tumour

Ultrasound Scans: CT or MRI
Scintigraphy-mIBG Biological studies

MycN, 1p-,17q+,...
Peripheral Blood Audiogram

Echocardiogram EEG Shaded squares hold data requested on the data base, others refer to surveillance of patients on study * No BM trephines at day 40 evaluation! If BM evaluation prior to PBSC harvest has to be linked with surgery due to local internal hospital organisation, steady state mobilisation after surgery should be considered. Final version of HR-NBL 1/ESIOP 14.03.06 32
5.4 Investigations after the Induction Phase until the End of Treatment
Careful timing is an important issue Contact the department of radiotherapy early to be able to start radiotherapy on time Plan R2 randomisation with results of post MAT investigations early 5.4.1 FLOW SHEET 2 FOR INVESTIGATIONS AFTER MAT AND PRIOR TO LOCAL IRRADIATION, DURING MRD TREATMENT PHASE AND END OF TREATMENT
13-cis RA Course Number Week 13-cis RA Randomised pts prior to ch14.18 anti GD2 course Weight Full blood counts Biochemistry (renal / liver function) GFR (EDTA) Urine catecholamines 2 BM aspirates + 2 BM trephines Primary Tumour: Ultrasound Scans: CT or MRI Scintigraphy- MIBG Audiogram Echocardiogram EEG Serum sample: R 2B pts
Post MAT & prior RX
1 1 3 1
2 4 7 2
3 8 11 3
4 12 15 4
5 16 19 5
6 20
End *
Shaded squares hold data requested on the data base, others refer to surveillance of patients on study * End of treatment or in case of early clinical progression
Repeat post MAT investigations if delay prior to ch 14.18 antiGD2 exceeds 2 months
5.5
5.5.1
Follow Up Investigations
TUMOUR ASSESSMENT/DETECTION In an asymptomatic patient, the following are recommended:
5.5.1.1 Imaging of the Primary Site (ultrasound or CT scan as appropriate) a) every 6 months for the first three years b) once a year up to 5 years
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5.5.1.2 Metastatic Assessment: a) If residual skeletal mIBG positive, repeat mIBG scan every 3 months until negative or progression. b) If stable over 1 year, repeat it on a yearly basis for up to 5 years. TOXICITY ASSESSMENT The toxicity assessment needs to be related to the randomised treatment received by the patient. All children require renal, audiological and fertility follow up. Those who have had extensive abdominal or pelvic radiotherapy may have prolonged thrombocytopenia. 5.5.2 5.5.2.1 Renal Follow-Up GFR assessment should be determined at the end of treatment. In children who can give a reliable 24 hour urine collection, endogenous creatinine clearance is acceptable. Where this is not possible then GFR estimation by DTPA or CrEDTA is preferred. Children who had an end of treatment GFR of less than 80ml/min/1.73m should have a repeat GFR and serum magnesium 5 years off treatment. It is known that children receiving platinum based compounds, the GFR does not decrease with time as it does after ifosfamide. However, tubular toxicity may persist or appear years after treatment. 5.5.2.2 Auditory Follow-Up Any child under the age of four during treatment should have pure tone audiometry when they have reached four years of age. Ototoxicity is usually permanent or irreversible. An adequate assessment at the end of treatment or before going to school is strongly advised. If the child has sudden severe hearing loss which is not only a high-frequency loss then serious otitis media should be excluded and the audiometry repeated after 6 months. 5.5.2.3 Cardiac Follow-Up In this protocol no anthracycline is used, thus no life-long late effects are anticipated. Cardiac follow-up as indicated for individual patients experiencing cardiac toxicity on treatment. 5.5.2.4 Etoposide: second malignancy follow up It is essential that any second malignancy occurring amongst the children treated with chemotherapy is registered urgently with the data centre. 5.5.2.5 Busulphan: Fertility In patients who have received busulphan and boys who received melphalan reduced fertility is to be expected. The results of this follow-up will be part of a long-term follow-up study. Any other toxicity in individual patients should be reported if found. (see chapter 25)
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6 Surgery
6.1 Introduction
Operation for high risk neuroblastoma is difficult and time consuming. There is some evidence that complete surgical excision does benefit the patient although it has been difficult to consider this variable separately because of the continuing development and assessment of chemotherapy variables.15;99-101 However, a recent analysis of a group of neuroblastoma patients by the Childrens Cancer Group found an association between incomplete resection of the primary tumour and relapse in that site87. Treatment included induction chemotherapy and surgery with or without local irradiation. Patients then received total body irradiation including megatherapy (MGT) and autologous bone marrow transplantation. Since then, US groups have focused on local control and have been using multimodal techniques. 86. Not withstanding the above, there are well established biological principles to support complete removal of the primary tumour, prior to MGT. The goal of induction chemotherapy is to clear metastatic disease and achieve maximum response at the primary site. In this way the number of viable cells capable of developing drug resistance is minimised. Complete surgical resection of the primary is clearly consistent with this aim. It is apparent that there is a large variation in the volume of tumour remaining after attempted excision. It is also apparent that, in some centres, almost all of the patients have almost all of their tumour excised, without unacceptable morbidity or mortality. In order to minimise the inherent variability of surgery there must be a commitment by those participating in the study to attempt complete excision. The principles of the vascular dissection required have been eloquently described.102 If a centre does not feel able to give this commitment then the patient should be transferred for surgical treatment to one which does.
6.2 Aims
(i) The aim of surgery in high risk neuroblastoma is to achieve complete excision of the tumour with minimal morbidity to improve local control. Chemotherapy is given to facilitate this. There is no place for surgery other than biopsy before induction chemotherapy, since the risks of operation are higher and the outcome is no better. (ii) This study will also collect data on the surgical procedure particularly on the completeness of excision (verification by early postoperative imaging CT/MRI- within 24h to 48h postoperatively).
6.3 Timing
Surgery should be undertaken after the end of induction chemotherapy and ideally after peripheral stem cell harvest. This will occasionally mean excising the primary tumour when metastatic disease is still present. The justification for this is that the primary will need to be excised at some point and that further metastatic response may be anticipated with additional treatment. Patients with metastatic disease achieving at least an overall partial response at metastatic sites on mIBG scanning with no detectable disease on cytomorphological evaluation of two bone marrow aspirates, may be randomised. If the primary tumour shows a partial response (PR) to induction chemotherapy, but is still judged inoperable, surgery may be further postponed.
35
6.4 Definition of Procedures

6.4.1 BIOPSY ( CHECK ALSO PATHOLOGY GUIDELINES) Biopsy should be the first procedure on all tumours. Sufficient tissue must be obtained, ideally from two different areas of the tumour, to allow histological diagnosis and biological studies. In particular, it is essential that sufficient material be obtained for the accurate determination of MycN status. Open biopsies are most satisfactory but many may feel this an unacceptable additional procedure given that the diagnosis can be made by less invasive means. Multiple needle core biopsies can provide sufficient tissue for diagnostic studies. Optimum treatment is critically dependent on correct tissue handling. It is mandatory that the pathologist is prepared to receive the samples. Fresh tissue should be delivered to the pathologist immediately under sterile conditions. Formalin or other preservatives must NOT be used. Fine needle aspiration is acceptable if there is no other alternative and are an effective source for additional tumour cells for biological studies. 6.4.2 COMPLETE EXCISION Complete excision is defined as the removal of all visible tumour, including the removal of abnormal lymph nodes and the sampling of normal lymph nodes. It is important to assess the likelihood of microscopic residual tumour even if macroscopic complete resection has been achieved. This can be aided by pathological examination of biopsies taken from the tumour bed as well as examination of the tumour margins.
6.4.3
EXCISION WITH MINIMAL RESIDUAL DISEASE (<5% of original and /or <5ml volume) Macroscopic residual disease remains after operation. The amount should be estimated by the surgeon in millilitres and as a percentage of the original tumour volume. INCOMPLETE EXCISION More than 5% or 5ml of tumour remain after attempted excision. The amount should be estimated by the surgeon in mls and as a percentage and evaluated by post-operative imaging.
6.4.4
6.5 Definition of Major Surgical Complications

Death within 30 days of operation, or obviously related to the operation at any time. Serious haemorrhage > 30% blood volume. Serious vascular injury leading to loss of tissue. Any spinal cord injury. Serious peripheral nerve injury leading to loss of function. Any organ failure. Please report any of the above complications as SAE immediately to the International Data Centre (St. Anna Childrens Hospital/CCRI, Vienna, Austria).
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6.6 Aspects of Surgical Procedures

SURGERY OF THE PRIMARY TUMOUR The aim of surgery is to remove the primary tumour completely. All suspicious tissue should be excised. Resection should be attempted after completion of induction chemotherapy, unless there is tumour progression or imaging suggests that complete excision is likely to be associated with a significant risk of death or serious mutilation, this latter situation is extremely rare. In those circumstances, the option of further chemotherapy or alternative therapy should be discussed with the national co-ordinator. Vascular encasement is not a contra-indication to surgery, indeed it is almost invariably present. LYMPH NODE EVALUATION Depending on the site of the primary tumour, lymph nodes from the following regions should be examined and removed if they appear abnormal: lateral cervical region: jugular chain and supraclavicular area; chest: mediastinal lymph nodes above and below the tumour; abdomen: coeliac nodes (infra diaphragmatic), mid-aortic (at renal level) and iliac region (bilaterally). 6.6.2 6.6.3 INTRASPINAL EXTENSION If feasible the extraspinal mass should be removed even though intraspinal disease remains. Macroscopic disease may be left in the intervertebral foramina, especially when there is a risk of leakage of spinal fluid and/or jeopardising the blood supply of the spinal cord. 6.6.4 NEPHRECTOMY Nephrectomy is not associated with a survival advantage and should be avoided if possible. Unilateral nephrectomy is acceptable if it is the only way to remove the primary tumour. In this case the surgeon should first make sure that the contralateral kidney is normal and its vessels are free from tumour. 6.6.5 6.6.1
TUMOUR INCISION After chemotherapy most tumours will be firm and compact, and spillage is therefore unlikely. Incision of the tumour is permissible if this aids excision. TUMOUR RELATION WITH GREAT VESSELS In order to gain further information on the accuracy of the pre-operative imaging, the intraoperative findings should be described in detail. Particular attention should be given to the technical difficulties encountered when the tumour is in contact with the vessels. 6.6.6
6.7 Risk Factors Related to Localisation

Data should be collected on the following for comparison with surgical complications. Neck Tumour encasing vertebral and/or carotid artery Tumour encasing brachial plexus roots Tumour crossing the midline Thorax Tumour encasing the trachea or principal bronchus Tumour encasing the origin and branches of the subclavian vessels Thoraco-abdominal tumour, peri-aortic fusiform tumour
Lower left mediastinal tumour, infiltrating the costo-vertebral junction between T9 and T12
Abdomen Adrenal tumour infiltrating the porta hepatis Suprarenal tumour infiltrating the branches of the superior mesenteric artery at the mesenteric root Suprarenal tumour surrounding the origin of the coeliac axis, and of the superior mesenteric artery Tumour invading one or both renal pedicles Fusiform tumour surrounding the infrarenal aorta Tumour encasing the iliac vessels Pelvic tumour crossing the sciatic notch
6.7.1
CLIPS Titanium or absorbable clips should be used if necessary to avoid interference with subsequent imaging.
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7 Pathology
7.1 General Remarks
The Neuroblastoma Pathology Guidelines (Appendix, chapter 18) for resectable and unresectable neuroblastic tumours have been produced for SIOP/Europe Neuroblastoma Group and were accepted as such by the board and group members as common rules and should serve as reference. The National reference pathologists will constitute a central review panel which will histologically review all tumour specimens derived from patients in this Study (responsible ESIOP NBL contact person: Klaus Beiske).
7.2 Recommendations for Handling of Tumour Material

Various biopsy techniques are applicable for collection of tissue material from unresectable tumours. TRU CUT BIOPSIES: preferably four biopsies (at least two biopsies in case of small lesions) from different areas of the tumour should be obtained. FINE NEEDLE ASPIRATIONS: at least two separate punctures/aspirations should be performed from each tumour. OPEN BIOPSY: two different areas of the tumour should be biopsied by the surgeon. Specimen size should be at least 1 cm3. The tumour material should be transferred immediately from the operation theatre to the local pathology department under sterile conditions. The splitting of the tumour material must be done by the pathologist as soon as possible after the operation. The time of splitting up the tumour should be stated. The paediatric oncologist in charge and/or the surgeon has to inform the local Pathologist and Biologist (local and/or national) in a timely manner about the new patient and the material to be expected. Further close co-operation between pathologists and biologists is strongly encouraged. Pathologists should inform the biologists if morphologically unfavourable looking areas are present in the paraffin embedded material but most likely not in the specimens selected for molecular-genetic/biologic investigations. These areas should also be specifically analysed using the paraffin material (see below).
7.3 Role of Pathologist

The handling of the tumour tissue should always be performed by the pathologist according to the Pathology Guidelines given in detail in the Appendix section (chapter 18). A copy of these guidelines should be available to all pathologists dealing with tumour material from neuroblastoma patients. Besides the important task of making morphological diagnoses and giving prognoses based on histopathological findings, another major task of the local pathologists is to choose the relevant tumour areas for molecular-genetic/biological analyses.
39
The material selected for molecular-genetic/biological investigations should be frozen as soon as possible and sent as fast as possible to the National Reference Biology Laboratory. Please refer to the Appendix (chapter 18) for details specific to your National Group. In case of queries please contact the National group co-ordinator. In all instances, tumour material from different tumour areas (nodules are of special interest!) ought to be taken for histologic and molecular-genetic/biologic examination. The reason for this recommendation is based on the observation of tumour heterogeneities at the genetic level (e.g. for the MycN and/or the chromosome 1p status) and/or at the histologic level (Ganglioneuroblastoma, nodular subtype according to the International Neuroblastoma Classification, INPC103), both of which have prognostic implications. To enable reliable interpretation of the molecular-genetic results, the exact tumour cell content of the specimen used for these investigations has to be determined. This is possible only if the pathologist evaluates the specimens adjacent to those used for moleculargenetic/biological analyses (for details see below). In case the tumour pieces selected for molecular-genetic/biologic investigations were not appropriate for getting reliable results, MycN, ploidy and chromosome 1p36.3 status can be determined on the paraffin embedded material. Laboratories which do not perform this kind of investigations have the possibility to send the paraffin blocks, ideally with H&E slides, to Drs. Peter and Inge Ambros, Vienna, Austria.
7.4 Further Important Aspects

The PATIENTS PERIPHERAL BLOOD (5-10ml plus EDTA [1:10] or ACD [1:5]) is needed for molecular-biologic studies as reference and should be sent to the reference biology laboratory together with the tumour specimens. Caution: All the recommendations for open biopsies, tru cut biopsies and fine needle aspirations (e.g. number of different tumour areas) concern only those patients for whom the risk of taking tumour material is considered to be reasonable. In some instances this risk has to weighed carefully (especially in case of patients with stage 4s neuroblastomas with coagulation disorders, but also in case of patients with large and fragile tumours). It is clear that if the procedure aiming at retrieving material carries an increased risk, it should not be performed! In this instance material should be obtained from secondaries e.g. cutaneous lesions, lymph nodes, or bone marrow if adequate, versus visceral specimen of tumour.
(National Co-ordinators: Add the address of your national reference laboratory here for clinicians in individual national centres)
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8 Biological Studies
Adequate material for biological studies is mandatory! MycN results have to reach the data centre within 10 days in patients with locoregional disease to be able to fulfil entry and eligibility criteria in case of MycN-amplification and within 6 weeks for patients with primary metastatic disease prior to the first randomisation.
8.1 Type of Biological Studies

Material has to be adequately handled and prepared in local centres and has to be transferred to the national reference laboratory for specific studies.
(National Co-ordinators: Add the address of your national reference laboratory here for clinicians in individual national centres)
8.1.1
FIRST PRIORITY INVESTIGATIONS WITHIN THIS PROTOCOL MycN copy number Ploidy Cytogenetics SECOND PRIORITY INVESTIGATIONS WITHIN THIS PROTOCOL 1p36 gain of 17q CHG (Comparative Genomic Hybridisation) for study of other chromosomal gains and losses Biology of disseminated tumour cells
8.1.2
8.2 Material for Biological Studies of Neuroblastoma

Samples should be sent to the National Reference Laboratory (list given in the Appendix section, chapter 18). 8.2.1 PRIMARY TUMOUR (MEDIUM) - Pathology, compulsory - MycN, compulsory (in RPMI) - 1p studies using FISH technique (RPMI) - Ploidy by FACS (RPMI) - Cytogenetics (RPMI) BLOOD SAMPLES - Constitutional DNA - Serum FROZEN TISSUE - Primary tumour (- 80) - Molecular biology DNA (- 80) RNA (- 80, sterile)
8.2.2
8.2.3
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8.3 Important Remarks

Analyses of MycN and tumour cell DNA content are obligatory investigations which have to be carried out in the National Reference Laboratories or other Reference Centres, i.e. laboratories participating in the European Neuroblastoma Quality Control Assessment (ENQUA) study. Results from other laboratories will not be used. The MycN copy number can be analysed by Southern blot (SB) and/or fluorescence based in situ hybridisation (FISH). Polymerase chain reaction is not recommended to be used without a second method. For SB and PCR, the tumour cell content has to be over 60 per cent. Further details and the terminology used for description of MycN results are given in the Appendix on Biological Guidelines (chapter 18). The DNA content can be determined by Flow Cytometry or Image Cytometry. The chromosome 1p36.3 status should be determined by FISH and PCR or SB. For SB and PCR, the tumour cell content has to be over 60 per cent. Further details and terminology used for description of chromosome 1p36.3 results, are given in the Appendix on Biological Guidelines (chapter 18). To enable reliable interpretation of the molecular-genetic results, the exact tumour cell content of the specimen used for these investigations has to be determined. This is possible only if the pathologist evaluates the specimens adjacent to those used for moleculargenetic/biological analyses (for details see below). An exact determination of the tumour cell content and correct tumour sampling are crucial prerequisites and absolutely necessary for obtaining reliable molecular-genetic/biological results (see chapter 18). To be eligible for the entry in the Study it is essential that the results for the MycN studies are available within 10 days in patients with locoregional disease of the tumour resection or the diagnostic biopsy (six weeks for patients with primary metastatic disease). A common terminology for reporting MycN and chromosome 1p36.3 results was developed by the ENQUA-group and is given in the Appendix on Biological Guidelines (chapter 18). For uniformity and comparability, it is strongly recommended to use these terms. A quality control study performed in 1998 made evident the pitfalls which are inherent to each method. A leaflet giving recommendations how to avoid or to reduce the most commonly observed problems and pitfalls will be circulated to all biology laboratories involved. Storage of the slides: The slides used for molecular-genetic/biological analyses and the blots should be stored according to national standards. It is recommended to store the FISH slides in the dark at 4C.
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Peripheral Stem Cell Harvest
9.1 Introduction
The aim of the PSC Harvest is to collect a sufficient number of blood progenitors in order to allow safe and prompt haematological recovery following the high dose chemotherapy (HDC) regimen and autograft. The target dose of collected CD34+ cells is 3 x 106 CD34 positive cells/kg for one MAT procedure like the BUMEL or CEM regimen used in this trial. In case of a circulating level still higher than 20 CD34 positive cells, a second collection time is recommended, regardless of the amount of first apheresis. Each collection should always be divided into two bags. If less than 3 x 106 CD34 /kg is obtained discuss with study co-ordinator. There is no ideal timing for PBSC harvest in patients with metastatic neuroblastoma, except that early collection increases the risk of tumour cell contamination and later ones of poor progenitor cell collection. The monitoring of both tumour cell contamination and progenitors in the graft using standard procedures will enable us to better define the impact of possible tumour cell contamination on EFS. It is recommended that all procedures should be carried out in institutions with particular expertise in stem cell harvest in small children.
9.2 Timing of Peripheral Stem Cell Harvest

The disease response status should be evaluated before performing the PBSC harvest procedure. The bone marrow staging on two aspirates should be cytomorphologically negative and skeletal response meet recommended criteria (partial remission according to INSS criteria). If the patient fulfils the response eligibility criteria for the R1 randomisation at least 3 x 106 CD34 + cells/kg BW should be harvested. There are various time points for stem cell collection, the decision about which ones to use is left to the discretion of centre, since this is closely related to individual local organisation . It is recommended to start early with harvest efforts. The first harvest option is after the end of induction, following recovery from aplasia after day 70 of COJEC chemotherapy. SUMMARY OF HARVEST OPTIONS Mobilisation with 5g/kg BW g-CSF (Filgrastim) s.c. after day 70 of COJEC Steady state mobilisation after COJEC but prior to surgery Steady state mobilisation after surgery Consider BM-Harvest No stem cells by day 120: Ineligible for R1. If the patient has to go off study because of an unsatisfactory response at this particular time point a different harvest procedure will be followed according to the type of phase II study he/she enters. Therefore both the mIBG and bone marrow response should be known before starting the first leukapheresis.
9.3 Recommended Procedure for PBSC Mobilisation and Day of Collection

There are at least two options for stem cell mobilisation : a) The use of G-CSF (filgrastim) 5 g/kg/day following the last cycle of chemotherapy, i.e. day 72, with monitoring of the presence of peripheral blood CD34+ cells until a sufficient number is reached (>20 CD34+ cells/microlitre are required to start the first leukapheresis). b) Steady state mobilisation after haematological recovery following the last course of chemotherapy (Steady state defined as >1 neutrophils and >100 platelets x 109/l giving GCSF (filgrastim) 10 g/kg/day for 5 days by subcutaneous injection. Collection should be started ideally on the 4th or 5th day.
The symptoms of fever and bone pain that may occur with G-CSF (filgrastim) administration can be reduced by regular paracetamol (24 mg/kg qds), but be aware of the possibility of masking fever due to infection. The number of collections depends on the quantity of peripheral blood stem cells harvested, evaluated by the number of CD34 positive cells. If the sample is insufficient, it can be repeated following surgery using the steady state method with G-CSF (filgrastim) at a dose of 10g/kg/day).
9.4 Vascular Access

PBSC may be collected using a central Broviac or Hickman line together with a large peripheral vein. Most patients will have a Broviac or Hickman catheter as central venous line for treatment and blood samples during the induction therapy. This line should be used as the inlet line in the leukapheresis procedure. As return line, i.e. a 22 20 G peripheral venous access (PVA) should be established for the procedure. Otherwise PVA (> 20 G) can be used as inlet line in combination with a second PVA (22 20 G). When necessary a central venous line should be established before leukapheresis according to the in- house standard techniques, i.e. a double lumen central venous catheter specifically designed for apheresis may be placed for the procedure. It is anticipated that most neuroblastoma patients will require placement of a temporary or tunnelled apheresis catheter.
9.5 Standard Procedure

Leukapheresis will be performed according to current recommended practice. However, the procedure should be performed by an operator who is experienced in paediatric procedures. In small children, i.e. less than 20 or 15kg, the extracorporeal volume of the machine used has to be considered. The priming procedure should be performed with human albumin 5% instead of saline solution and a further priming step with packed red blood cells, irradiated and leukocyte depleted, should be carried out. In children weighing less than 10 kg, it is recommended that the cell separator is primed with irradiated, white cell depleted, packed red cells resuspended in 5% albumin and diluted with saline to match the patient's haematocrit. Once an adequate harvest is obtained the cells should be frozen in at least two separate bags, pending use after BUMEL or CEM. After leukapheresis a sample of each buffy coat should be analysed for CD34+ cell content and tumour cell contamination.
9.6 Freezing Procedure and Storage

The buffy coat should be frozen after mixing with DMSO (concentration after mixing 10%) in a rate controlled freezer. Samples should be stored in the liquid phase of nitrogen. Two small samples (i.e. 1 ml) should also be frozen and stored under the same conditions for backup examinations and quality controls.
9.7 Purging of PBSC Harvests

No in vitro purging of stem cells is envisaged. It is strongly recommended that stem cell harvest and MAT/PSCR are carried out in institutions with particular expertise in this procedure. Consideration for referral to such an institution is advised and should be planned early.
44
10 Chemotherapy Regimen Details

Any organ dysfunction which could lead to an important alteration of drug elimination should be discussed with the national trial co-ordinator.
10.1 COJEC Induction

This induction treatment will be applied over ten weeks and proceed regardless of neutrophil or platelet counts and controlled infection. Three different courses are given every 10 days. Consult study co-ordinators if glomerular filtration rate < 80 ml/min/1.73m. COURSE A starts on days 0 and 40, COURSE B on days 10, 30, 50 and 70 and COURSE C on days 20 and 60. COURSE A consists of vincristine 1.5 mg/m (maximum dose 2 mg), carboplatin 750 mg/m and etoposide 2 x 175mg/m. COURSE B uses vincristine 1.5 mg/m and cisplatin 80 mg/m/ctn over 24 hours. COURSE C consists of vincristine 1.5 mg/m (maximum dose 2 mg), etoposide 2 x 175mg/m and cyclophosphamide 2 x 1050mg/m. Dose /day R0G-CSF CBDCA Vp16 VCR CDDP CYC DAY COURSE CYCLE HARVEST SURGERY STAGING Primary site MRI or CT, ultrasound, mIBG, Bone Marrow: 2 aspirates/ 2 biopsies Bone Marrow: 2 aspirates, primary site ultrasound Primary site: control of surgical success (method optional ultrasound, MRI, CT: 2 weeks after surgery ; if positive repeat it but not prior to day 60 post MAT) - Disease assessment according to the International Neuroblastoma Staging and Response Criteria. The following guidelines are aimed at enabling the prescription of the chemotherapy to be easier. Infusion rates are slightly higher than required to infuse the chemotherapy over the stated time due to the fact that there will invariably be interruptions in the infusion. 5g/kg 750mg/m 175mg/m 1.5mg/m 80mg/m 1050mg/m 0 days days days days days days days days
3-8 12-18 23-28 32- 38 43- 48 52-58 63-68 72 till harvest
ctn
ctn
ctn
ctn
10
20
30
40
50
60
70
80
90
100 110
A
1
B
2
C
3
B
4
A
5
B
6
C
7
B
8
H H Sx H
R1 MAT
45
COURSE A START ON DAY 0 AND 40

DAYS of course A VINCRISTINE CARBOPLATIN ETOPOSIDE DRUG VINCRISTINE CARBOPLATIN Time 0 hrs 0 hrs 1st 2nd
4h
4h
Administration
(maximum dose 2 mg) as a single iv bolus
Dose(mg/m) 1.5 mg/m 750 mg/m
standard size bags infused over 1 hour 1 - 250mg of carboplatin in 50 mls of 5% dextrose >250 - 500mg of carboplatin in 100 mls of 5% dextrose >500 -1000mg of carboplatin in 250 mls of 5% dextrose standard size bags infused over 4 hours 0 - 40mg of etoposide in 100 mls of 0.9% saline >40 - 50mg of etoposide in 150 mls of 0.9% saline >50 - 100mg of etoposide in 250 mls of 0.9% saline >100 - 200mg of etoposide in 500 mls of 0.9% saline >200 - 300mg of etoposide in 750 mls of 0.9% saline >300 - 600mg of etoposide in 1000 mls of 0.9% saline
ETOPOSIDE
1 hrs
175 mg/m
0.5 VINCRISTINE
0.75
0.6
0.9
0.7
1.05
0.8
1.2
0.9
1.35
SURFACE AREA SQUARE METRES 1.0 1.1 1.2 1.3 1.4 1.5 1.6
1.5 1.65 1.8 1.95 2
1.7
1.8
1.9
2.0
m
mg
2MG DOSE MAXIMUM
0.5 CARBOPLATIN
Reconstituted Carboplatin solution 10mg/ml Glucose 5% injection Infusion rate ml/h 375 37.5 12 50
0.6
450 45 15 60
0.7
525 52.5 18 70
0.8
600 60 20 80
0.9
675 67.5 22 90
1.0
750 75 25 100
SURFACE AREA SQUARE METRES 1.1 1.2 1.3 1.4 1.5 1.6
825 82.5 28 110 900 90 30 120 975 97.5 32 130 1050 105 35 140 1125 1200 112.5 120 38 150 40 160
1.7
1275
1.8
1350
1.9
1425
2.0
1500
m mg ml ml ml/h
127.5 135 42 170 45 180
142.5 150 48 190 50 200
0.5
ETOPOSIDE (VP16) Sodium Chloride 0.9% injection Infusion rate ml/h 88 250 65
0.6
106 300 75
0.7
122 350 90
0.8
140 400 100
0.9
158 450 115
1.0
175 500 130
192 550 140 210 600 155 228 650 165 246 700 180 262 750 190 280 800 205
1.7
298 850 220
1.8
316 900 230
1.9
332 950 245
2.0
350 1000 255
m
mg
ml ml/h
Patients participating in Randomisation R0

Patient randomised to receive G-CSF (filgrastim) during COJEC induction will be given 5g/kg/day s.c. in between Courses A and B starting 24h after last chemotherapy, and to be stopped the day prior to commencing the next Course with an interval of at least 24 hours between the last G-CSF (filgrastim) injection and the start of chemotherapy, i.e. days 3 to 8 inclusive, and days 43 to 48 inclusive. Physicians are discouraged from using G-CSF (filgrastim) during induction outside the controlled study except for life threatening infections.
46
COURSE B START ON DAYS 10 - 30 - 50 - 70

DAY VINCRISTINE CISPLATIN DRUG VINCRISTINE Time 0 hrs 0 hrs Dose(mg/m) 1.5 mg/m 1
24h
Administration (maximum dose 2 mg) iv bolus pre-hydration for cisplatin
infused at 200 ml/m/hr for 3 hours 0.9% sodium chloride with 20 mmol/l potassium chloride
Mannitol 20% 0 hrs Mannitol 20% 2.5 hrs CISPLATIN 3 hrs
short infusion in a dose of 40ml/m short infusion in a dose of 40ml/m 80 mg/m/24 hours in a mini-bag alongside the hydration for 24h
1- 50mg cisplatin in 100mls of 0.9% sodium chloride > 50-100mg cisplatin in 150mls of 0.9% sodium chloride >100-200mg cisplatin in 200mls of 0.9% sodium chloride
Commence hydration in parallel with cisplatin

1.5 l /m/24 hrs of 0.9% sodium chloride 1.5 l/m/24 hrs of 5% glucose 60 mmol/m/ 24 hrs of potassium chloride 2.5 mmol/ m/24 hrs of calcium gluconate 10 mmol /m/24hrs of magnesium sulphate
Mannitol 20% 9 hrs 27 hrs
if diuresis falls below 400 ml/m/6 hours, short infusion in a dose of 40ml/m, repeat thereafter whenever indicated
Commence post cisplatin hydration

1.5 l/m/ 24 hours of 0.9% sodium chloride 1.5 l/m/24 hours of 5% glucose 60 mmol/ m/24 hours of potassium chloride 2.5 mmol/ m calcium gluconate 10 mmol /m/24hrs of magnesium sulphate
51 hrs
Complete therapy
During prehydration, the cisplatin infusion together with its parallel hydration and postcisplatin hydration, a careful record of fluid input and output should be kept to prevent hydration overload and ensure diuresis. Cisplatin is given as a continuous infusion over 24h combined with forced mannitol diuresis: Mannitol 20% in a dose of 40ml/m should be administered 3 hours and 30 minutes prior to starting cisplatin and thereafter if diuresis falls below 400 ml/m/6 hours (during the cisplatin infusion furosemide should be avoided because of its enhancing effect on ototoxicity). The addition of magnesium during cisplatin treatment is recommended at a daily dose of 180mg/m/day during the induction period but may need to be adjusted following strict monitoring of Mg levels. Mannitol and magnesium are not to be given con-currently as mannitol
47
and magnesium are not compatible. The addition of calcium, potassium and phosphate may also be modified according to serum levels.

Patients randomised to receive G-CSF (filgrastim) during COJEC induction will be given 5g/kg/day s.c. in between Courses A, B and C starting 24h after the last chemotherapy and stopping the day prior to commencing the next Course with an interval of at least 24 hours between the last G-CSF (filgrastim) injection and the start of chemotherapy. Physicians are discouraged from using G-CSF (filgrastim) during the induction outside the controlled study except for life threatening infections. Vincristine dosage 1.5mg/m (to a maximum of 2mg) as a single bolus
0.5 VINCRISTINE
0.75
0.6
0.9
0.7
1.05
0.8
1.2
0.9
1.35
1.5 1.65 1.8 1.95 2
1.7
1.8
1.9
2.0
m
mg
2MG DOSE MAXIMUM
Pre-hydration scheme day one, to be infused over 3 hours

Pre-hydration fluid: Sodium chloride injection B.P. 0.9% Strong potassium chloride solution B.P. (73) 2mmol/ml 500ml 10ml SURFACE AREA SQUARE METRES 1.1 1.2 1.3 1.4 1.5 1.6
220 240 260 280 300 320
0.5
Infusion rate ml/h 100
0.6
120
0.7
140
0.8
160
0.9
180
1.0
200
1.7
340
1.8
360
1.9
380
2.0
400
m ml/h
DAY1 (VERSION WITH CISPLATIN IN THE 24H INFUSION): Hydration scheme for cisplatin infusion to be infused over 24 hours day 1
0.5 CISPLATIN
Sodium chloride injection B.P. 0.9% Glucose 5% injection Strong potassium chloride solution B.P. (73) 2mmol per ml Magnesium sulphate injection 50% 4.15mmol in 2ml Calcium Gluconate injection B.P. 10% 2.2mmol in 10ml Infusion rate ml/h 40 750 750 15
0.6
48 850 850 18
0.7
56 1000 1000 21
0.8
64 1200 1200 24
0.9
72 1350 1350 27
1.0
80
88 1600 1600 33 96 1700 1700 36 104 2000 2000 39 112 2000 2000 42 120 2250 2250 45 128 2400 2400 48
1.7
136 2500 2500 51
1.8
144 2700 2700 54
1.9
152 2850 2850 57
2.0
160 3000 3000 60
1500 1500 30
m mg ml ml ml ml
2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5
10
10
11
12
13
14
15
16
17
18
19
20
ml
75 90 100 120 140 150 170 180 210 210 230 250 260 280 300 300
ml/h
DAY 2 Hydration scheme for cisplatin infusion to be infused over 24 hours day 1
0.5
Sodium chloride injection B.P. 0.9% Glucose 5% injection Strong potassium chloride solution B.P. (73) 2mmol per ml Magnesium sulphate injection 50% 4.15mmol in 2ml Calcium Gluconate injection B.P. 10% 2.2mmol in 10ml Infusion rate ml/h 750 750 15
0.6
850 850 18
0.7
1000 1000 21
0.8
1200 1200 24
0.9
1350 1350 27
1.0
1600 1600 33 1700 1700 36 2000 2000 39 2000 2000 42 2250 2250 45 2400 2400 48
1.7
2500 2500 51
1.8
2700 2700 54
1.9
2850 2850 57
2.0
3000 3000 60
1500 1500 30
m ml ml ml ml
2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5
10
10
11
12
13
14
15
16
17
18
19
20
ml
75 90 100 120 140 150 170 180 210 210 230 250 260 280 300 300
ml/h
48
COURSE C START ON DAYS 20 - 60

DAY VINCRISTINE CYCLOPHOSPHAMIDE ETOPOSIDE Time 0 hrs 0 hrs 1st 2nd
4h
Administration
4h
DRUG Vincristine Etoposide
Dose(mg/kg) 1.5 mg/m 175 mg/m
(maximum dose 2 mg) iv bolus

standard size bags infused over 4 hours 0 - 40mg of etoposide in 100 mls of 0.9% saline >40 - 50mg of etoposide in 150 mls of 0.9% saline >50 - 100mg of etoposide in 250 mls of 0.9% saline >100 - 200mg of etoposide in 500 mls of 0.9% saline >200 - 300mg of etoposide in 750 mls of 0.9% saline >300 - 600mg of etoposide in 1000 mls of 0.9% saline i.v. bolus as iv bolus followed by post-cyclophosphamide infusion for 24 hours with : 1.2 g / m / 24 hrs mesna 1.5 l /m / 24 hrs of 0.9% sodium chloride 1.5 l /m / 24 hrs 5% glucose 60 mmol/m/24 hours of potassium chloride standard size bags infused over 4 hours as above as iv bolus followed by post-cyclophosphamide infusion for 24 hours with : 1.2 g / m / 24 hrs mesna 1.5 l /m / 24 hrs of 0.9% sodium chloride 1.5 l /m / 24 hrs 5% glucose 60 mmol/m/24 hours of potassium chloride
Mesna Bolus 4hrs Cyclo4 hrs phosphamide
200mg/m 1.05 gm/m
Etoposide
28 hrs
175 mg/m 1.05 gm/m
Cyclo32 hrs phosphamide
56 hrs
Complete therapy
During post-cyclophosphamide infusion a careful record of fluid input and output should be kept to prevent fluid overload and ensure an adequate diuresis. If diuresis falls less than 400 ml/m/6 hours, furosemide 0.5 1.0 mg/kg should be given.
49
Vincristine dosage 1.5mg/m (to a maximum of 2mg) as a single bolus

1.5 1.65 1.8 1.95 2
0.5 VINCRISTINE
0.75
0.6
0.9
0.7
1.05
0.8
1.2
0.9
1.35
1.6
1.7
1.8
1.9
2.0
m
mg
2MG DOSE MAXIMUM
Etoposide 175mg/m in sodium chloride 0.9% injection (infused over 4 hours)

0.5
ETOPOSIDE (VP16) Sodium Chloride 0.9% injection Infusion rate ml/h 88 250 65
0.6
106 300 75
0.7
122 350 90
0.8
140 400 100
0.9
158 450 115
175 500 130 192 550 140 210 600 155 228 650 165 246 700 180 262 750 190 280 800 205
1.7
298 850 220
1.8
316 900 230
1.9
332 950 245
2.0
350 1000 255
m
mg
ml ml/ h
Cyclophosphamide 1050mg/m as a slow bolus

0.5 CYCLOPHOSPHAMIDE AS A SLOW BOLUS 525
0.6
630
0.7
735
0.8
840
0.9
945
1050 1155 1260 1365 1470 1575 1680
1.7
1785
1.8
1890
1.9
1995
2.0
2100
m mg
Scheme for post-cyclophosphamide mesna rescue

0.5 Glucose 5% Injection
Sodium Chloride Injection B.P. 0.9% Strong potassium chloride solution B.P. (73) 2mmol/ml Mesna (g) Infusion Rate ml/hr 750 750 15
0.6
850 850 18
0.7
1000 1000 21
0.8
1200 1200 24
0.9
1350 1350 27
1500 1500 30 1600 1600 33 1700 1700 36 2000 2000 39 2000 2000 42 2250 2250 45 2400 2400 48
1.7
2500 2500 51
1.8
2700 2700 54
1.9
2850 2850 57
2.0
3000 3000 60
m mg ml ml
0.5 75
0.6 90
0.7 100
0.8 120
0.9 140
1.0 150
1.1 170
1.2 180
1.3 210
1.4 210
1.5 230
1.6 250
1.7 260
1.8 280
1.9 300
2.0 300
ml/ h

Patients randomised to receive G-CSF (filgrastim) during COJEC induction will be given 5g/kg/day s.c. in between Courses A, B and C starting 24h after the last chemotherapy and stopping the day prior to commencing the next Course with an interval of at least 24 hours between the last G-CSF (filgrastim) injection and the start of chemotherapy. G-CSF (filgrastim) is never to be administered on the days of chemotherapy. Physicians are discouraged from using G-CSF (filgrastim) during the induction outside the controlled study except for life threatening infections. After the last Course B G-CSF (filgrastim) is to be given to all patients and continued until the criteria to start the peripheral stem cell harvest procedure are fulfilled.
50
10.2 Check List prior to MAT

Investigations necessary prior to MAT are outlined in Section 5.3. ELIGIBILTY FOR MAT Study entry criteria fulfilled. COJEC induction treatment given (strictly no anthracyclines, no investigational anti-tumour chemotherapy) Complete re-staging of disease after completion of induction therapy. CR or PR at metastatic sites after induction treatment, i.e. at least 50% reduction in skeletal mIBG positivity and not more than 3 positive, but improved spots on mIBG as well as cytomorphological CR in 2 BM aspirates. Organ functions (liver, kidney, heart, lungs, ears) fulfilling criteria prior to MAT. ALT, bilirubin < 3 x normal Creatinine clearance and/or GFR 60 ml/min/1.73m and serum creatinine < 1.5mg/dl. Call study co-ordinator for MAT dose modifications if GFR < 60 ml/min/1.73m and serum creatinine 1.5mg/dl. Shortening fraction 28%, or ejection fraction 55%, no clinical congestive heart failure. Normal chest X-ray and oxygen saturation. Patients of childbearing potential must practice an effective method of birth control while participating in this study. Sufficient stem cells available. Minimum required: 3 x10 6 CD34 cells /kg body weight, if a BM harvest was unavoidable at least 3 x 10 8 MNC /kg body weight. Written informed consent for R1, and for minors agreement by parents or legal guardian. Randomisation done
Any negative answer will render the patient ineligible. INELIGIBILITY FOR MAT Patients with neoplastic or non-neoplastic disease of any major organ system that would compromise their ability to withstand the pre-transplant conditioning regimen. This will include: Patients with a response less than PR following induction therapy. Patients with a mixed response, stable disease or persistent tumour in the bone marrow by cytomorphologic evaluation come off protocol therapy for entry on to available Phase I and Phase II studies. Patients with uncontrolled (culture or biopsy positive) infections requiring intravenous antivirals, antibiotics, or antifungals. Patients on prolonged antifungal therapy are still eligible if they are culture negative and biopsy negative in residual radiographic lesions, and they meet the other organ function criteria. Patients who are pregnant or lactating. HIV seropositive patients.
51
10.3 Monitoring for Ototoxicity

In children over the age of three pure-tone audiometry should be carried out as near to the beginning of treatment as possible, preferably before starting cisplatin. This should be classified according to the Brock criteria.104 In children under the age of three an experienced audiologist should be able to get good results using distraction techniques if the child is not in pain. The results from distraction techniques may be graded according to the Brock criteria. Table: Brock Grading System for Cisplatin-Induced Bilateral High-Frequency Hearing Loss104
BILATERAL HEARING LOSS 1 < 40 dB at all frequencies > 40 dB at 8,000 Hz only > 40 dB at 4,000 Hz and above > 40 dB at 2,000 Hz and above > 40 dB at 1,000 Hz and above
GRADE
DESIGNATION None Mild Moderate Marked Severe
0 1 2 3 4
Notes 1 The results are obtained by pure-tone audiometry, from the "better" ear. 2 < 40 dB at lower frequencies
52
10.4 Monitoring of Renal Function

10.4.1 EVALUATION OF RENAL FUNCTION Renal function should be evaluated at the time of diagnosis and prior to MAT. It should be assessed by the glomerular filtration rate and serum magnesium. The GFR should be measured by a radio-isotope method preferably with Cr51 EDTA otherwise DPTA clearance. The GFR needs to be adjusted for surface area giving a result in ml/min./1.73m. The same method should be used in the same child throughout the treatment. 10.4.2 BACKGROUND OF CARBOPLATIN DOSING The antitumour and toxic effects of carboplatin have been related to its pharmacokinetics in several studies in both children and adults.105-108 This has led to the concept of basing carboplatin dosing on a target area under the plasma concentration-time curve (AUC) and the development and validation of dosing formulae based on renal function in adult and paediatric patient populations.109-111 Renal function based dosing is derived from the observation that the clearance of carboplatin is almost exclusively (up to 80%) via the renal route, with the remainder being due to irreversible binding to plasma/cellular macromolecules. Furthermore the mechanism of carboplatin clearance is glomerular filtration and a formulary, that predicts the absolute dose of carboplatin required to achieve a target AUC, has been developed. Pharmacokinetic studies in children have also shown a large degree of interpatient variability in the AUC achieved in children for a given surface area-based dose. In a prospective, randomised cross-over study carboplatin dosing based on renal function resulted in more reproducible and reliable drug exposure (as determined by AUC), than dosing based on surface area.112;113 The mean observed AUCs after renal function and surface area based dosing were 98% and 95% of the target AUCs, respectively. The variation in the observed AUC was significantly less after renal function based dosing (F test, p=0.02), such that 74% of the courses had an AUC within +/- 20% of the target value, versus 49% for courses after dosing according to the body surface area. The development of limited sampling strategies allows the daily monitoring of carboplatin AUC with minimum patient inconvenience when the drug is administered over a number of days. 114;115 Real-time pharmacokinetic monitoring of carboplatin is currently used to guide dosing in both the SIOP study for the treatment of poor prognosis patients with Stage IV soft tissue sarcoma, and the UKCCSG CNS 2000-01 study for the treatment of recurrent CNS primitive neuroectodermal tumours in children. This approach involves limited sampling on the first day of carboplatin treatment. The clearance and estimated AUC of the drug on day 1 are then determined from carboplatin ultrafiltrate (free platinum) samples using a two compartment model and Bayesian analysis based on a previous data set of patients who received similar treatment. Dosing on day 2 can then be adjusted to achieve the desired target AUC and monitoring continued on a daily basis throughout the course of treatment. Results from initial studies, with patients studied at a number of different centres in the UK, show the feasibility of real-time monitoring of carboplatin pharmacokinetics with adaptive dosing.116 In those patients where dose adjustment was considered clinically appropriate, pharmacokineticallyguided dosing resulted in a total AUC within 2% of the target, suggesting that carboplatin exposure can be optimised in individual patients. Therefore in this protocol carboplatin dosing is GFR based using the Newell formula105 for all patients with a target AUC of 4.1 mg/ml.min/day. GFR based dosing for all patients will limit the variation in AUC values predicted based on the current carboplatin dosing schedule. In addition there is a potential for pharmacokinetic monitoring with dose adjustments on days 2-4 based on day 1 sampling data. Real-time pharmacokinetic monitoring of carboplatin
would ideally be carried out in all patients with limited sampling carried out on day 1 of dosing and the dose adjusted for days 2-4 to achieve an exposure as close as possible to the desired target AUC of 16.4 mg/ml.min (4.1 mg/ml.min/day) and may be done in centres with required established know-how.
10.4.3 CALCULATION OF THE CARBOPLATIN DOSE FOR CEM-MAT Dosing must be based on renal function, with a target AUC of 4.1 mg/ml.min, for all patients on the CEM regimen. The two problem areas regarding this would appear to be a) the use of complex equations and potential for dosing errors, and b) the lack of a common method for evaluating renal function throughout Europe. A two pronged approach will be used to solve these problems: a) The use of tables for determining the daily dose of carboplatin based on a measure of GFR and body weight will allow renal function-based dosing to be used without the need for complex equations. This approach has been successfully used in a number of UKCCSG protocols for optimising carboplatin dosing and works well. b) For those centres with the facility to measure 51Cr-EDTA clearance in patients, the 51Cr-EDTA half-life (t) should ideally be used to determine carboplatin dosing using the Newell formula (Newell et al. J. Clin. Oncol., 11: 2314-23; 1993) (Method 1). For centres using other methods of GFR determination, the GFR value can be used to calculate the carboplatin dose using a modified version of the Calvert formula (Method 2).
Method 1. If 51Cr-EDTA clearance is used for determination of GFR, the dose of carboplatin can be calculated from the 51Cr-EDTA half-life (t) value and patients body weight using the tables as provided below . These tables give the daily dose of carboplatin calculated to give an AUC of 4.1 mg/ml.min by the Newell formula.111 Method 2. For any other method of GFR determination, the dose of carboplatin can be calculated from the uncorrected GFR (ml/min) value and patients body weight using the tables as provided below. These tables give the daily dose of carboplatin calculated to give an AUC of 4.1 mg/ml.min by the modified version of the Calvert formula.109 It is strongly discouraged to use creatinine clearance.
Below two sets of tables are included with 4 tables for each method to cover the range of body weights and GFR values expected within the patient population. To avoid overdosing in patinets with normal renal function, i.e. GFR>100ml/min/1.73m, the upper dose limit is a CBDCA dose of 425 mg/m.
54
METHOD 1
AUC 4.1 mg/ml.min Half life EDTA (min) 70 75 80 82 89 97 104 111 118 125 132 139 146 153 167 181 194 207 221 234 247 260 273 286 299 312 325 337 350 363 375 388 400 413 425 437 450 462 474 77 84 91 98 104 111 118 124 131 137 144 157 170 182 195 208 220 232 245 257 269 281 293 305 317 329 341 353 365 376 388 400 411 423 435 446 73 79 86 92 98 105 111 117 124 130 136 148 160 172 184 196 208 219 231 243 254 266 277 288 300 311 322 333 344 356 367 378 389 400 411 422
Body Wt (kg) 5 5,5 6 6,5 7 7,5 8 8,5 9 9,5 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
30 182 198 214 230 245 261 277 292 307 323 338 368 398 427 457 486 515 543 572 600 629 657 685 712 740 768 795 823 850 877 904 931 958 985 1011 1038
35 157 171 184 198 212 225 239 252 265 279 292 318 343 369 394 419 444 469 494 519 543 567 591 615 639 663 687 711 734 758 781 804 828 851 874 897
40 138 150 163 175 187 199 210 222 234 245 257 280 303 325 348 370 392 414 436 457 479 500 522 543 564 585 606 627 648 668 689 710 730 751 771 791
45 124 135 145 156 167 178 188 199 209 220 230 251 271 291 311 331 351 371 390 410 429 448 467 486 505 524 543 562 580 599 617 636 654 673 691 709
50 112 122 132 142 151 161 171 180 190 199 209 227 246 264 282 300 318 336 354 371 389 406 424 441 458 475 492 509 526 543 560 577 594 610 627 643
55 102 112 121 130 139 147 156 165 174 182 191 208 225 242 258 275 291 308 324 340 356 372 388 404 420 436 451 467 482 498 513 529 544 559 574 590
60 94 103 111 120 128 136 144 152 160 168 176 192 208 223 239 254 269 284 299 314 329 344 359 373 388 402 417 431 446 460 474 488 503 517 531 545
65 88 96 103 111 119 126 134 142 149 156 164 179 193 208 222 236 250 264 278 292 306 320 333 347 361 374 388 401 414 428 441 454 467 481 494 507
85 69 75 81 87 93 99 105 111 117 123 129 140 152 163 175 186 197 208 219 230 241 252 263 273 284 295 305 316 327 337 348 358 369 379 389 400
90 65 71 77 83 89 94 100 106 111 117 122 133 144 155 166 177 187 198 208 219 229 240 250 260 270 280 291 301 311 321 331 341 351 361 371 380
95 62 68 74 79 85 90 95 101 106 111 117 127 138 148 158 169 179 189 199 209 219 229 238 248 258 268 277 287 297 306 316 325 335 344 354 363
100 60 65 70 76 81 86 91 96 102 107 112 122 132 142 151 161 171 181 190 200 209 219 228 237 247 256 265 275 284 293 302 311 320 329 339 348
105 57 62 67 72 77 82 87 92 97 102 107 117 126 136 145 155 164 173 182 192 201 210 219 228 237 246 255 263 272 281 290 299 307 316 325 333
110 55 60 65 70 74 79 84 89 93 98 103 112 121 130 140 149 158 166 175 184 193 202 210 219 228 236 245 253 262 270 279 287 296 304 312 321
115 53 58 62 67 72 76 81 85 90 95 99 108 117 126 134 143 152 160 169 177 186 194 203 211 219 228 236 244 252 260 269 277 285 293 301 309
120 51 56 60 65 69 74 78 82 87 91 96 104 113 121 130 138 146 155 163 171 179 187 196 204 212 220 228 236 243 251 259 267 275 283 290 298
55
METHOD 1
Body Wt (kg) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
30 1038 1064 1091 1117 1144 1170 1196 1222 1248 1274 1300 1326 1352 1378 1403 1429 1455 1480 1506 1531 1557 1582 1607 1632 1658 1683 1708 1733 1758 1783 1808 1833 1858 1883 1908 1932
35 897 920 943 966 988 1011 1034 1056 1079 1102 1124 1146 1169 1191 1213 1235 1258 1280 1302 1324 1346 1368 1390 1411 1433 1455 1477 1499 1520 1542 1563 1585 1607 1628 1650 1671
40 791 812 832 852 872 892 912 932 952 972 992 1012 1031 1051 1071 1090 1110 1129 1149 1168 1188 1207 1226 1246 1265 1284 1303 1323 1342 1361 1380 1399 1418 1437 1456 1475
45 709 727 745 764 782 800 818 835 853 871 889 907 924 942 960 977 995 1012 1030 1047 1065 1082 1099 1117 1134 1151 1169 1186 1203 1220 1237 1254 1272 1289 1306 1323
50 643 660 676 693 709 726 742 758 774 791 807 823 839 855 871 887 903 919 935 951 966 982 998 1014 1029 1045 1061 1076 1092 1108 1123 1139 1154 1170 1185 1201
55 590 605 620 635 650 665 680 695 710 725 739 754 769 784 798 813 828 842 857 871 886 900 915 929 944 958 973 987 1001 1016 1030 1044 1058 1073 1087 1101
60 545 559 573 587 601 614 628 642 656 670 683 697 711 724 738 751 765 778 792 805 819 832 846 859 872 886 899 912 926 939 952 965 978 992 1005 1018
65 507 520 533 546 559 572 585 597 610 623 636 649 661 674 687 699 712 724 737 750 762 775 787 800 812 824 837 849 861 874 886 898 911 923 935 947
85 400 410 420 431 441 451 461 471 482 492 502 512 522 532 542 552 562 572 582 592 602 612 622 632 641 651 661 671 681 690 700 710 720 729 739 749
90 380 390 400 410 420 429 439 449 458 468 478 487 497 506 516 526 535 545 554 564 573 582 592 601 611 620 629 639 648 657 667 676 685 694 704 713
95 363 373 382 391 401 410 419 428 438 447 456 465 474 484 493 502 511 520 529 538 547 556 565 574 583 592 601 610 619 628 637 645 654 663 672 681
100 348 357 366 374 383 392 401 410 419 428 437 445 454 463 472 480 489 498 506 515 524 532 541 550 558 567 575 584 593 601 610 618 627 635 644 652
105 333 342 351 359 368 376 385 393 402 410 419 427 436 444 453 461 469 478 486 494 503 511 519 528 536 544 552 561 569 577 585 593 601 610 618 626
110 321 329 337 346 354 362 370 378 387 395 403 411 419 427 435 443 451 460 468 476 484 492 500 507 515 523 531 539 547 555 563 571 579 586 594 602
115 309 317 325 333 341 349 357 365 373 380 388 396 404 412 420 427 435 443 451 458 466 474 481 489 497 504 512 520 527 535 543 550 558 565 573 580
120 298 306 314 321 329 337 344 352 360 367 375 382 390 398 405 413 420 428 435 443 450 457 465 472 480 487 495 502 509 517 524 531 539 546 553 561
56
METHOD 1
Body Wt (kg) 5 5,5 6 6,5 7 7,5 8 8,5 9 9,5 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
125 49 54 58 62 67 71 75 80 84 88 92 101 109 117 125 133 141 149 157 165 173 181 189 197 205 212 220 228 235 243 251 258 266 273 281 288
130 48 52 56 60 65 69 73 77 81 85 89 97 105 113 121 129 137 145 152 160 168 175 183 191 198 206 213 220 228 235 243 250 257 265 272 279
135 46 50 54 58 63 67 71 75 79 83 87 94 102 110 118 125 133 140 148 155 163 170 177 185 192 199 207 214 221 228 235 242 250 257 264 271
140 45 49 53 57 61 65 69 72 76 80 84 92 99 107 114 121 129 136 143 151 158 165 172 179 186 193 201 208 215 222 228 235 242 249 256 263
145 43 47 51 55 59 63 67 70 74 78 82 89 96 104 111 118 125 132 139 146 153 160 167 174 181 188 195 202 209 215 222 229 236 242 249 256
150 42 46 50 54 57 61 65 68 72 76 79 87 94 101 108 115 122 129 136 142 149 156 163 170 176 183 190 196 203 210 216 223 229 236 242 249
155 41 45 49 52 56 59 63 67 70 74 77 84 91 98 105 112 119 125 132 139 145 152 159 165 172 178 185 191 198 204 211 217 223 230 236 243
160 40 44 47 51 54 58 61 65 68 72 75 82 89 96 102 109 116 122 129 135 142 148 155 161 168 174 180 187 193 199 205 212 218 224 230 237
180 36 40 43 46 50 53 56 59 62 65 69 75 81 87 93 99 105 111 117 123 129 135 141 147 153 159 165 170 176 182 188 193 199 205 210 216
185 36 39 42 45 48 52 55 58 61 64 67 73 79 85 91 97 103 109 115 121 127 132 138 144 150 155 161 167 172 178 184 189 195 200 206 212
190 35 38 41 44 47 51 54 57 60 63 66 72 78 84 89 95 101 107 113 118 124 130 135 141 147 152 158 163 169 175 180 186 191 196 202 207
195 34 37 40 43 46 50 53 56 59 61 64 70 76 82 88 93 99 105 110 116 122 127 133 138 144 149 155 160 166 171 177 182 187 193 198 203
200 34 37 40 43 46 49 52 54 57 60 63 69 75 80 86 92 97 103 108 114 119 125 130 136 141 147 152 157 163 168 173 179 184 189 194 200
205 33 36 39 42 45 48 51 53 56 59 62 68 73 79 84 90 95 101 106 112 117 123 128 133 139 144 149 154 160 165 170 175 180 186 191 196
210 32 35 38 41 44 47 50 52 55 58 61 66 72 77 83 88 94 99 104 110 115 120 126 131 136 141 146 152 157 162 167 172 177 182 187 193
215 32 35 37 40 43 46 49 52 54 57 60 65 71 76 81 87 92 97 103 108 113 118 123 129 134 139 144 149 154 159 164 169 174 179 184 189
57
METHOD 1
Body Wt (kg) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
125 288 296 303 311 318 326 333 340 348 355 363 370 377 384 392 399 406 414 421 428 435 442 450 457 464 471 478 485 493 500 507 514 521 528 535 542
130 279 287 294 301 308 315 323 330 337 344 351 358 365 372 379 387 394 401 408 415 422 429 436 443 450 456 463 470 477 484 491 498 505 512 519 525
135 271 278 285 292 299 306 313 320 327 334 341 347 354 361 368 375 382 389 395 402 409 416 423 429 436 443 450 456 463 470 476 483 490 496 503 510
140 263 270 277 283 290 297 304 311 317 324 331 338 344 351 358 364 371 377 384 391 397 404 410 417 424 430 437 443 450 456 463 469 476 482 489 495
145 256 262 269 276 282 289 295 302 309 315 322 328 335 341 348 354 361 367 374 380 386 393 399 406 412 418 425 431 437 444 450 456 463 469 475 482
150 249 255 262 268 275 281 288 294 300 307 313 320 326 332 339 345 351 357 364 370 376 382 389 395 401 407 414 420 426 432 438 445 451 457 463 469
155 243 249 255 261 268 274 280 287 293 299 305 311 318 324 330 336 342 348 355 361 367 373 379 385 391 397 403 409 415 421 427 433 439 445 451 457
160 237 243 249 255 261 267 273 280 286 292 298 304 310 316 322 328 334 340 346 352 358 364 370 376 382 387 393 399 405 411 417 423 429 435 440 446
180 216 222 227 233 239 244 250 255 261 266 272 278 283 289 294 300 305 311 316 322 327 333 338 343 349 354 360 365 371 376 381 387 392 397 403 408
185 212 217 223 228 234 239 245 250 256 261 266 272 277 283 288 294 299 304 310 315 320 326 331 336 342 347 352 358 363 368 374 379 384 389 395 400
190 207 213 218 224 229 234 240 245 251 256 261 267 272 277 282 288 293 298 304 309 314 319 325 330 335 340 345 351 356 361 366 371 377 382 387 392
195 203 209 214 219 225 230 235 240 246 251 256 261 267 272 277 282 287 293 298 303 308 313 318 324 329 334 339 344 349 354 359 364 370 375 380 385
200 200 205 210 215 220 226 231 236 241 246 251 257 262 267 272 277 282 287 292 297 302 308 313 318 323 328 333 338 343 348 353 358 363 368 373 378
205 196 201 206 211 217 222 227 232 237 242 247 252 257 262 267 272 277 282 287 292 297 302 307 312 317 322 327 332 337 342 346 351 356 361 366 371
210 193 198 203 208 213 218 223 228 233 238 243 248 253 258 262 267 272 277 282 287 292 297 302 307 311 316 321 326 331 336 341 345 350 355 360 365
215 189 194 199 204 209 214 219 224 229 234 239 243 248 253 258 263 268 273 277 282 287 292 297 301 306 311 316 321 325 330 335 340 344 349 354 359
58
METHOD 2: It is strongly discouraged to use creatinine clearance

AUC 4.1 mg/ml.min Body Wt (kg) 5 5,5 6 6,5 7 7,5 8 8,5 9 9,5 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 20 89 90 91 92 92 93 94 95 95 96 97 98 100 101 103 104 106 107 109 110 112 113 114 116 117 119 120 122 123 125 126 128 129 131 132 25 110 111 111 112 113 114 114 115 116 117 117 119 120 122 123 125 126 128 129 131 132 133 135 136 138 139 141 142 144 145 147 148 150 151 153 30 130 131 132 133 133 134 135 136 136 137 138 139 141 142 144 145 147 148 150 151 153 154 155 157 158 160 161 163 164 166 167 169 170 172 173 35 151 152 152 153 154 155 155 156 157 158 158 160 161 163 164 166 167 169 170 172 173 174 176 177 179 180 182 183 185 186 188 189 191 192 194 40 171 172 173 174 174 175 176 177 177 178 179 180 182 183 185 186 188 189 191 192 194 195 196 198 199 201 202 204 205 207 208 210 211 213 214 45 192 193 193 194 195 196 196 197 198 199 199 201 202 204 205 207 208 210 211 213 214 215 217 218 220 221 223 224 226 227 229 230 232 233 235 50 212 213 214 215 215 216 217 218 218 219 220 221 223 224 226 227 229 230 232 233 235 236 237 239 240 242 243 245 246 248 249 251 252 254 255 55 233 234 234 235 236 237 237 238 239 240 240 242 243 245 246 248 249 251 252 254 255 256 258 259 261 262 264 265 267 268 270 271 273 274 276 GFR (ml/min) 60 65 253 254 255 256 256 257 258 259 259 260 261 262 264 265 267 268 270 271 273 274 276 277 278 280 281 283 284 286 287 289 290 292 293 295 296 274 275 275 276 277 278 278 279 280 281 281 283 284 286 287 289 290 292 293 295 296 297 299 300 302 303 305 306 308 309 311 312 314 315 317 70 294 295 296 297 297 298 299 300 300 301 302 303 305 306 308 309 311 312 314 315 317 318 319 321 322 324 325 327 328 330 331 333 334 336 337 75 315 316 316 317 318 319 319 320 321 322 322 324 325 327 328 330 331 333 334 336 337 338 340 341 343 344 346 347 349 350 352 353 355 356 358 80 335 336 337 338 338 339 340 341 341 342 343 344 346 347 349 350 352 353 355 356 358 359 360 362 363 365 366 368 369 371 372 374 375 377 378 85 356 357 357 358 359 360 360 361 362 363 363 365 366 368 369 371 372 374 375 377 378 379 381 382 384 385 387 388 390 391 393 394 396 397 399 90 376 377 378 379 379 380 381 382 382 383 384 385 387 388 390 391 393 394 396 397 399 400 401 403 404 406 407 409 410 412 413 415 416 418 419 95 397 398 398 399 400 401 401 402 403 404 404 406 407 409 410 412 413 415 416 418 419 420 422 423 425 426 428 429 431 432 434 435 437 438 440 100 417 418 419 420 420 421 422 423 423 424 425 426 428 429 431 432 434 435 437 438 440 441 442 444 445 447 448 450 451 453 454 456 457 459 460 105 438 439 439 440 441 442 442 443 444 445 445 447 448 450 451 453 454 456 457 459 460 461 463 464 466 467 469 470 472 473 475 476 478 479 481 110 458 459 460 461 461 462 463 464 464 465 466 467 469 470 472 473 475 476 478 479 481 482 483 485 486 488 489 491 492 494 495 497 498 500 501
59
METHOD 2: It is strongly discouraged to use creatinine clearance

AUC 4.1 mg/ml.min Body Wt (kg) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 20 134 135 137 138 140 141 143 144 145 147 148 150 151 153 154 156 157 159 160 162 163 165 166 168 169 171 172 174 175 176 178 179 181 182 184 185 25 154 156 157 159 160 162 163 164 166 167 169 170 172 173 175 176 178 179 181 182 184 185 187 188 190 191 193 194 195 197 198 200 201 203 204 206 30 175 176 178 179 181 182 184 185 186 188 189 191 192 194 195 197 198 200 201 203 204 206 207 209 210 212 213 215 216 217 219 220 222 223 225 226 35 195 197 198 200 201 203 204 205 207 208 210 211 213 214 216 217 219 220 222 223 225 226 228 229 231 232 234 235 236 238 239 241 242 244 245 247 40 216 217 219 220 222 223 225 226 227 229 230 232 233 235 236 238 239 241 242 244 245 247 248 250 251 253 254 256 257 258 260 261 263 264 266 267 45 236 238 239 241 242 244 245 246 248 249 251 252 254 255 257 258 260 261 263 264 266 267 269 270 272 273 275 276 277 279 280 282 283 285 286 288 50 257 258 260 261 263 264 266 267 268 270 271 273 274 276 277 279 280 282 283 285 286 288 289 291 292 294 295 297 298 299 301 302 304 305 307 308 55 277 279 280 282 283 285 286 287 289 290 292 293 295 296 298 299 301 302 304 305 307 308 310 311 313 314 316 317 318 320 321 323 324 326 327 329 GFR (ml/min) 60 298 299 301 302 304 305 307 308 309 311 312 314 315 317 318 320 321 323 324 326 327 329 330 332 333 335 336 338 339 340 342 343 345 346 348 349 65 318 320 321 323 324 326 327 328 330 331 333 334 336 337 339 340 342 343 345 346 348 349 351 352 354 355 357 358 359 361 362 364 365 367 368 370 70 339 340 342 343 345 346 348 349 350 352 353 355 356 358 359 361 362 364 365 367 368 370 371 373 374 376 377 379 380 381 383 384 386 387 389 390 75 359 361 362 364 365 367 368 369 371 372 374 375 377 378 380 381 383 384 386 387 389 390 392 393 395 396 398 399 400 402 403 405 406 408 409 411 80 380 381 383 384 386 387 389 390 391 393 394 396 397 399 400 402 403 405 406 408 409 411 412 414 415 417 418 420 421 422 424 425 427 428 430 431 85 400 402 403 405 406 408 409 410 412 413 415 416 418 419 421 422 424 425 427 428 430 431 433 434 436 437 439 440 441 443 444 446 447 449 450 452 90 421 422 424 425 427 428 430 431 432 434 435 437 438 440 441 443 444 446 447 449 450 452 453 455 456 458 459 461 462 463 465 466 468 469 471 472 95 441 443 444 446 447 449 450 451 453 454 456 457 459 460 462 463 465 466 468 469 471 472 474 475 477 478 480 481 482 484 485 487 488 490 491 493 100 462 463 465 466 468 469 471 472 473 475 476 478 479 481 482 484 485 487 488 490 491 493 494 496 497 499 500 502 503 504 506 507 509 510 512 513 105 482 484 485 487 488 490 491 492 494 495 497 498 500 501 503 504 506 507 509 510 512 513 515 516 518 519 521 522 523 525 526 528 529 531 532 534 110 503 504 506 507 509 510 512 513 514 516 517 519 520 522 523 525 526 528 529 531 532 534 535 537 538 540 541 543 544 545 547 548 550 551 553 554
60
METHOD 2: It is strongly discouraged to use creatinine clearance.

AUC 4.1 mg/ml.min Body Wt (kg) 5 5,5 6 6,5 7 7,5 8 8,5 9 9,5 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 110 458 459 460 461 461 462 463 464 464 465 466 467 469 470 472 473 475 476 478 479 481 482 483 485 486 488 489 491 492 494 495 497 498 500 501 503 115 479 480 480 481 482 483 483 484 485 486 486 488 489 491 492 494 495 497 498 500 501 502 504 505 507 508 510 511 513 514 516 517 519 520 522 523 120 499 500 501 502 502 503 504 505 505 506 507 508 510 511 513 514 516 517 519 520 522 523 524 526 527 529 530 532 533 535 536 538 539 541 542 544 125 520 521 521 522 523 524 524 525 526 527 527 529 530 532 533 535 536 538 539 541 542 543 545 546 548 549 551 552 554 555 557 558 560 561 563 564 130 540 541 542 543 543 544 545 546 546 547 548 549 551 552 554 555 557 558 560 561 563 564 565 567 568 570 571 573 574 576 577 579 580 582 583 585 135 561 562 562 563 564 565 565 566 567 568 568 570 571 573 574 576 577 579 580 582 583 584 586 587 589 590 592 593 595 596 598 599 601 602 604 605 140 581 582 583 584 584 585 586 587 587 588 589 590 592 593 595 596 598 599 601 602 604 605 606 608 609 611 612 614 615 617 618 620 621 623 624 626 145 602 603 603 604 605 606 606 607 608 609 609 611 612 614 615 617 618 620 621 623 624 625 627 628 630 631 633 634 636 637 639 640 642 643 645 646 GFR (ml/min) 150 155 622 623 624 625 625 626 627 628 628 629 630 631 633 634 636 637 639 640 642 643 645 646 647 649 650 652 653 655 656 658 659 661 662 664 665 667 643 644 644 645 646 647 647 648 649 650 650 652 653 655 656 658 659 661 662 664 665 666 668 669 671 672 674 675 677 678 680 681 683 684 686 687 160 663 664 665 666 666 667 668 669 669 670 671 672 674 675 677 678 680 681 683 684 686 687 688 690 691 693 694 696 697 699 700 702 703 705 706 708 165 684 685 685 686 687 688 688 689 690 691 691 693 694 696 697 699 700 702 703 705 706 707 709 710 712 713 715 716 718 719 721 722 724 725 727 728 170 704 705 706 707 707 708 709 710 710 711 712 713 715 716 718 719 721 722 724 725 727 728 729 731 732 734 735 737 738 740 741 743 744 746 747 749 175 725 726 726 727 728 729 729 730 731 732 732 734 735 737 738 740 741 743 744 746 747 748 750 751 753 754 756 757 759 760 762 763 765 766 768 769 180 745 746 747 748 748 749 750 751 751 752 753 754 756 757 759 760 762 763 765 766 768 769 770 772 773 775 776 778 779 781 782 784 785 787 788 790 185 766 767 767 768 769 770 770 771 772 773 773 775 776 778 779 781 782 784 785 787 788 789 791 792 794 795 797 798 800 801 803 804 806 807 809 810 190 786 787 788 789 789 790 791 792 792 793 794 795 797 798 800 801 803 804 806 807 809 810 811 813 814 816 817 819 820 822 823 825 826 828 829 831 195 807 808 808 809 810 811 811 812 813 814 814 816 817 819 820 822 823 825 826 828 829 830 832 833 835 836 838 839 841 842 844 845 847 848 850 851 200 827 828 829 830 830 831 832 833 833 834 835 836 838 839 841 842 844 845 847 848 850 851 852 854 855 857 858 860 861 863 864 866 867 869 870 872
61
METHOD 2 : It is strongly discouraged to use creatinine clearance.

AUC 4.1 mg/ml.min Body Wt (kg) 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 110 504 506 507 509 510 512 513 514 516 517 519 520 522 523 525 526 528 529 531 532 534 535 537 538 540 541 543 544 545 547 548 550 551 553 554 115 525 526 528 529 531 532 533 535 536 538 539 541 542 544 545 547 548 550 551 553 554 556 557 559 560 562 563 564 566 567 569 570 572 573 575 120 545 547 548 550 551 553 554 555 557 558 560 561 563 564 566 567 569 570 572 573 575 576 578 579 581 582 584 585 586 588 589 591 592 594 595 125 566 567 569 570 572 573 574 576 577 579 580 582 583 585 586 588 589 591 592 594 595 597 598 600 601 603 604 605 607 608 610 611 613 614 616 130 586 588 589 591 592 594 595 596 598 599 601 602 604 605 607 608 610 611 613 614 616 617 619 620 622 623 625 626 627 629 630 632 633 635 636 135 607 608 610 611 613 614 615 617 618 620 621 623 624 626 627 629 630 632 633 635 636 638 639 641 642 644 645 646 648 649 651 652 654 655 657 140 627 629 630 632 633 635 636 637 639 640 642 643 645 646 648 649 651 652 654 655 657 658 660 661 663 664 666 667 668 670 671 673 674 676 677 145 648 649 651 652 654 655 656 658 659 661 662 664 665 667 668 670 671 673 674 676 677 679 680 682 683 685 686 687 689 690 692 693 695 696 698 GFR (ml/min) 150 155 668 670 671 673 674 676 677 678 680 681 683 684 686 687 689 690 692 693 695 696 698 699 701 702 704 705 707 708 709 711 712 714 715 717 718 689 690 692 693 695 696 697 699 700 702 703 705 706 708 709 711 712 714 715 717 718 720 721 723 724 726 727 728 730 731 733 734 736 737 739 160 709 711 712 714 715 717 718 719 721 722 724 725 727 728 730 731 733 734 736 737 739 740 742 743 745 746 748 749 750 752 753 755 756 758 759 165 730 731 733 734 736 737 738 740 741 743 744 746 747 749 750 752 753 755 756 758 759 761 762 764 765 767 768 769 771 772 774 775 777 778 780 170 750 752 753 755 756 758 759 760 762 763 765 766 768 769 771 772 774 775 777 778 780 781 783 784 786 787 789 790 791 793 794 796 797 799 800 175 771 772 774 775 777 778 779 781 782 784 785 787 788 790 791 793 794 796 797 799 800 802 803 805 806 808 809 810 812 813 815 816 818 819 821 180 791 793 794 796 797 799 800 801 803 804 806 807 809 810 812 813 815 816 818 819 821 822 824 825 827 828 830 831 832 834 835 837 838 840 841 185 812 813 815 816 818 819 820 822 823 825 826 828 829 831 832 834 835 837 838 840 841 843 844 846 847 849 850 851 853 854 856 857 859 860 862 190 832 834 835 837 838 840 841 842 844 845 847 848 850 851 853 854 856 857 859 860 862 863 865 866 868 869 871 872 873 875 876 878 879 881 882 195 853 854 856 857 859 860 861 863 864 866 867 869 870 872 873 875 876 878 879 881 882 884 885 887 888 890 891 892 894 895 897 898 900 901 903 200 873 875 876 878 879 881 882 883 885 886 888 889 891 892 894 895 897 898 900 901 903 904 906 907 909 910 912 913 914 916 917 919 920 922 923
62
10.5 BUMEL MAT Regimen

The BUMEL MAT - regimen consists of oral administration of busulphan (600mg/m2/course) and the short i.v. infusion of melphalan (140mg/m2).
BUMEL MAT
DRUG
BUSULPHAN
DOSE
150 mg/m2/day* [ 37.5 mg/m2 per dose p.o.]
DAY
-7
-6
-5
-4
-3
-2
-1
MELPHALAN
140 mg/m2 i.v. short infusion (15) at least 24 h after last busulphan dose 3l/m/day = 125 ml/m2/hr 0,025 0,1 mg/kg/day total dose i.v. as ctn infusion or divided in 3 doses p.o./day 300 mg/m/day p.o. [150 mg/m/day p.o. BID] Minimum: 3x 10 6 /kg/CD34 + i.v.
Continues until day 1 (24 hours after melphalan) Continuous infusion day -7 until day 0 (day 0 = day of stem cell return ) If the child is excessively drowsy then reduce dose Day 8 until day +80 post P SCR (ensure intake even in presence of mucositis)
Hydration Clonazepam
Ursodiol Peripheral Stem Cells
BUSULPHAN Tablets at 2mg [25mg capsules now produced by Novolabs/UK] Give every 6h starting for a total of 16 doses. Each dose is 37.5mg/m (corresponding to 150mg/m/day). This starts at 1800 hours on day 7 and continues until 1200 hours on day 3. The child should fast 2 hours before and 30 minutes after administration. *Dose adjustment if patient less than 12kg to 480mg/m instead of 600mg/m total dose, i.e. 4x 120mg/m/day [30mg/m per dose]
Since to date no paediatric trial has sufficiently established correct dosing of iv preparations in children, use of iv busulphan is prohibited within this phase III study setting due to the risks of over and underdosing in children. Only after sufficient data is established, an amendment to this phase III protocol may be envisaged.
TOTAL DOSE = 140 mg/m/day and at least 24 h after last busulphan dose. There will be no melphalan dose adjustment based on kg or GFR in BUMELMAT. Preparation: (ALKERAN for intravenous administration, 50 mg vials, Welcome) Melphalan is reconstituted at room temperature, from the lyophilised powder with 10 ml of the solvent diluent provided, by agitating until complete dissolution. The resultant solution contains 5mg in 1 ml anhydrous Melphalan. Administration: Either give undiluted or further dilute in normal saline to a maximum concentration of 0.4mg/ml. Short IV infusion through the central venous catheter over 10 to 15 minutes. Melphalan should be given within an hour of reconstitution. If this time is exceeded, a new batch of melphalan must be prepared. The diluent contains propylene glycol, which has been reported to cause hypotension and arrhythmias when infused intravenously in large doses. Care
MELPHALAN
Final Version of HR-NBL 1/ESIOP 14.03.06
63
should be taken to prevent skin contact or inhalation of aerosolised particles of drug. Hydration: Start hydration at a rate of 125ml/m2/hr three hours prior to melphalan administration. Continue until at least 12 hours post melphalan. Start with sodium chloride 0.9% (compatible with melphalan). Change to Dextrose 2.5%, Sodium Chloride 0.45% post melphalan administration. It is essential to establish a urine output of 4ml/kg/hour pre-melphalan and for two hours post-melphalan administration. Give increased fluids and/or furosemide to achieve this urine output. STEM CELLS : should not be re-infused until at least 12 hours after the end of the melphalan infusion. Ancillary Treatments During busulphan treatment no anti-emetics are indicated according to the French experience when the individual dose is administered in capsule form (has to be provided if possible by the local pharmacy). Anti-emetics should be given i.v. approximately 30 minutes prior to the melphalan injection and again scheduled post-melphalan, for a minimum of 24 hours after the last melphalan dose. Anti-emetic therapy may be administered according to institutional policy, i.e. Ondansetron 5mg/m p.o. or i.v. every 12 hours as anti-emetic (max. single dose 8mg) Adequate hydration is crucial prior to and following melphalan administration due to bladder irritation from high urine concentrations of the drug. Minimal urine output immediately prior to and 24 hours following melphalan administration should be more than 90 ml/m/hr. To achieve this urine output, give i.v. hydration at 125 ml/m/hr. Ursodiol: The administration twice per day during the entire prophylactic period, even in the case of mucositis, from day -8 until day 80 post stem cell reinfusion is of major importance. G-CSF (filgrastim) 5 g/kg/day IV will be given daily beginning on Day +5. G-CSF (filgrastim) will continue until a stable increase of WBC > 5 x 109/l or ANC >0.5 x 109/l All blood products (packed red blood cells, platelets) must be irradiated with 15Gy and be leucocyte depleted (ideally CMV negative). It is recommended that patients receive red packed blood cells to maintain haemoglobin > 8.0g/dl. Stop Co-trimoxazole prophylaxis from day 0 until at least day +10 or until WBC 1.0 x 109/l. Prophylactic antifungal treatment with ketoconazole, itraconazole or fluclonazole should be avoided, because of the increased risk of VOD with these drugs in particular in association with busulphan. For proven fungal infection amphotericin would be used. Antibiotics and antivirals should be given in line with the institutional policy whenever indicated but any prophylactic use should be prudent in view of side effects and drug interactions.
10.6 CEM MAT Regimen

The CEM MAT-regimen uses three drugs: the dose of carboplatin must be based on renal function with a target area under the concentration versus time curve [AUC] of 16.4mg/ml.min/course strictly based on renal function by glomerular filtration rate, etoposide 350mg/m/course and melphalan 210mg/m/course. Carboplatin and etoposide are given in parallel as a continuous infusion over 4 days. Melphalan is given in split doses on three consecutive days as a 15 minute intravenous infusion. Reinfusion of peripheral stem cells 72 hours after the end of the carboplatin and etoposide infusion .
64
Patients with a GFR >100ml/min/1.73m will receive full dose etoposide and melphalan. Patients with a GFR below 100ml/min/1.73m need reduced CEM-MAT doses otherwise they may experience major toxicities. A GFR measurement must be performed prior to carboplatin dosing and should be utilised to calculate the dosage of carboplatin for all patients by one of the two methods as outlined in detail in sections 10.4 and Appendix 20.1. METHOD 1: If 51Cr-EDTA clearance is used for determination of GFR, the dose of carboplatin can be calculated from the 51Cr-EDTA half-life (t) value and patients body weight using the tables provided in section 10.4. These tables give the daily dose of carboplatin calculated to give an AUC of 4.1 mg/ml.min by the Newell formula.111 METHOD 2: For any other method of GFR determination, the dose of carboplatin can be calculated from the uncorrected GFR (ml/min) value and patients body weight using the tables provided in section 10.4. These tables give the daily dose of carboplatin calculated to give an AUC of 4.1 mg/ml.min by the modified version of the Calvert formula.109 It is strongly discouraged to use creatinine clearance.
10.7 Patients with Normal Renal Function

If GFR is 100 ml/min/1.73 m patients will be dosed for etoposide and melphalan by surface area (by weight if 12 kg) according to the table below:
CEM MAT (for normal renal function)

DRUG
CARBOPLATIN
DOSE
DAY
-7
24h 24h
-6
24h
-5
24h
-4
24h
-3
-2
-1
AUC 4.1mg/ml.min /day, actual dose based on GFR rate 338 mg/m/day x 4 days as ctn i.v. [total dose= 1352mg/m/course] Patients 12 kg : 11.3 mg/kg/day x 4 days as ctn i.v. [total dose = 45.2 mg/kg/course]
ETOPOSIDE
24h
24h
24h
MELPHALAN 70 mg/m/day on 3 days at hour 0 [total dose= 210mg/m/course] Patients 12 kg: 2.3 mg/kg/day on 3 days at hour 0 [total dose = 6.9 mg/kg/course] 3l/m/day = 125 ml/m2/hr 300 mg/m/day p.o. [150 mg/m/day p.o. BID] Minimum: 3x 10 6 /kg/CD34 + i.v.
Hydration Ursodiol Peripheral Stem Cells
Continues until day 1 Day 8 until day +80 post P SCR (ensure intake even in the presence of mucositis)
65
CARBOPLATIN
Actual dose is based on the GFR rate and weight aiming at an AUC of 4.1 mg/ml.min/day over 4 days. Dose may be evaluated according to method 1 or method 2 as outlined above and the tables adjoined. Details in section 10.4 and appendix 20.1. To avoid overdosing in patients with normal renal function , i.e. GFR>100ml/min/1.73m, the upper dose limit is a CBDCA dose of 425 mg/m.
ETOPOSIDE
TOTAL DOSE = 1352mg / m/ 4 days 338 mg/m/day as 24 hour continuous infusion x 4 days (patients 12 kg will receive 11.3 mg/kg/day x 4 days, total dose = 45.2 mg/kg/4 days). Etoposide is given by continuous intravenous infusion Day - 7 through Day -4 at the same time that carboplatin is given. Maximum etoposide concentration is 0.4 mg/ml. Etoposide should not be mixed with carboplatin, but can be infused concomitantly with it through the same central venous catheter ideally using two different lumina or, in case of a single lumen catheter, a "Y" connector. A controlled rate infusion pump is to be used for each lumen or arm of the "Y". TOTAL DOSE
MELPHALAN
= 210 mg/m/course 70 mg/m/day x 3 days. Give at hour 0 of Day -7, -6 and 5 before SCR, (patients 12 kg will receive 2.3 mg/kg/day x 3 days, total dose = 6.9 mg/kg/3days). Preparation: (ALKERAN for intravenous administration, 50 mg vials, Welcome) Melphalan is reconstituted at room temperature, from the lyophilised powder with 10 ml of the solvent diluent provided, by agitating until complete dissolution. The resultant solution contains 5mg in 1 ml anhydrous melphalan. Administration: Either give undiluted or further dilute in normal saline to a maximum concentration of 0.4mg/ml. Short IV infusion through the central venous catheter over 10 to 15 minutes. Melphalan should be given within an hour of reconstitution. If this time is exceeded, a new batch of melphalan must be prepared. The diluent contains propylene glycol, which has been reported to cause hypotension and arrhythmias when infused intravenously in large doses. Care should be taken to prevent skin contact or inhalation of aerosolised particles of drug. Hydration: Start hydration at a rate of 125ml/m/hr three hours prior to melphalan administration. Continue until at least 12 hours post melphalan. Start with sodium chloride 0.9% (compatible with melphalan). Change to Dextrose 2.5%, Sodium Chloride 0.45% post melphalan administration. It is essential to establish a urine output of 4ml/kg/hour pre-melphalan and for two hours postmelphalan administration. Give increased fluids and/or furosemide to achieve this urine output.
Ancillary Treatments Anti-emetics should be given i.v. approximately 30 minutes prior to the melphalan injection and again be scheduled post-melphalan, for a minimum of 24 hours after the last melphalan dose. Anti-emetic therapy may be administered according to institutional policy, i.e. Ondansetron 5mg/m p.o. or i.v. every 12 hours as antiemetic (max. single dose 8mg). Adequate hydration is crucial prior to and following melphalan administration due to bladder irritation from high urine concentrations of the drug. Minimal urine output immediately prior to and 24 hours following melphalan administration should be more than 90 ml/m/hr. To achieve this urine output, give i.v. hydration at 125 ml/m/hr.
Final Version of HR-NBL 1/ESIOP 14.03.06 66
Ursodiol: The administration twice per day during the entire prophylactic period even in the case of mucositis from day -8 until day 80 post stem cell reinfusion is of major importance. G-CSF (filgrastim) 5 g/kg/day IV will be given daily beginning on Day +5. G-CSF (filgrastim) will continue until a stable increase of WBC > 5 x 109/l or ANC > 0.5 x 109/l is achieved. All blood products (packed red blood cells, platelets) must be irradiated with 15 Gy and be leucocyte depleted (ideally CMV negative). It is recommended that patients receive red packed blood cells to maintain haemoglobin > 8.0g/dl. Stop Co-trimoxazole prophylaxis from day 0 until at least day +10 or until WBC 1.0 x 109/l. Prophylactic antifungal treatment with ketoconazole, itraconazole or fluconazole should be avoided, because of the increased risk of VOD with these drugs in particular in association with busulphan. Antibiotics and antivirals should be given in line with institutional policy whenever indicated but any prophylactic use should be prudent in view of side effects and drug interactions.
10.8 Patients with Abnormal Renal Function

If the GFR is <100 ml/min/1.73m, the dose levels of etoposide and melphalan will be decreased in these patients compared to patients with a normal GFR (see above). The dose of carboplatin will be adjusted based on the GFR result, no additional changes in the etoposide or melphalan doses will be made.
CEM MAT (for abnormal renal function)

DRUG
CARBOPLATIN
DOSE
DAY
-7
24h 24h
-6
24h 24h
-5
24h 24h
-4
24h 24h
-3
-2
-1
AUC 4.1mg/ml.min /day, actual dose based on GFR rate 200 mg/m/day x 4 days as ctn i.v. [total dose= 800 mg/m/course] Patients 12 kg : 6.7mg/kg/day x 4 days as ctn i.v. [total dose = 26.8 mg/kg/course]
ETOPOSIDE
MELPHALAN 60 mg/m/day on 3 days at hour 0 [total dose= 180mg/m/course] Patients 12 kg: 2mg/kg/day on 3 days at hour 0 [total dose = 6mg/kg/course] 3l/m/day = 125 ml/m2/hr 300 mg/m/day p.o. [150 mg/m/day p.o. BID] Minimum: 3x 10 6 /kg/CD34 + i.v.
Hydration Ursodiol Peripheral Stem Cells
Continues until day 1 Day 8 until day +80 post P SCR (ensure intake even in the presence of mucositis)
67
CARBOPLATIN
ETOPOSIDE
Actual dose is based on the GFR rate and weight aiming at an AUC of 4.1 mg/ml.min/day over 4 days. Dose may be evaluated according to method 1 or method 2 as outlined above and the tables adjoined. Details in section 10.4 and appendix 20.1. TOTAL DOSE = 800mg/m/ 4 days 200 mg/m/day as 24 hour continuous infusion x 4 days (patients 12 kg will receive 6.7mg / kg / day x 4 days, total dose = 26.8mg / kg / 4 days). Etoposide is given by continuous intravenous infusion Day -7 through Day -4 at the same time that carboplatin is given. Maximum etoposide concentration is 0.4 mg/ml. Etoposide should not be mixed with carboplatin but can be infused concomitantly with it through the same central venous catheter using a "Y" connector; a controlled rate infusion pump is used for each arm of the "Y".
TOTAL DOSE
MELPHALAN
= 180mg/m/course 60 mg/m/day x 3 days. Give at hour 0 of Day -7, -6 and 5 before SCR, (patients 12 kg will receive 2mg/kg/day x 3 days, total dose = 6mg/kg/3days). Preparation: (ALKERAN for intravenous administration, 50 mg vials, Welcome) Melphalan is reconstituted at room temperature, from the lyophilised powder with 10 ml of solvent diluent provided, by agitating until complete dissolution. The resultant solution contains 5mg in 1 ml anhydrous melphalan. Administration: Either give undiluted or further dilute in normal saline to a maximum concentration of 0.4mg/ml. Short IV infusion through the central venous catheter over 10 to 15 minutes. Melphalan should be given within an hour of reconstitution. If this time is exceeded, a new batch of melphalan must be prepared. The diluent contains propylene glycol, which has been reported to cause hypotension and arrhythmias when infused intravenously in large doses. Care should be taken to prevent skin contact or inhalation of aerosolised particles of drug. Hydration: Start hydration at a rate of 125ml/m2/hr three hours prior to melphalan administration. Continue until at least 12 hours post melphalan. Start with sodium chloride 0.9% (compatible with melphalan). Change to Dextrose 2.5%, Sodium Chloride 0.45% post melphalan administration. It is essential to establish a urine output of 4ml/kg/hour pre-melphalan and for two hours post melphalan administration. Give increased fluids and/or furosemide to achieve this urine output.
Ancillary Treatments Anti-emetics should be given i.v. approximately 30 minutes prior to the melphalan injection and again be scheduled post melphalan, for a minimum of 24 hours after the last melphalan dose. Anti-emetic therapy may be administered according to institutional policy, i.e. Ondansetron 5mg/m p.o. or i.v. every 12 hours as antiemetic (max. single dose 8mg). Adequate hydration is crucial prior to and following melphalan administration due to bladder irritation from high urine concentrations of the drug. Minimal urine output immediately prior to and 24 hours following melphalan administration should be more than 90 ml/m/hr. To achieve this urine output, give i.v. hydration at 125 ml/m/hr. Ursodiol: The administration twice per day during the entire prophylactic period, even in the case of mucositis, from day -8 until day +80 post stem cell reinfusion is of major importance. G-CSF (filgrastim) 5 g/kg/day IV will be given daily beginning on Day +5. 5g/kg GCSF (filgrastim) will continue until a stable increase of WBC > 5 x 109/l or ANC > 0.5 x 109/l.
All blood products (packed red blood cells, platelets) must be irradiated with 15 Gy and be leucocyte depleted (ideally CMV negative). It is recommended that patients receive red packed blood cells to maintain haemoglobin > 8.0g/dl. Stop Co-trimoxazole prophylaxis from day 0 until at least day +10 or until WBC 1.0 x 109/l. Prophylactic antifungal treatment with ketoconazole, itraconazole or fluconazole should be avoided, because of the increased risk of VOD with these drugs in particular in association with busulphan. Antibiotics and antivirals should be given in line with institutional policy whenever indicated but any prophylactic use should be prudent in view of side effects and drug interactions.
10.9 Stem Cell Reinfusion

10.9.1 PREMEDICATION/MONITORING Discontinue all other IV fluids where possible and replace them with 0.9% sodium chloride 4 hours prior to and after the stem cell infusion. Fifteen minutes prior to the stem cell infusion, premedicate with acetaminophen (10mg/kg p.o.) and diphenhydramine (1 mg/kg i.v.). Ambubag, diphenhydramine and epinephrine at bedside. Place patient on cardiac monitor during infusion and for 1-2 hours following completion. Discontinue all other IV fluids where possible during stem cell infusion to avoid volume overload. Hydrate for 24 hours post stem cell infusion with 3000 ml/m/day total IV fluids. 10.9.2 DOSAGE/TIMING PERIPHERAL STEM CELLS A minimum of 3 x 106 CD34 cells/kg (optimum 5 x 106 /kg) must be available. A maximum of 10 x 106 CD 34 cells/kg should be used. Or BONE MARROW A minimum of 3 x 108 mononuclear bone marrow cells/kg or > 8 x 10 4 CFU-GM/kg should be used. REINFUSION OF STEM CELLS Stem cells will be infused intravenously on Day 0 with specified rest, according to the MAT regimen, following completion of chemotherapy, within 1 hours of thawing.
69
11 Radiotherapy
11.1 Indication
In this protocol all patients will receive radiotherapy to the initial tumour site regardless of the extent and/or result of surgery. Some patients may be considered unsuitable for radiotherapy by reason of the site of primary tumour and the volume which would require irradiation. In these situations please discuss with your paediatric clinical oncologist (radiotherapist) and contact trial Co-ordinators for discussion. Discussion about administration of radiotherapy should include consideration of referral to a centre with more extensive experience.
11.2 Timing of Radiotherapy

Radiotherapy will be given after MAT/PSCR and prior to the start of 13-cis RA treatment. After BUMEL MAT the interval must be greater than 60 days after stem cell transplantation, due to the risk of Busulphan-enhanced radiotoxicity. A negative interaction between radiotherapy and 13-cis RA has been described (Philadelphia 1996), so these should not be used together.
11.3 Fields and Dose of Radiotherapy

CT Planning Planning should be based on preoperative imaging. A planning CT scan at this time will allow the GTV to be identified accurately. Postoperatively the surgical and pathological notes will also be taken into account. The CTV includes a delineated preoperative GTV as indicated on preoperative imaging and areas of persistent lymph node enlargement after induction chemotherapy and in addition all areas of microscopic disease as indicated from the surgical report and the pathological examination. The PTV takes into account uncertainties of positioning and includes an additional 0.5 to 1.0 cm margin. So, the entire safety margins will approximate 2 cm in each direction. Bean shaping with customised blocks or MLC should be used to reduce unnecessary irradiation of normal tissues, but care should be taken to ensure symmetry of subsequent growth by treating the full width of vertebral bodies. Distant metastatic sites will not be irradiated systematically. Dose Doses will be specified according to ICRU recommendations i.e. at the intersection point of the mid axis of each beam when multiple fields are used or at the mid-plane if two fields only are used. Variation of dose across the target volume should not exceed +/- 5%. The dose should be treated to 21 Gy in 14 fractions of 1.5 Gy over not more than 21 days. Visible residual disease following the high dose chemotherapy regimen should not be boosted. Conventional 1.5 Gy per fraction, 5 fractions per week. All fields will be treated daily. High energy photons from a linear accelerator 6 to 10 MeV
Volume
Fractionation
Energy
70
11.4 Normal Tissue Tolerance

Normal tissues within or adjacent to the treated volume may be dose limiting. Doses to normal tissue will be kept as low as reasonably achievable consistent with adequate treatment of the PTV and homogeneous treatment of vertebrae. The following recommendations should be considered : Liver The dose to the whole liver should not exceed 19 Gy. 21 Gy is acceptable for one lobe. A dose of 21 Gy is acceptable for any length of spinal cord. If one kidney has been removed surgically the dose to the remaining kidney should not exceed 12 Gy. A dose of 21 Gy is acceptable for up to half a kidney if the patient has two kidneys. A free of milk and gluten diet is recommended since. There will be an inevitable effect on the epiphyses of vertebrae within the field of irradiation. Care should be given to maintain the symmetry by irradiation of the whole vertebra. < 5 Gy if possible Normal tissue tolerance is unlikely to be exceeded.
Spinal cord Kidney
Bowels Bone
Gonads Other sites
11.5 Quality control

In the event of loco-regional failure the planning films should be reviewed by the Radiotherapy Panel to see if the recurrence is within or outside of the field. The following will be requested for radiotherapy quality control assessment: the CT/MRI scan used for the definition of the tumour volume, planning and simulator films, portal films, computer assisted dose distribution parameters and dose/volume histograms for the tumour and surrounding critical organs (lungs, kidney, heart).
71
12 13-cis Retinoic Acid Therapy

12.1 Treatment Schedule for 13-cis RA
Aim to begin at day 80, but not later than day 120 after MAT/PBSCR and local radiation, if the ANC > 0.5 x 109/l, liver function < CTC grade 2 toxicity, and renal function, calcium, uric acid and triglycerides 2 x normal values. Common toxicity criteria (CTC) can be found in the Appendix on toxicity (chapter 26). Patients will receive cis-retinoic acid 160 mg/m/day divided into two equal doses given orally twice a day for 14 days, followed by a 14 day rest for a total of six cycles (six months). Patients 12 kg will be given 5.33 mg/kg/day divided into two equal doses given orally twice a day. Doses will need to be rounded to the nearest 10 mg. Capsules come as 10, 20, and 40 mg sizes, and can be emptied into a high fat food such as ice cream or peanut butter to administer. Treatment Schedule for 13-cis RA W1 RA W13 RA W2 RA W14 RA W15 rest W3 rest W4 rest W5 RA W16 rest RA RA W6 RA W17 W18 W19 rest W7 rest W8 rest W9 RA W20 rest RA RA W10 RA W21 W22 W11 W12
12.2 Suggested Supportive Care

Topical Vit E should be applied to the lips twice a day during cis-retinoic acid therapy if cheilitis develops. Patients should avoid direct sun exposure while on cis-retinoic acid. Patients should avoid exposure to vitamin A products during cis-retinoic acid therapy.
12.3 Criteria prior to Each Cycle of 13-cis RA

ALT < 5 x normal Skin toxicity no greater than grade 1 Serum triglycerides < 300 mg/dl No haematuria and/or proteinuria on urinalysis Serum creatinine < 1.5 mg/dl
12.4 Dose Modifications

a. A dose reduction of 25% (to 120 mg/m/day or 4 mg/kg/day if child weighs < 12kg) for subsequent cycles should be made for the occurrence of any grade 3 or 4 CTC toxicity. EXCLUDING: grade 3 or 4 haematologic, grade 3 hepatic, grade 3 nausea, grade 3 vomiting, or grade 3 fever. If the same grade 3 or 4 toxicity recurs after a 25% dose
72
reduction, then decrease the dose by another 20% (to 100 mg/m/day or 3.33 mg/kg/day if child weighs 12 kg). If the same grade 3 or 4 toxicity recurs after two dose reductions, then discuss with study co-ordinator before continuing further therapy. b. It has been reported (rarely) that some patients treated with 13-cis-retinoic acid develop new areas of abnormal uptake on bone scan. This is likely to be due to increased bone resorption. If such changes occur during the cis-retinoic acid phase in the absence of any other evidence of tumour recurrence, discuss with study co-ordinator before reporting as disease progression. If the criteria to begin the next cycle are not met by the date the cycle is due to begin, delay the cycle for one week. If the criteria are still not met, treat at 25% dose reduction (120 mg/m/day or 4 mg/kg/day if child weighs 12 kg). An additional dose reduction to 100 mg/m/day (3.33 mg/kg/day if child weighs 12 kg) should occur if criteria are not met within one week after due date for subsequent cycles. If serum creatinine increases by > 50% in any cycle, measured GFR should be carried out prior to commencing the next cycle. If GFR is < 50 ml/min/1.73 m, then call the study co-ordinator for dose adjustment. If patient develops haematuria, proteinuria, and/or hypertension during any cycle of therapy, withhold medication and contact study co-ordinator. For localised cheilitis, apply topical vitamin E to lips for subsequent cycles. If this does not control symptoms sufficiently to allow sufficient oral intake, then decrease dose by 25% to 120 mg/m/day or 4 mg/kg/day if child weighs 12kg. If serum triglycerides are > 300 mg/dl when next cycle is due, delay starting therapy for two weeks. If still > 300 mg/dl, then start patient on medical therapy for serum triglyceride reduction and begin cycle at previous cis-retinoic acid dosage. If serum triglycerides are < 300 mg/dl by time subsequent cycle is due, then continue at same dosage cis-retinoic acid. If triglycerides are still > 300 mg/dl after one cycle on medical therapy, then reduce cis-retinoic acid dosage by 25% for subsequent cycles.
c.
d.
e. f.
g.
73
13 Immunotherapy with Ch14.18 Anti-GD2 mAB

13.1 Indication
The monoclonal antibody ch 14.18 will be given only to patients fulfilling R2 criteria and being randomised to the immunotherapy arm. Prior to the start of the antibody treatment, patients have to be in good health without clinical or laboratory signs of infection. In case of infection postpone cycle.
Patients will be randomised for additional immunotherapy with the chimeric 14.18 antiGD2 antibody at a dose of 20mg/m/day over 5 days every 4 weeks for 5 courses. The first course will start three weeks after initiation of 13-cis RA. Treatment schedule for 13-cis RA and the ch14.18 anti-GD2 mAb W1 RA W13 RA W2 RA W14 RA W3 rest W4 W5 W6 RA W17 RA W18 RA W7 rest W8 W9 W10 RA W21 RA W22 RA W11 Rest W12 GD2
13.2 Ch14.18 Anti-GD2 mAb Treatment Schedule
13.3 Mode of Action

After binding to neuroblastoma cells, the ch14.18 anti-GD2 mAb induces killing of tumour cells by complement dependent (CDC) and antibody dependent cytotoxic lysis (ADCC). Background is given in detail in Section 3.2.6.
13.4 Mode of Administration of ch14.18 anti-GD2 mAb Treatment

Admit to hospital on the day/or the evening before starting the ch14.18 anti-GD2 mAb infusion Preferably the central venous line will still be in place G-CSF (filgrastim) should not be given prior to any ch14.18 anti-GD2 mAb cycle Intravenous immunoglobulins 200- 400mg/kg over 2 to 4 hours should be administered prior to the day 1 infusion of each ch14.18 antiGD2 -cycle. (1x per cycle)
Anti-histamine medication prior to each ch14.18 anti-GD2 mAb infusion on day 1 through day 5 should be administered as allergic prophylaxis (if not clinically indicated avoid steroids). DOSE/CYCLE 100mg/m/cycle DOSE/DAY 20mg/m/day ADMINISTRATION as 8 hour infusion in 100ml NaCl 0.9% + 5 ml human albumin 20%
ch14.18 anti-GD2 mAb
Hydration
2000ml/m Gluc 5% ml NaCl ml KCL

74
13.5 Toxicity
13.5.1 PAIN During the infusion intensive visceral pain and pain in the extremities has to be anticipated. Therefore concomitant morphine treatment is a necessity. Pain symptoms are usually quickly reversible once the ch14.18 anti-GD2 mAb infusion is stopped. 13.5.2 ANAPHYLACTIC REACTIONS Allergic reactions are seen quite frequently, with associated symptoms such as tachycardia, coughing attacks, fever and elevation of CRP (reactive protein C) levels. 13.5.3 REVERSIBLE PUPIL PALSRY Pupil palsry was previously observed in early dose escalation studies, but was reported to resolve within a few weeks.78
13.6 Special Supportive Care during Ch14.18 Anti-GD2 mAb Treatment

13.6.1 MANDATORY MEDICATION FOR PAIN DURING THE ANTIBODY INFUSION: Morphine hydrochloride (mandatory) a) bolus rate (2 hours prior to starting the ch14.18 antiGD2 infusion) b) infusion rate during ch14.18 antiGD2 infusion (8 hours) c) interval infusion rate (14 hours the first day, thereafter 4 hours, try to stop for 10h if tolerated)
0.05 mg/kg/h 0.03mg/kg/h 0.01mg/kg/h
MORPHINE INFUSION SCHEDULE

Prepare 10mg morphine in 40ml Gluc 5% (0.25mg=1ml) duration pre-infusion rate Infusion rate during ch14.18 antiGD2 inf. Interval infusion rate Total dose mg/kg/24h 2h 8h 14 h (4 h) mg/kg/h 0.05 mg/kg/h 0.03 mg/kg/h 0.01 mg/kg/h ml/kg/h mg/kg (0.25mg=1ml) 0.2 ml/kg/h 0.1 mg/kg 0.12 ml/kg/h 0.24 mg/kg 0.04 ml/kg/h 0.14 (0.04) mg/kg 0.48 (0.38) mg/kg
The aim is to achieve absence of pain. Dosing may need to be adapted to obtain complete analgesia . The individual dose may vary widely. According to WHO recommendations the concomitant use of at least one non-opioid analgesic is encouraged. Further analgesics (optional) a) Paracetamol 10 15mg/kg x dose orally every 4 hrs or b) Ibuprofen 5 mg/kg x dose orally every 6 12 hrs or c) Metamizol 10 15 mg/kg x dose orally every 4 hrs
If a patient still experiences pain, stop the ch14.18 antiGD2mAb infusion for 30 minutes, add a non-opioid analgesic and/or give a morphine bolus or increase the permanent morphine infusion rate. In the German experience morphine infusions of up to 1.2mg/kg/h over 24 hours were required, however such high doses were rarely needed in the Austrian phase II experience (unpublished data) even with the use of an higher ch14.18 antiGD2 mAb dose. 13.6.2 MANDATORY SURVEILLANCE DURING ANTIBODY INFUSION Monitor (24h) ( equipped with pulsoximetry ) Blood pressure: every 10 min for the first 30 minutes, then every 30 minutes
13.6.3 STANDARDISED TREATMENT OF ANAPHYLACTIC REACTIONS This part serves only as a suggestion and may be adapted to each centres guidelines. It is the treating physicians responsibility to judge the severity of the reaction, but it is recommended to introduce medication stepwise, i.e. allergic skin reaction may be well controlled with antihistamines alone. Stop ch14.18 antiGD2 infusion for at least 30 minutes Volume substitution, i.e. 5% albumin 10ml/kg x doses Clemastin: 0.6 mg/ 10 kg per dose i.v.
If not successful, next step: Methylprednisolone: 8-10mg/kg per dose i.v. (i.e.: Urbanson , 5ml= 250mg) If not successful, next step: Norepinephrine (i.e. Arterenol 1:1000, 1ml=1000g) 1ml Arterenol 1: 1000 + 9ml NaCl 0.9%: 0,2-1ml i.v. If the reaction resolves easily, continue the ch14.18 antiGD2 infusion. If the reaction is severe, eventually stop the infusion for the day, but try again the next day with appropriate premedication and reduce the antibody dose to 50% for one day. Try to increase to 75% the day thereafter if the 50% regime was well tolerated. Any dose reduction has to be documented and reported on the case report forms sent to the study centre.
13.6.4 SCIENTIFIC ADDITIONAL EXAMINATIONS A 1 ml serum sample needs to be frozen and stored before the first and 4 weeks after the last cycle of immunotherapy, for the determination of human anti- chimeric antibodies (HAChA) and anti-idiotypic antibodies.
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14 Use of G-CSF
14.1 G-CSF (filgrastim) during Induction Treatment (Supportive Care Randomisation R0)
14.1.1 BACKGROUND ON THE USE OF G-CSF (FILGRASTIM) DURING INDUCTION TREATMENT The prophylactic administration of G-CSF (filgrastim) might be expected to shorten the period of neutropenia, and thus reduce the associated morbidity and mortality, improve patient compliance and permit increased dose intensity over a given time period (either by shortening the time intervals between chemotherapy or by enabling the envisaged time intervals in between chemotherapy courses to be respected). In support of this, randomised trials in adults have revealed that primary prophylaxis with G-SF reduces the incidence of chemotherapy-associated febrile neutropenia in patients at especially high risk of infection (40%)117
118;119
In children, clinical study evidence suggests that although not all patients benefit from universal use of G-CSF with chemotherapy, prophylaxis with G-CSF does reduce the incidence of febrile neutropenia and confirmed infections in some patients receiving high intensity, dose intensive chemotherapy for high-risk, advanced stage tumours including acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL) and some solid tumours.120-123 A randomised study was reported by the French Society of Pediatric Oncology testing the potential benefits of the use of G-CSF (filgrastim) at a dose of 5g/kg/day starting at day 7 of each course within the protocol NB87. The duration of neutropenia was reduced after all these courses, its incidence was reduced for all courses except the first course of CADO. Neither the incidence of febrile neutropenia or that of documented infections was changed, on the other hand the duration of the use of antibiotics was reduced. The use of G-CSF (filgrastim) allowed the intervals between courses, after the courses of etoposide-cisplatin, to be respected but had no effect on the timing of cycles after CADO. According to these results the use of G-CSF (filgrastim) following courses of cisplatin-etoposide appeared necessary in order to respect delays between the courses and improve the dose intensity ratio.124 In a retrospective analysis of two sequential groups Kushner et al showed that G-CSF (filgrastim) after high-dose CAVA in cycles 1,2,4 or 6 (cyclophosphamide 4.2g/m, doxorubicin 60mg/m, vincristine) or cisplatin (200mg/m) and etoposide (600mg/m) after cycles 3,5 and 7 in 58 children with stage 4 neuroblastoma. Recovery after CAV and cisplatin/etoposide to an ANC > 0.5 x 109/l was significantly earlier with G-CSF(filgrastim). However, G-CSF (filgrastim) had no impact on the number of febrile episodes in this analysis. Bacterial and fungal infections were slightly less frequent after high-dose CAV.125 The recommendations for the primary use of G-CSF given in The European Journal of Paediatrics in 1998126 are that it is only used in children following dose-intensive therapy for advanced stage tumours such as neuroblastoma, soft tissue sarcoma and osteosarcoma. In all cases, further randomised trial evidence is required. In summary, many of the above trials are either lacking power to demonstrate a possible effect of G-CSF (filgrastim) or were not randomised within a comparable cohort of patients. Thus it appears justified and of interest to try to answer this question in a clear, prospective randomised setting within a trial recruiting a sufficient number of children to answer the
question: is there a clinical benefit for patients receiving prophylactic G-CSF during intensive induction regimens. COJEC is of special interest since persistent aplasia over a duration of at least 70 days is expected with an important number of febrile episodes necessitating hospitalisation. Since the attitude towards hospitalisation might differ in a multi-centre setting, the number of documented febrile episodes necessitating the use of antibiotics was felt to be the most objective parameter to be tested. 14.1.2 RATIONALE AND AIMS OF R0 RANDOMISATION Since COJEC induces a period of CONTINUOUS APLASIA OVER 70 DAYS, i.e. WBC 1x10 9/l and/or ANC 0.5 1x109/l the capacity of G-CSF (filgrastim) to reduce the incidence of febrile neutropenic episodes during this dose intensive schedule will be investigated. Major Scientific Questions of R0 Randomisation: 1) Can G-CSF (filgrastim) significantly reduce the number of febrile neutropenic episodes by one third or at least by one episode during COJEC induction in comparison to ENSG5 data which reports a median number of 4 febrile episodes per patient? 2 a) What is the influence on the feasibility and quality of the peripheral stem cell harvest when G-CSF (filgrastim) is applied over an extended period such as COJEC induction? 2 b) Is there any evidence that it depletes the stem cell pool? The use of G-CSF (filgrastim) within the COJEC schedule will require close monitoring of patient data, strict application of the rules as given below and activation of the stopping rules in the case of undesired effects. HAEMATOLOGICAL AND CLINICAL ENDPOINTS Primary Endpoint The reduction of the mean number of febrile neutropenic episodes (at least two spikes of oral or axillary temperature > 38C ) by at least one compared to the control group . Secondary Endpoints Incidence of blood-born bacterial or fungal infections Incidence of infection associated toxic deaths Incidence and duration of severe neutropenia (ANC < 0.5 x 109/l) Time to recover to a platelet count 100 x 109/l Number of platelet transfusions Signs of stem cell depletion - Insufficient number of CD34+ cells at harvest immediately after induction period - Insufficient number of CD34+ cells at harvest after surgery out of steady state The time frame (or) dose intensity of chemotherapy Rates of remission with responses defined by international criteria Results of R0 will be known early in the study period (depending on the recruitment rate after 1 to 2 years). In the case where the primary endpoint of R0 is met and in the case where no stopping rule was activated, G-CSF (filgrastim) will be given to all patients during the COJEC induction for the rest of this study.
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Use of G-CSF (filgrastim) outside R0 Primary prophylaxis The use of G-CSF (filgrastim) as primary prophylaxis throughout the induction period is not recommended outside the controlled R0 randomisation. This is because cell recovery dynamics within this rapid schedule are clearly different from any other 3 weekly chemotherapy schedule and established rules and guidelines for the latter may not be applicable to this rapid schedule. G-CSF was not used as primary prophylaxis in the ENSG5 randomised study using COJEC. A particular concern is that the prolonged stimulation of the stem cell pool could have an adverse effect on the stem cell harvest. Therefore the use of G-CSF outside the controlled setting is not recommended. Secondary prophylaxis with G-CSF (filgrastim) The use of G-CSF (filgrastim) as secondary prophylaxis, i.e. administration after one neutropenic event in all subsequent cycles, is not recommended except after severe or life threatening infections for the same reasons as outlined for primary prophylaxis outside of R0. Intervention therapy with G-CSF (filgrastim) Treating physicians are encouraged to use G-CSF (filgrastim) during induction for severe or life threatening infections as intervention therapy 117 118;119together with antibiotics in children at particular risk, i.e. proven Pseudomonas or fungal infections, or when there is multi-organ dysfunction present, or pneumonia present. MODE OF ADMINISTRATION G-CSF (filgrastim) will be given according to clear guidelines in this study. Patients randomised to receive G-CSF (filgrastim) will be given a single daily subcutaneous injection of 5 g/kg/day G-CSF (filgrastim) beginning 24 hours after the last chemotherapy dose. G-CSF (filgrastim) must be stopped at least 24 hours prior to the next chemotherapy administration in between COJEC courses. G-CSF (filgrastim) is never to be administered on the day of, or on the day before, or the day following chemotherapy. The administration of G-CSF (filgrastim) will be discontinued if the ANC 10x109/l. After the last Cycle G-CSF (filgrastim) will be continued until PBSC harvest is completed. See guidelines for G-CSF (filgrastim) dosing during PBSC harvest (see chapter 9).
14.2 G-CSF for PBSC Mobilisation

Routine G-CSF (filgrastim) for the mobilisation of PBSCs is recommended for children needing autologous PBSC transplantation. G-CSF (filgrastim) can be administered following myelosuppressive therapy at a dose of 5g/kg per day until a value of 0.02 x 109/l circulating CD34+ cells or higher is reached for the start of the PBSC collection. Alternatively, G-CSF (filgrastim) can be administered alone at a dose of 10g/kg per day for 4 days with collection on day 5, or for 5 days with collection on days 5 and 6.
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14.3 G-CSF after MAT

14.3.1 BACKGROUND Adult guidelines strongly recommend the use of G-CSF to shorten the period of neutropenia after autologous BMT. Data on G-CSF use in children as an adjunct to autologous BMT are limited and no randomised trial has been carried out. However, two open studies, with historical controls, have indicated the usefulness of G-CSF (filgrastim) in this setting.127;128 There is no evidence that beneficial effects can be observed when G-CSF is given during the first days after autologous transplantation. Beginning the treatment after the first week seems adequate. Recent data on G-CSF (filgrastim) in the setting of PBSC transplantation is given in the table below summarizing numerous studies depicting an advantage for the use of G- and /or GMCSF. There is however much less evidence in children to suggest that routine CSF administration to recipients of autologous PBSCs is effective in accelerating neutrophil recovery. In a nonrandomised study of 98 children G-CSF (filgrastim) had no effect on neutrophil recovery following autologous PBSC transplantation.129 Author Klumpp 130 Tarella 131 Benedetti 132 Spitzer 133 Bensinger
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R ? 1995 1998 1997 1994 1994 + + + + -
Pts Dose of No. G-CSF / GM-CSF 41 G-CSF 5 g/kg 40 G-CSF 5 g/kg 40 G-CSF 5 g/kg + Epo 37 G-CSF 7.5 g/kg and 54 33
GM-CSF 5 g/kg + Epo GM-CSF 2.5 g/kg G-CSF 5 g/kg or GM-CSF 250g/m G-CSF 50g/m
Treatment duration
D1- 0.5x109/l for 3 days D1-D12 D1->1.5x109/l for 2 days D1- 1.0x109/l D1- 0.5x10 /l D5-1.0x10 /l for 2 days D1-0.5x109/l
9 9
ANC> 500/l Days +/-GCSF 10.5 vs 16 10 vs 14 9 (10) vs 11 10 vs 16 12 vs 14 10 vs 14 9.7 vs 13.5 10 vs 14
Days in Hospital 18 vs 24 10 vs 16 18 (16) vs 20 19 vs 21 ? 13 vs 16 12 vs 15 ?
pvalue S S S S S S S S
McQuaker 135 1997 Lee 1998 136 1998
31 G-CSF 300g 20 G-CSF 50g/m
Shimazaki 137 1994
R: Randomised? Pts No.: Number of Patients A recent prospective, randomized phase III AEIOP study of G-CSF (filgrastim) following allogeneic, autologous bone marrow and peripheral blood progenitor cell transplantation in children was reported by S. Dallorso et al for the Italian group (Dallorso, S. et al. 2000. Acta Haematologica 103, S1, p.92.). Among the PBSC transplant group the majority of 76 children had solid tumours. The time to reach ANC 0.5x109/l was 11 days in the treatment group versus 14 days in the control group (p=0.0001). No difference was depicted for platelet recovery, transfusional support, duration of fever, antibiotic therapy and length of hospitalisation. However, it is difficult to draw definite conclusions from this study on the possible impact of GCSF (filgrastim) in view of the number of patients and their treatment heterogeneity associated with various protocols.
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A very recent French randomized trial compared 2 schedules of G-CSF (lenogastrim) after autologous peripheral stem cell transplantation (APSCT) to a third group without Lenogastrim (D. Valteau-Couanet, et al for the Socit Franaise de Greffe de Moelle (SFGM) (Valteau-Couanet, D. et al. 2001. Bone Marrow Transplant 27, S1, abstr. OS107). Both children and adults either with haematological malignancies or solid tumours underwent consolidation with high-dose chemotherapy (HDC) TBI and APBSCT and received after randomisation 150 g/m/d of intravenous Lenograstim starting on day 1 post SCT (G1), day 5 post SCT (G5) or no Lenograstim (G0). Randomisation was stratified on the conditioning regimen (Busulphan vs TBI vs no Busulphan and no TBI) and the CD34+ cells count (> 3 x106/kg vs < 3 x 106/kg). Between September 1998 and December 1999, 240 patients entered this trial, 239 are evaluable, 80, 80 and 79 in the G5, G1 and G0 groups respectively. Patients in the 3 groups were comparable in terms of age, underlying malignancy, previous treatment, conditioning regimen and number of CD34+ cells. Median duration of neutropenia (<0.5 x 10x9/l) was significantly shorter in the 2 groups receiving G-CSF (Lenograstim): 10 days (5-15) and 9 days (4-20) in G5 and G1 groups respectively compared to the G0 group : 13 days (7-36) (p<0.0001). Neutropenia duration was significantly longer (p < 0.01) after TBI containing regimens, but as the other regimens was significantly reduced when G-CSF (Lenograstim) was administered after APBSCT. Median duration of G-CSF (Lenograstim) administration was significantly shorter in the G5 group 8 days (4-15) compared to the G1 group 11 days (6-34) (p<0.0005). The median duration of thrombocytopenia did not differ in the 3 groups. It was 5 (1-65), 7 (1-21) and 6 (1-34) in the G5, G1 and G0 groups, respectively. The incidence of infectious complications, visceral toxicity and duration of hospitalisation were similar in the 3 groups. The study concluded that Administration of G-CSF (Lenograstim), post autologous PBSCT, was associated with a significantly more rapid recovery from neutropenia without impairing platelet recovery. No significant difference was observed between the groups where G-CSF (Lenograstim) was administered on day 1 versus day 5. No difference in non-haematological toxicities was observed between the 3 groups. A cost-efficacy study is in progress. Therefore the administration of G-CSF (filgrastim) starting at day 5 post SCT is recommended in this study.
14.3.2 MODE OF ADMINISTRATION Based on the results of the above French trial, G-CSF (filgrastim) administration following stem cell transplant will be given to all patients according to the established standard: 5g/kg G-CSF (filgrastim) from DAY 5 post PBSCR until a stable increase of WBC > 5 x 109/l or ANC > 0.5 x 109/l on two consecutive blood cell counts with a 48h interval.
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15 Supportive Care Guidelines

All the treatment described in this protocol, in particular the induction and MAT, is intensive and aggressive and will be followed by severe bone marrow depression. Hence, treatment according to this protocol should be restricted to institutions who are familiar with the administration of intensive aggressive combination chemotherapy and where the full range of supportive care is available.
15.1 Anti-Emetics
Antiemetic therapy should be administered according to institutional guidelines, e.g. Ondansetron 5 mg/m (maximum single dose 8 mg) p.o./i.v. every 12 hours for 5 days.
15.2 Hydration
Sufficient hydration (2 to 3 l/m) with appropriate electrolyte supplementation must be provided during chemotherapy. Monitoring of blood pressure, cardiac and respiratory rates, body weight, and diuresis is mandatory. The application of diuretics may become necessary in the case of oedema or hypertension.
15.3 Blood Component Therapy

Due to the risk of graft versus host reactions in patients on chemotherapy (especially in the case of high-dose therapy) all blood products (except fresh frozen plasma) should be irradiated with at least 15 Gy prior to transfusion, according to national policies. The use of leukocyte filters for leukocyte depletion (CMV negativity) is advised. Red blood cells Keep the haemoglobin level above 8g/dl. Platelets Platelet substitution is advised when the platelets are < 10 x 109/l, and/or there is clinical evidence of bleeding.
15.4 Central Lines

The use of central lines is strongly recommended. Especially in HDT patients, multi-lumen central lines are essential for PSC sampling and supportive care.
15.5 Treatment of Infections

The HR-NBL 1/ESIOP study is a very intensive protocol which is likely to result in prolonged episodes of neutropenia and infection. All participating institutions must be familiar with managing such problems according to the accepted general principles of supportive care. During episodes of fever and neutropenia patients need to be admitted to hospital for adequate diagnostic measures and appropriate treatment.
15.6 Pneumocystis Pneumonitis Prophylaxis

Pneumocystis carinii pneumonitis prophylaxis is mandatory, but may be given according to the recommendations of each national group. Patients should start at the time of diagnosis up to 6 months after MAT.
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During COJEC Patients should be considered for prophylactic sulfamethoxazole/trimethoprim (5 mg TMP/kg/day divided in two equal doses and given orally 3 days a week) . For sulfa-intolerant patients it is recommended that inhaled pentamidine or a preparation of trimethoprim only is used as prophylaxis. PCP prophylaxis using a pentamidine nebuliser at three-weekly intervals can be encouraged for children who are able to co-operate with jet inhalation (necessary to be effective) which is usually only the case for children of school age. During MAT Stop prophylactic treatment with SMZ/TMP from day 0 until at least day +10 or until WBC 1.0 x 10x9/l after stem cell reinfusion.
15.7 Nutrition
According to the ENSG5 experience patients on the COJEC schedule require close monitoring of patient weight, food intake and tolerance. The early start of supplementary nutrition is highly recommended. Once a 10% weight loss occurs, the institution of naso-gastric alimentation with a high caloric nutritional formula and/or parenteral nutrition via the central venous line according to institutional standards is recommended.
15.8 Additional Supportive Care during the Acute Phase of MAT

Protective isolation per local institutional guidelines. Prophylactic antifungal treatment with ketokonazole, itraconazole or fluconazole should be avoided, because of the increased risk of VOD with these drugs in particular in association with busulphan. Prophylaxis against HSV, VZV and/or CMV will be given in seropositive patients as per institutional guidelines. Infections : Documented or suspected infections during the cytopenic phase will be treated with appropriate antibiotics, anti-fungals, and/or antivirals as determined by the treating physician. Prophylactic use of the latter, in view of side effects and drug interactions, is strongly advised against.
15.9 Prophylaxis, Diagnosis and Management of Hepatic Veno Occlusive Disease

In spite of the published results of low dose heparin prophylaxis against hepatic venoocclusive disease (HVOD),138 the administration of heparin did not, in the French experience, improve the incidence or the severity of HVOD after administration of a busulphan containing regimen. A randomised study demonstrated that prophylaxis with ursodiol decreased the incidence of this complication in patients receiving allogeneic bone marrow transplantation after a busulphan-cyclophosphamide preparative regimen. The incidence of HVOD was 40% in the placebo group and 15% in ursodiol recipients.139 Since July 1998, ursodiol has been administered orally to all patients treated with a busulphan containing regimen without concomitant administration of heparin in the Transplant Unit of Institut Gustave Roussy with a significant decrease in the incidence and severity of HVOD.
Recently V. Lapierre et al. reported on further IGR studies, suggesting that G-CSF mobilized PBSCR results in an increased incidence of anti-HLA-immunization and further confirms that the use of such a graft alters allo-immune Ab responses. It was recommended to alter the transfusion policy and not use ABO-incompatible platelet transfusions. This may help to further reduce HVO-incidence. (Lapierre,V. et al. 2001.Marrow Transplant 27, S.1, abstr.PRS314) 15.9.1 PROPHYLAXIS OF HVOD NO HEPARIN URSODIOL Schedule of administration: Oral administration, twice a day Dosage : 300mg/m2/day, i e 150 mg/m2/ per dose in the morning and evening Presentation in France: URSOLVAN : 200 mg capsules DELURSAN : 250 mg tablets The tablets can be crushed and mixed with stewed fruit, yoghurt. It tastes bad. In the case of vomiting occurring within half an hour of administration, it should be administered again. Treatment duration: The administration twice a day during the entire prophylactic period even in the case of mucositis from day 8 until day +80 post stem cell reinfusion is of major importance according to Essell et als publication139. 15.9.2 DIAGNOSIS OF HVOD Clinical features According to MacDonald140 VOD is clinically defined by the combination of at least two of the three following criteria: liver enlargement and/or pain in the right hypochondrium jaundice ascites and/or unexplained weight gain exceeding 2.5% of baseline value Pleural effusion may be observed, and can contribute to respiratory distress. Fever even if rarely described in the literature is frequent, nevertheless an extensive infection screen should be carried out, and repeated in order to eliminate an infectious cause of the above signs.
Biological features
Hepatic function is often disturbed, with hyperbilirubinaemia in more than 90% of cases. Elevated transaminases occur in 60-70%. Coagulation abnormalities are less frequent, reduction in Factor VII & X are most frequent, and may occur before any clinical signs. A decrease in urinary sodium output is an early finding and constant (< 10 mmol/l), its normalisation is often the first sign of clinical recovery. Moderate abnormalities in renal function may be observed, often as a result of the restricted fluid regimen imposed. There is a significant increase in platelet transfusion requirements.141
Ultrasonography
The ultrasound findings are liver and spleen enlargement, ascites, gall bladder wall thickening, reduction of hepatic vein diameter, enlargement of the portal vein diameter and visualisation of the paraumbilical vein. Doppler ultrasound can be useful to confirm the diagnosis. The results can be predictive and of prognostic relevance.142
Histological features
Hepatic biopsy should not be performed on these children who are often in a perilous clinical state, except in the rare situation where there is doubt about the diagnosis. Histologic features consist of concentric subendothelial thickening and luminal narrowing of terminal hepatic venules or small sublobular veins by either oedematous reticulum fibres or collagen.143 15.9.3 MANAGEMENT OF HVOD
Symptomatic Treatment
These children are usually very unwell and require supportive care until the hepatic lesions improve. The following measures may assist in this: Fluid restriction (60 ml/kg/day), adjusted according to renal function. Strict sodium restriction, note that platelet transfusions contain considerable amounts of sodium. Spironolactone (5 mg/kg/day), 5 days a week (with a 2 day break because of the long half life, reducing problems related to the accumulation of metabolites). This diuretic treatment is not always effective. Furosemide is ineffective, and may exacerbate renal failure. It may sometimes be useful with blood product transfusions. Platelet transfusions should be given, to maintain the platelet count in excess of 20 x 109/l. This level may be difficult to maintain even with twice daily transfusions. Opiate analgesia may be required for abdominal pain. Abdominal paracentesis to drain ascites should only be carried out where the volume of ascites is causing serious respiratory distress, or when pain cannot be controlled by analgesia. However pain may be related to the enlarged liver rather than the ascites. Albumin administration is not recommended, but might be considered when the albumin level is profoundly low (< 15g/l).
15.10 Renal Function Monitoring

15.10.1GLOMERULAR FUNCTION GFR Serum creatinine should be monitored prior to each chemotherapy course. Glomerular function is to be assessed according to national / group guidelines, applying either isotope clearance, or calculated creatinine clearance (only acceptable during induction phase, not allowed to be used for creatine clearance determination prior to MAT). According to Schwartz's formula,144 creatinine clearance (Ccrea) can be calculated from single serum samples:
C crea = F x Height [cm] [ml/min/1.73m] Crea serum [mg/dl]
where F is proportional to body muscle mass, hence depending on age and gender: Male, 1-16 years F = 0.55 Female, 1-21 years F = 0.55 Male, 16-21 years F = 0.70 Normal values [ml/min/1.73m]: 1 year: 120
16 Statistics and Biometrical Methodology

The main scientific aims of this trial address therapeutic options to improve control of disease in two sequential randomisations (R1 and R2). In addition a randomised supportive care question (R0) is asked during the induction phase. Participation in R0 is not a prerequisite to be eligible on the study nor for R1.
16.1 End Points

16.1.1 PRIMARY ENDPOINTS Randomisation 1: MAT-question (BUMEL vs CEM) The primary endpoint is the 3-year event free survival (EFS) calculated from the date of the first randomisation. The following will be considered as events: disease progression, death from any cause second neoplasm. Patients lost to follow-up without event will be censored at the date of their last follow up evaluation. The aim is to compare the 3-year EFS of the two MAT regimens. Randomisation 2: Immunotherapy-Question (Ch14.18 antiGD2 mAb) The primary endpoint is 3-year event free survival calculated from the date of the second randomisation. The following will be considered as events: disease progression, death from any cause second neoplasm. Patients lost to follow-up without event will be censored at the date of their last follow up evaluation. The aim is to test the effect of immunotherapy on 3-year EFS. Randomisation 0: Supportive Care question (G-CSF) The primary endpoint is the mean number of febrile episodes per cycle during COJEC induction. 16.1.2 SECONDARY ENDPOINTS 16.1.2.1 Secondary endpoints of the main trial The response rate following induction chemotherapy on the basis of bone marrow and mIBG evaluations. Response will be assessed according to the International Neuroblastoma Response Criteria after 4 and 8 induction chemotherapy cycles and MAT. The 5 year EFS and overall survival. The toxicity of the two MAT regimens Response rates and EFS will be related to potential prognostic factors including: Biological factors (MycN amplification, 1p deletion, ploidy, 17 q+, CD44 and Trk-A of neuroblastoma cells in the bone marrow and/or the primary tumour). Serological factors (serum concentrations at diagnosis of LDH, ferritin, neurone specific enolase). Urinary catecholamines at diagnosis (VMA, HVA, Dopamine)
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16.1.2.2 Secondary endpoints for R0: the rate of failure to harvest sufficient CD34+cells/kg BW at peripheral stem cell harvest the number of platelet transfusions per patient the proportion of patients with severe bleeding episodes the time to recover 100 000 platelets after day 70 (as prerequisite for surgery) of patients with and without G-CSF
16.2 Randomisation
Randomisation and data handling will be undertaken in the trial office. The study committee and investigators will be unaware of patients treatment assignments. MAIN TRIAL OFFICE: Childrens Cancer Research Institute (CCRI) / Unit for Applied Clinical Research and Statistics (UACRS) St. Anna Childrens Hospital, Kinderspitalg 6 , A-1090 Vienna/Austria Phone: 0043-1-40170-475, Fax: 0043-1-40170 437 E-mail: HRNBL1@ccri.univie.ac.at 16.2.1 RANDOMISATION 1: MAT-QUESTION 16.2.1.1 Randomisation requirements patients registration and full eligibility criteria for entry onto the study at diagnosis confirmed stage (high-risk locoregional disease or primary distant metastatic disease) biological results available eligibility criteria met for randomisation 1 signed informed consent for R1 16.2.1.2 Timing of randomisation All patients eligible for the first randomisation will be randomised at the end of induction after completion of full re-staging as defined and provided sufficient stem cells harvested To either receive the BUMEL or the CEM MAT regimen. 16.2.1.3 Mode of randomisation Randomisation will be done by minimisation* taking account of the following factors: Age For patients with an age at diagnosis of between one and two years a superior prognosis to those over the age of two is expected. Therefore age at diagnosis needs to be considered in the trial design. Stage Patients will be stratified considering highrisk locoregional disease (stage 2 and 3 with MycN amplification) and primary distant metastatic disease. MycN status National group for a random assignment of approximately equal numbers of patients from each of the strata.
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16.2.2 RANDOMISATION 2: IMMUNOTHERAPY-QUESTION 16.2.2.1 Randomisation requirements Participation in randomisation 1 eligibility criteria met for randomisation 2 signed informed consent for R2 16.2.2.2 Timing of randomisation All patients eligible for the second randomisation will be randomised after completion of disease restaging after MAT prior to local radiation . 16.2.2.3 Mode of randomisation Randomisation will be done by minimisation* with the following factors: National group Allocated treatment in the first randomisation 16.2.3 RANDOMISATION 0: G-CSF-QUESTION 16.2.3.1 Randomisation requirements patients registration and full eligibility on study at diagnosis confirmed stage (risk locoregional disease or primary distant metastatic disease) signed informed consent for R0 16.2.3.2 Timing of randomisation Once a patient is registered and fully eligible on study at diagnosis with confirmed stage (risk locoregional disease or primary distant metastatic disease) randomisation R0 will be performed without delay. . 16.2.3.3 Mode of randomisation Randomisation will be done by minimisation* taking into account the following factors Stage stratification according to highrisk locoregional disease (stage 2 and 3 with MycN amplification) and primary distant metastatic disease. National group for a random assignment of approximately equal numbers of patients from each of the strata.
* see Pocock, Stuart J. 1997. Clinical trials: a practical approach. New Yor: John Wiley & Sons
16.3 Patient Number Estimates

Based on the annual accrual rates of previous national studies the estimated accrual of high-risk patients per year according to the national co-ordinators are as follows: Austria 5pts, Belgium 6pts, France 40pts, Israel 15 pts, Italy 40 pts, Nordic Countries (Denmark, Sweden, Norway) 10pts, Portugal 6 pts, Spain 14pts, Switzerland 3pts, United Kingdom 40 pts. [Germany 55 pts after the German trial is closed by the end of 2001, i.e. one year after the official start of the HR-NBL-1/ESIOP study].Thus a total accrual of approximately 175 pts/year is expected NOT INCLUDING Germany.
16.4 Power Considerations

RANDOMISATION R1: MAT QUESTION About 75 % of the expected annual recruitment of 175 patients will enter the first randomisation, i. e. about 130 randomisations/year. The 3-year EFS in the CEM-group (without immunotherapy) is estimated to be 45 %. This study aims to show an improvement of 10 % for the BUMEL-group (3-year EFS of 55%). With a recruitment period of 6 years (1050 pts entered and 780 randomised) and a minimum follow up of 1.5 years the power to show a 10% difference is 89% (bilateral formulation of the logrank test and =5%)145 irrespective of whether there is a benefit from immunotherapy.
16.4.1
16.4.2
RANDOMISATION R2: IMMUNOTHERAPY QUESTION 60% of the patients entered into the trial are expected to be eligible for the 2nd randomisation. This means that there will be about 105 randomisations for the immunotherapy question/year and a total of 630 randomisations in a recruitment period of 6 years. For the study to be able to show a 10% improvement with a two sided alternative hypothesis and =5 % there is power of 81% irrespective of whether there is a 10% difference in 3-year EFS in favour of BUMEL or not.145
RANDOMISATION R0: G-CSF QUESTION About 75 % of the expected annual recruitment of 175 patients will enter randomisation 0, i.e. about 130 randomisations/year. The mean number of febrile episodes per cycle of COJEC without G-CSF is estimated to be 0.5, i.e. 4 febrile episodes in 8 cycles. This study aims to show an improvement of 0.125 to 0.375 febrile episodes, i. e. an average reduction of 1 febrile episode in 8 cylces of chemotherapy. It is anticipated that the standard deviation () is approximately 0.3. To achieve a power of 80% with a type I error rate of 5% a sample size of 184 patients is needed. Since the estimation of with 0.3 may be imprecise, an internal pilot study is planned. An initial sample of 92 subjects is chosen, corresponding to half of the previously fixed sample size and is used as an internal pilot to estimate more precisely. This will allow to re-estimate the final sample size required 146. If is confirmed the final sample size will not be significantly changed. If the standard deviation is found to be substantially lower, fewer patients are needed If = 0.21 only half of the previously estimated sample size is needed, i.e. approximately 92 patients only. In this case no further recruitment would be necessary. If =0.4 about twice as many patients would be needed, i.e. resulting in a need for 368 patients. If the sample size becomes too large against expectations, the DMC will re-evaluate this issue and may abandon R0 randomisation if no result can be reached within the main trial.
16.4.3
16.5 Analysis
16.5.1 FINAL ANALYSES The final analysis will be performed 18 months after the inclusion of the last patient. The first step of the analysis will be to verify that there is no significant interaction for the EFS between the two factors (randomisations). This will be done in a Cox regression restricted to the patients included in both randomisations. Interactions are considered to be very unlikely. To evaluate the difference of EFS between the randomised arms all randomised patients will be analysed and the comparison of treatment regimens will be performed according to intention to treat. The 3-year EFS will be estimated by the Kaplan-Meier method. The standard errors of these estimates will be calculated according to the method described by Peto et al.147
16.5.1.1 Randomisation 1: MAT Question (BUMEL vs CEM) The difference in EFS will be analysed using the log rank-test (two-sided =5%). The log rank statistic will be adjusted on the prognostic factors age and stage and on national groups. (see Mantel, N. and Haenszel, W. 1959. Journal of the National Cancer Institute, 22, 719 748) 16.5.1.2 Randomisation 2: Immunotherapy Question (Ch 14.18 antiGD2 mAb) To evaluate the effect of immunotherapy the log rank-test will be used (bilateral =5%). The log-rank statistic will be adjusted on the prognostic factors age and stage and on national groups as well as on the assigned treatment for MAT (levels: CEM, BUMEL). (see Mantel, N. and Haenszel, W. 1959. Journal of the National Cancer Institute, 22, 719 748) 16.5.1.3 Randomisation 0: Supportive care question (G-CSF ) The difference in the number of febrile episodes per cycle will be analysed using the modification of the two sample t-test described by Denne and Jennison.146 16.5.2 INTERIM ANALYSES The study will be monitored by an independent committee according to a group sequential monitoring plan. In order to stop the trial early and reject the null hypothesis of no difference between the randomised arms the alpha spending function approach of Lan and DeMets (see Lan, K.K.G. and DeMets, D.L. 1983. Biometrika 70, 659-663) with OBrien and Fleming type148 spending function will be followed to conclude at each sequential analysis.. The boundaries proposed in that rule require very strong evidence of an effect to terminate at the first interim test, whereas the criteria at the final test are quite close to those for a fixed sample design (i.e., a design with no interim testing). Three interim analyses are planned after observing 25%, 50% and 75% of the number of expected events. Below is an approximate schedule of the time of these analyses. Approximate Time Table 1st Randomisation (BUMEL vs. CEM MAT) p-value events approximate time of the analyses 0.001 120 3 years First analysis 0.0039 240 4.5 years Second analysis 0.0184 360 6 years Third analysis 0.0412 480 7.5 years Final analysis Approximate Time Table 2nd Randomisation (Immunotherapy) p-value events approximate time of the analyses 0.001 97 3 years First analysis 0.0039 194 4.5 years Second analysis 0.0184 291 5 years 9 months Third analysis 0.0412 388 7.5 years Final analysis The schedule of the planned interim analyses is only a guideline and calculated under the assumption of exponential EFS and constant inclusion rate. The first interim analysis is scheduled 3 years after the study start, the second after 4.5, the third after 6 years. The results of the interim analyses will be reviewed by the DMSC (Independent Data Monitoring/ Safety Committee). The recommendation to close or continue a trial is essentially a medical one and the statistical stopping rules will be used as guidelines. Nevertheless, if a -value falls below the specific thresholds it is anticipated that the interim analysis will be
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discussed with the Trial Management Committee (TMC). The final decision or recommendation to modify or terminate entry to the whole or one part of the trial rests with the TMC. It is anticipated, even if the first randomisation is discontinued, that the trial will continue for the second randomisation on immunotherapy with the recommended MAT arm only. Similarly it is anticipated that if the second randomisation is discontinued the trial should be continued for the first randomisation (MAT question).
16.6 Monitoring Guidelines for Severe Toxicities

Severe toxicities will be monitored continuously and summarised for group members on a 2-monthly basis. Therefore toxic deaths, surgical events, and MAT related events must be reported immediately. The study centre must also be informed within 1 week about all surgical interventions performed and about patients who are going off study due to progression during the induction chemotherapy. This is needed to be able to judge toxicity rates correctly. 16.6.1 RELATED TO INDUCTION PHASE 1. Toxic deaths occurring after diagnosis during induction treatment If the Kaplan Meier estimate of toxic deaths at 90 days exceeds 5 % then the toxic deaths will be formally reviewed to check if any modification to the induction therapy is required. 2. Failure to harvest sufficient CD34+cells/kg BW at peripheral stem cell harvest The rate of failure to harvest sufficient CD34+cells/kg BW at peripheral stem cell harvest will be monitored after 30, 60, 90, 120 and 200 patients have finished induction treatment. If the failure rate exceeds 15 %, the data will be formally reviewed to check if any modification to the induction therapy is required. 3. Particular issues for R0 randomisation Interim analyses related to R0 randomisation will be done after 30, 60 and 90 patients have finished induction treatment comparing the G-CSF (filgrastim) group versus the control group. - The rate of failure to harvest sufficient CD34+cells/kg BW at peripheral stem cell harvest exceeds 10 % in the control group and 20% in the G-CSF (filgrastim) group, the data will be formally reviewed to check if any modification to the induction therapy is required. 16.6.2 RELATED TO SURGERY We assume that about 75% of the 1050 patients will undergo surgery, i.e. about 780 operations. If the Kaplan Meier estimated surgical event rate exceeds 5 % within 15 days after surgery then the surgical events will be formally reviewed to check if any modification to the surgical procedure is required. 16.6.3 RELATED TO MAT The absolute severe adverse MAT related event rate observed in each arm of the first randomisation will be compared to a reference rate in order to detect an absolute excess of toxic events. The need for ventilation or haemofiltration in an ICU setting or death are considered adverse MAT related events. If the Kaplan-Meier estimate of the MAT related event rate within 100 days exceeds 10 % the information will be forwarded to the DMC. The DMC will then make a recommendation, after consultation with the study co-ordinators and statisticians, as to how the study should proceed.
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APPENDICES SECTION 17 APPENDIX STAGING CRITERIA ACCORDING TO INSS

17.1 Diagnosis of Neuroblastoma
A diagnosis of neuroblastoma is established if : 1. An unequivocal pathological diagnosis is made from tumour tissue by light microscopy (with or without immunohistology, electron microscopy, increased urine or serum catecholamines or metabolites ) OR 2. Bone marrow contains unequivocal tumour cells (e.g. syncytia or immunocytologically positive clumps of cells) and increased urine or serum catecholamines or metabolites *. Notes If histology is equivocal, karyotypic patterns or abnormalities in tumour cells characteristic of other tumours [e.g. t(11;22)], then exclude a diagnosis of neuroblastoma, whereas genetic features characteristic of neuroblastoma (1p deletion, MycN amplification) would support this diagnosis. * Catecholamines and metabolites include dopamine, HVA and/or VMA; levels must be > 3 SD above the mean for age (measured in mmol per mmol creatinine)# to be considered increased, and at least two of these metabolites must be measured. # Timed urine collections are difficult in young children so normalisation per mmol creatinine does away with the necessity for a timed collection and also avoids the problem of false negatives due to dilute urine.
17.2 Assessment of Extent of Disease

Tumour Site Primary Tumour Metastatic Sites Bone marrow Bilateral posterior iliac crest marrow aspirates and trephine (core) bone marrow biopsies required to exclude marrow involvement. A single positive site documents marrow involvement. Core biopsies must contain at least 1 cm of marrow (excluding cartilage) to be considered adequate MIBG scan; 99Tc scan is required if MIBG scan negative or unavailable, and plain radiographs of positive lesions are recommended. Clinical examination (palpable nodes), confirmed histologically. CT scan for non-palpable nodes (3D measurements) CT and/or MRI scan* with 3D measurements AP and lateral chest radiographs. CT/MRI necessary if chest radiograph positive, or if abdominal mass/nodes extend into the chest Recommended Tests CT and/or MRI scan* with 3D measurements. MIBG scan if available.
Bone Lymph nodes Abdomen/liver Chest
Notes * Ultrasound examination considered suboptimal for accurate 3D measurements The MIBG scan is applicable to all sites of disease.
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17.3 INSS Staging

Stage 1 Localised tumour with complete gross excision, with or without microscopic residual disease; representative ipsilateral lymph nodes negative for tumour microscopically (nodes attached to and removed with the primary tumour may be positive). Localised tumour with incomplete gross excision; representative ipsilateral non-adherent lymph nodes negative for tumour microscopically. Localised tumour with or without complete gross excision, with ipsilateral non-adherent lymph nodes positive for tumour. Enlarged contralateral lymph nodes must be negative microscopically. Unresectable unilateral tumour, infiltrating across the midline*, with or without regional lymph node involvement; or localised unilateral tumour with contralateral regional lymph node involvement; or midline tumour with bilateral extension by infiltration or lymph node involvement. Any primary tumour with dissemination to distant lymph nodes, bone, bone marrow, liver and/or other organs.(except as defined for Stage 4S). Localised primary tumour (as defined for Stage 1, 2A or 2B), with dissemination limited to liver, skin, and/or bone marrow . (limited to infants < 1 year of age)
Stage 2A Stage 2B Stage 3
Stage 4 Stage 4S
Notes * Multi-focal primary tumours (e.g. bilateral adrenal primary tumours) should be staged according to the greatest extent of disease, as defined above, and be followed by a subscript "M" (e.g. Stage 3M). The midline is defined as the vertebral column. Tumours originating on one side and crossing the midline must infiltrate to or beyond the opposite side of the vertebral column. Marrow involvement in Stage 4S should be minimal, i.e. less than 10% nucleated cells on bone marrow biopsy or quantitative assessment of nucleated cells on marrow aspirate. More extensive marrow involvement should be considered Stage 4. The MIBG scan (if done) should be negative in the marrow for Stage 4S.
17.4 INRC Definition of Response

Response CR VGPR PR Primary tumour* No tumour Decreased by 90-99% Metastatic Sites* No tumour; catecholamines normal No tumour; Residual 99Tc bone changes allowed Decreased by > 50% All measurable sites decreased by > 50% Bones and bone marrow : Number of positive sites decreased by > 50%; no more than 1 positive bone marrow site allowed. No new lesions; > 50% reduction of any measurable lesion (primary or metastases) with < 50% reduction in any other; < 25% increase in any existing lesion. No new lesions; < 50% reduction but < 25% increase in any existing lesion. Any new lesion; increase of any measurable lesion by > 25% ; previous negative marrow positive for tumour.
MR NR PD Notes CR VGPR PR MR NR PD *
Complete Response Very Good Partial Response Partial response Mixed Response No Response Progressive Disease Evaluation of primary and metastatic disease as outlined in Table 2
One positive marrow aspirate or biopsy is allowed for PR if this represents a decrease in the number of positive sites at diagnosis.
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18 APPENDIX Pathology and Biology Guidelines for resectable and unresectable Neuroblastic Tumours and Bone Marrow Examination Guidelines
Peter F. Ambros, Ph.D. and Inge M. Ambros, M.D., for the SIOP Europe Neuroblastoma Pathology, Biology and Bone Marrow Group In co-operation with: Gabriele Amann M.D. Klaus Beiske M.D., Ph.D. Jean Benard Ph.D. Maria Boavida Nick Bown Bruno de Bernardi M.D. Emanuele d'Amore M.D. Huib Caron M.D., Ph.D. Valerie Combaret Ph.D. Jerome Couturier M.D. Olivier Delattre M.D. Laurence Faulkner M.D. Claudio Gambini M.D. Nicole Gross Per Kogner M.D. Ruth Ladenstein, M.D. John Lunec Ph.D. Tommy Martinsson Ph.D. Jean Michon M.D. Rosa Noguera Andrew D. J. Pearson M.D. Michel Peuchmaur M.D. Frank Speleman Ph.D. Gian Paolo Tonini Ph.D. Nadine Van Roy Ph.D. Lisa Walaas M.D. (Austria, g.amann@akh-wien.ac.at) (Norway, klaus.beiske@labmed.uio.no) (France, benard@igr.fr) (Spain, ffjpb@mail.telepac.pt) (United Kingdom, Nicholas.Bown@newcastle.ac.uk) (Italy, brunodebernardi@ospedale-gaslini.ge.it) (Italy, eda@ux1.unipd.it) (The Netherlands, H.N.Caron@amc.uva.nl) (France, COMBARET@lyon.fnclcc.fr) (France, jerome.couturier@curie.net) (France, Olivier.Delattre@curie.fr) (Italy, lfaulkn@tin.it) (Italy, claudiogambini@ospedale-gaslini.ge.it) (Switzerland, Nicole.Gross@chuv.hospvd.ch) (Sweden, Per.Kogner@ks.se) (Austria, ladenstein@ccri.univie.ac.at) (United Kingdom, John.Lunec@newcastle.ac.uk) (Sweden, tommy.martinsson@clingen.gu.se) (France, jean.michon@curie.fr) (Spain, Rosa.Noguera@uv.es) (UK, A.D.J.Pearson@ncl.ac.uk) (France, michel.peuchmaur@rdb.ap-hop-paris.fr) (Belgium, franki.speleman@rug.ac.be) (Italy, tonini@cba.unige.it) (Belgium, nadine.vanroy@rug.ac.be) (Norway, FAX: ++47-23071410)
General Remarks
The tumour should be transferred from the operating room to the local pathology department under sterile conditions as quickly as possible. Tumour handling and sectioning must always be performed by the pathologist (as soon as possible; the time of tumour sample were taken should be stated) following the scheme presented in these Guidelines. At least two macroscopically different tumour areas (if present) should be chosen for molecular-genetic/biological analyses.
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The patients peripheral blood (5-10ml, with EDTA or heparin or according to the requirements of the laboratory) is needed for molecular-biological studies as reference and should be sent to the reference biology laboratory together with the tumour specimens. The material selected for molecular-genetic/biologic investigations should be sent as quickly as possible to the National Reference Biology Laboratory. Please refer to the Appendix (chapter 18) for details specific to your National Group. In case of queries please contact the National group co-ordinator. The paediatric oncologist in charge and/or the surgeon has to inform in a timely manner the local pathologist and biologist (local and/or national) about the new patient and the material to be expected.
PATHOLOGY GUIDELINES
18.1 Remarks and Recommendations for Pathology
The handling of the tumour tissue should always be performed by the pathologist who, besides the important task of making morphologic diagnoses and giving prognoses based on histopathologic findings, should choose the relevant tumour areas for moleculargenetic/biological analyses as another major task. This procedure is a sine qua non to enable reliable interpretation of the molecular-genetic results for which the exact tumour cell content of the specimen used for these investigations has to be determined. This is possible only if the pathologist evaluates the specimens adjacent to those used for molecular-genetic/biological analyses (for details see below). In all instances, concerning either tumour resection or biopsies, tumour material from different tumour areas (nodules are of special interest!) ought to be taken for histologic and molecular-genetic/biological examination. The reason for this recommendation is based on the observation of tumour heterogeneities at the genetic level (e.g. for the MYCN and/or the chromosome 1p status) and/or at the histologic level (ganglioneuroblastoma, nodular subtype according to the International Neuroblastoma Classification, INPC,103 both of which have prognostic implications. Close co-operation between pathologists and biologists is therefore strongly recommended. Pathologists should inform the biologists if morphologically unfavorable looking areas are present in the paraffin embedded material but most likely not in the specimens selected for molecular-genetic/biological investigations. These areas should also be specifically analysed using the paraffin material. In case the tumour pieces selected for molecular-genetic/biological investigations were not appropriate for getting reliable results, MYCN and chromosome 1p36.3 status and ploidy can be determined on the paraffin embedded material.
18.2 Sectioning and Securing Tumour Material in Case of Resected Tumours

The following procedure is recommended (it is necessary for the pathologist to have help from a person, i.e. from a technician or the paediatric oncologist): Cut the tumour along the largest diameter and take at least two samples from morphologically different-appearing areas (1x1x1cm) if such are present. Tissue from a suspected nodule must always be sampled. Identify the samples specifically with capitals (A, B, etc.), or whatever system is the practice of each laboratory, and cut each of them into four pieces which are marked with numbers (e.g. tumour specimen A 1-4, specimen B 1-4). More material
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can be processed in the same way (C, D, etc.), but material from two different areas is the minimum. Check carefully for the presence of nodules! (See also Figure 1). Samples A1 and B1: make 10 touch preparations (at least 5) from a freshly cut surface. The slides are air-dried and unfixed and, if necessary, they can be stored at 20C (storage of the slides for one week at room temperature does not adversely affect the following analyses); for fluorescence based in situ hybridisation (FISH) and image cytometry (ICM). After making the touch preparations, these pieces should be fixed in formalin for routine histologic examination. This also should include the determination and indication of the tumour cell content versus content of normal cells, such as Schwann cells, lymphocytes, fibrovascular stroma etc.; amount of necrosis should be indicated as well. This information is crucial for the interpretation of the FISH, ICM and cytogenetic results! Samples A2,3 and B2,3: snap freeze as soon as possible in separate vials in liquid nitrogen or at 70C carbon dioxide. Before using these for further analyses, making cryosections for the determination of the tumour cell content is mandatory. Samples A4 and B4: put in sterile culture medium (RPMI 1640) for preparation of tumour cell suspensions which may serve for cytogenetics, assessment of MYCN and chromosome 1p status (on cytospin preparations), evaluation of ploidy, drug sensitivity, etc. Tumour cell content should be checked by immunocytology on the cytospin preparations using appropriate antibodies (see below). The samples should be forwarded as soon as possible! After this procedure, the rest of the surgical specimen can be fixed in formalin and worked-up according to standard guidelines. The whole central 4mm section of the tumour at the plane of the largest diameter should be embedded. A minimum of one tumour section per cm should be embedded from the whole specimen including central and peripheral areas of the tumour. Surgical margins must be reliably and reproducibly identifiable. or: 1 3 2 4 2 1 3 4 A, B etc.
A , B etc.
Figure 1. Tumour samples (at least 1x1x1cm) are designated with the letters A, B, etc. There are two methods of sectioning. From piece 1 touch preparations are made before formalin fixation; in the variant on the right hand side, one of the central sections is designated as piece 1. Pieces 2, 3 (and 4) are snap frozen. Piece 4 is put in RPMI 1640 for classic cytogenetic investigation and/or making cytospin preparations. In case of small specimens, touch preparations and snap freezing are the first priorities.
18.3 Securing Tumour Material in Case of Unresectable Tumours

The general recommendations given above should be followed. The sectioning procedure depends on the amount of tumour material available for diagnostic histology and moleculargenetic/biological investigations. Touch preparations can almost always be performed before fixing and embedding the tumour material (for tru cut and fine needle aspirations see below). All the recommendations for open biopsies, tru cut biopsies and fine needle aspirations (e.g. number of different tumour areas) concern only those patients for whom the risk of taking tumour material is considered to be reasonable. This risk has to be
weighed carefully; for example in the case of patients with stage 4s neuroblastomas with coagulation disorders, or patients with large and fragile tumours. It is clear that if the procedure to retrieve material is risky, it should not be performed! One should think about a less dangerous procedure, e.g. biopsy of subcutaneous metastases or bone marrow aspiration, instead of a dangerous biopsy from the primary tumour. Tru cut biopsies (A, B, etc.), at least 1cm long, 0.1cm thick.
Formalin
snap freeze
Figure 2. Sectioning of biopsy cylinder for histologic and molecular-genetic/biological investigations. The exact sizes of pieces used for the individual investigations should be discussed between pathologist and biologist.
18.3.1 OPEN BIOPSIES In case of open biopsy, two different areas of the tumour should be biopsied by the surgeon. Specimen size should equal at least 1cm. In accordance with the recommendations given above, the tumour specimens are labelled with capitals (A, B etc.), are cut into 4 pieces, and the procedure given in the paragraph above can be followed. In case of smaller specimens, the material can be cut into only three pieces (piece 1 for touch preparations and histology; piece 2 for snap freezing; piece 3 for RPMI 1640) or into two pieces (piece 1 for touch preparations and histology and piece 2 for snap freezing). 18.3.2 TRU CUT BIOPSIES General remarks. If only tru cut biopsies are feasible, preferably four samples (at least two biopsies in case of small lesions) from different areas of the tumour should be obtained. The recommended needle size is 18G. Tru cut biopsies are usually ultrasound-guided and performed by radiologists, but the pathologist should be present during the procedure and immediately assess the quality of the specimens (macroscopically). This is in order to save trouble and frustration due to delivery of mainly necrotic material (see below). It is important that the tumour cell content be determined in the biopsied material used for molecular-genetic/biological analyses. Handling of tru cut biopsies. The pathologist/radiologist should not try to scrape off the tissue from the biopsy needle using a scalpel or other devices, but put the needle into a tube/glass with sterile RPMI 1640 or Hanks or PBS solution and shake gently until the specimen is released into the fluid. This procedure helps to avoid artefacts due to crushing and squeezing. In addition, needle biopsies tend to stick to each other which may hamper the subsequent division of the material. Therefore, each biopsy should be kept in a separate tube in the above mentioned sterile solution. Assessment of quality. Specimens should be at least 1 cm long and 0.1 cm thick. Tiny and fragmented material cannot be approved. If there is any doubt regarding the quality of the material, shake the glass with the specimen thoroughly once or twice. If the specimen breaks into pieces, it often indicates tissue necrosis. The pathologist should not hesitate to ask for a new biopsy. It is impossible to judge macroscopically, whether a needle biopsy contains tumour cells or mainly stroma. Therefore, an ideal procedure would be, if feasible, to have tru cut biopsies preceded by fine
needle aspiration which (a) informs about the tumour cell content of the elected area after only a few minutes, and (b) produces cell suspensions for ploidy measurement and large numbers of cytological specimens for FISH analyses. Securing of tru cut biopsies. Either of two acceptable methods can be recommended for the securing of tru cut biopsies.
Method 1) All pieces obtained are identified with capitals (A, B, etc.) and transferred from the tubes into separate Petri dishes, pouring the entire contents of the tube into the dish. Each sample is divided carefully into two parts using a scalpel (Figure 2). This procedure should be done without using tweezers to avoid crush artefacts. One part of each piece is carefully transferred into a tube with formalin using a Pasteur pipette (plastic, for single use) to avoid crush artefacts. The other half of each piece is snap frozen. Put the specimen carefully on the wall of a freezing tube in order to avoid a curved orientation of the piece and put the tube into liquid nitrogen. The pathologist and the biologist should discuss and agree on the size of the parts they need for histology and for the biological analyses. Alternatively, one whole biopsy specimen could be used for histology and the second (third, etc.) specimen can be split. Before the material is transferred to the biologist, the representativity (tumour cell content) must always be checked and documented by the pathologist either on frozen sections or on touch preparations (immunocytology). Method 2) If four pieces are available, two pieces are transferred from the isotonic solution or the RPMI 1640 into 4% buffered formalin in separate glasses and embedded in separate paraffin blocks (marked A and B) for morphologic and immunohistochemical analysis. Two pieces are gently transferred by tweezers into separate, flat-bottomed plastic tubes, carefully overlaid by a waterbased embedding compound for preparation of frozen tissue sections (try to keep the specimen localised at the bottom level of the vial!) and must be snap frozen as soon as possible in liquid nitrogen or at 70C carbon dioxide for molecular-genetic/biological analyses and immunocytochemistry. Ten touch preparations are made as described below. If only two pieces are available for analysis, one is transferred into formalin and embedded in paraffin and the other piece is snap frozen as described above followed by making 10 touch preparations. Procedure for making touch preparations
It is well known that preparations containing whole nuclei (touch or cytospin preparations) are superior for FISH analyses. However, fresh unfixed needle biopsies only rarely yield enough cells when used for touch preparations. This problem can be solved by using the snap frozen material (see above) for making touch preparations. In method 1), the frozen tumour piece can be directly touched on warm glass slides, without thawing the tissue. After this, a frozen section for tumour cell determination should be performed. In method 2) the plug of embedding compound, containing the biopsy material at the bottom, is carefully removed from the tube with a forceps by warming the tube wall between two fingers, and mounted in a microtome at 20C. Frozen sections are cut starting from the bottom of the frozen plug, stained with H&E and screened in a microscope until sufficient areas of morphologically intact tumour cells are found (representativity). It is important to perform frozen sections prior to the preparation of imprints because the cutting procedure removes any embedding compound which might have covered the tissue, merge with the tumour cells and thus impair the quality of the imprints. The cold cut surface of the plug is used for making 10 touch preparations on warm (room temperature) glass slides, but the plug should be carefully refrozen in time, because complete thawing destroys the morphology and renders the material inappropriate for any in situ-analyses at the single cell level. However, such specimens can still serve as sources for DNA/RNA and protein extraction
(Southern blot, Polymerase chain reaction, comparative genome hybridisation, Northern blot, Western blot). Tru cut biopsies eventually frozen in plastic tubes without a water based embedding compound, are well suited for investigations based on DNA/RNA and protein extraction. However, it might pose a serious problem to use small, frozen and non-embedded pieces of e.g. stroma-poor, fragile tumour tissue for preparation of imprints. Without the mechanical support of the embedding compound, they thaw rapidly, become crushed or even lost and unavailable for the analysis of morphology and tumour cell content. In this case frozen, non-embedded tru-cut biopsies are transferred into a water-based compound and frozen sections are cut before preparing imprints. Further comment. If the frozen material does not contain a sufficient number of tumour cells, FISH and ploidy analyses can be assessed on nuclei extracted from the paraffin-embedded biopsy material. 18.3.3 FINE NEEDLE ASPIRATION CYTOLOGY (FNAC) General remarks. In case of unresectable tumours, the pathologists and biologists can also be confronted with tumour material derived from fine needle aspiration (FNA). In general, at least two separate punctures/aspirations should be performed from each tumour. Depending on the size of the tumour, the needle should be moved backward and forward into different areas under constant aspiration in order to sample from more than one region of the lesion (important for representativity!). FNA is also recommended to be done before tru cut biopsies are taken (for determination of the representativity of elected tumour area!). The recommended needle size is: external diameter of 0.6-0.7 mm; 22-23 Gauge. The FNA sampling is usually performed in close co-operation between radiologist and cytopathologist. Due to the results of palpation and ultrasound examination of the tumour, FNA is either performed directly by the cytopathologist (palpable tumours) or by means of ultrasound guidance (non-palpable tumours). In this case, the radiologist guides the needle into the lesion, while the cytopathologist aspirates. Handling It should be considered that the ultrasound gel when aspirated, might ruin the morphology of the cells. Therefore, the skin must be wiped carefully before puncture. An adequate aspiration will often contain 104-106 cells. The aspirate should always be utilised for preparation of smears and cell suspensions. Depending on the amount of aspirate, one droplet is placed on each of at least four slides and ordinary monolayer smears are produced and air-dried. Subsequently, the needle and the rest of aspirate are flushed through with 1 ml sterile phosphate buffered saline and the cell suspension is kept in the Eppendorf vial. One smear from each aspiration is stained by the DiffQuik method which takes 3 min to render the smear light-microscopically evaluable (preferentially done by an assisting cytotechnician!). A preliminary report on adequacy of the sample is given to the clinician and the radiologist, and the cytopathologist decides whether the FNA has to be repeated. Recommendations for subsequent analyses Smears are well-suited for morphologic evaluation (May-Grnwald-Giemsa stain), immunocytology, FISH (MYCN and 1p status etc.), and image cytometry, e.g. for static ploidy analysis. Cell suspensions can be used for flow-cytometry analysis of ploidy, tissue culture for e.g. cytogenetics, preparation of cytospins, which are air-dried over night and used for the same purposes as smears.
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Quality assessment A series of cytospin preparations often contains a more equal (i.e. predictable) number of tumour cells as compared to a series of individually produced touch preparations and smears. One smear/cytospin of each series/aspiration has to be analysed by morphology/immunocytology in order to estimate the number of tumour cells. This information must be sent to the reference biology laboratory. Cytospins, smears and cell suspensions may be stored frozen for future analysis. 18.3.4 BONE MARROW ASPIRATIONS (SEE BELOW) In case of a sufficient high tumour cell number infiltrating the bone marrow, these cells can also be used for determination of the genetic composition of the disseminated neuroblastic tumour cells. If the tumour cell content exceeds 60 per cent, the MYCN copy number can also be determined by SB. Lower infiltrations should always be analysed by FISH for evaluating the MYCN and 1p status. In unclear situations, a GD2 staining preceding the FISH is highly recommended.
18.4 Histology/Cytology Report

Surgically resected tumours. Morphologic classification: The tumour should be classified according to the International Neuroblastoma Classification.103 The mitotic rate and calcifications should also be indicated. Surgical margins of resection: There should be a comment regarding whether there are tumour cells infiltrating the resection margins or not, without making any conclusion as to whether the tumour residual is microscopic or macroscopic. Histologic report on the specimens A1, B1 etc.: This report must clearly indicate the estimated percentage of tumour cells, i.e. neuroblastic/ganglionic cells, versus Schwann cells and other normal cells contained in the samples used for the biological studies. A copy of the report should then be submitted to the molecular biologist. Biopsies. In case of limited biopsy material, it has to be kept in mind that the tumour material obtained is not necessarily representative of the whole tumour. For example, the biopsy could be taken from either a neuroblastic nodule or the ganglioneuromatous area of a nodular ganglioneuroblastoma. In such critical cases, the use of the following term, according to the INPC, is recommended: neuroblastic tumour, unclassifiable. This term relates to a tumour which belongs unequivocally to the peripheral neuroblastic tumour entity, but which cannot be allocated with certainty into one of the four basic categories which are neuroblastoma (Schwann cell stroma-poor), ganglioneuroblastoma intermixed (Schwann cell stroma-rich), ganglioneuroma (Schwann cell stroma-dominant), ganglioneuroblastoma nodular (Schwann cell stroma-rich/-dominant and stroma-poor). Other terms recommended by the INPC to be used for tumours giving rise to problems in classification, are: neuroblastoma (Schwann cell stromapoor), NOS. This term is used for tumours with an unequivocal categorisation, but the subtype, i.e. undifferentiated, poorly differentiated, differentiating, cannot be assessed due to poor quality of the sections, extensive haemorrhage, necrosis, crush artefacts, etc. (see INPC). Ganglioneuroblastoma, NOS is used for a tumour with a stroma-rich/-dominant appearance containing areas of extensive calcification which may obscure a stroma-poor nodule. Fine Needle Aspiration Cytology (FNAC). If only FNAC material is available for primary diagnosis, the report including number, morphology (differentiation status of tumour cells, Schwann cells, necrotic debris) and immunophenotype of the cells analysed must be submitted to the biologist.
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18.5 Tumour Material Obtained after Cytotoxic Therapy

Sectioning of the tumour material in resected tumours or biopsies after cytotoxic therapy can be done following the same guidelines as for tumours resected or biopsied at diagnosis before cytotoxic therapy. However, for sampling, it must be remembered that necrotic areas and also calcifications can be massive. Therefore, it is essential to state exactly the percentage of viable tumour cells versus normal cells (see above), and to indicate the amount of necrosis and calcification. It is known that both chemo- and radiotherapy can induce marked morphologic changes and can also induce cytodifferentiation and maturation (with development of a Schwann cell stroma), but most likely do not change the original genetic characteristics of the tumour. (see also INPC) Therefore, assignment to the prognostic subgroups must not be made, although the tumours can be classified morphologically according to the INPC. The statement on preoperative therapy has to be included in the diagnosis.
18.6 Regional Lymph Node Examination

Biopsy of regional nodes is highly recommended whenever feasible despite their appearance. The histology report should include information on site and number of positive nodes, type of metastatic spread, i.e. presence of micrometastases (less than 2 mm), intranodal parcelled metastases, intranodal massive metastases, nodal metastases with extracapsular extension in localisations not adherent to the resected tumour specimen, and morphologic description of the tumour infiltrate.
18.7 Immunohistology/-cytology
Differential Diagnosis. In some cases, i.e. neuroblastomas, undifferentiated subtype according to the INPC, the differential diagnosis can present difficulties. In these instances, the use of the following antibodies is recommended: MIC2 (CD99), actin, desmin, low molecularweight cytokeratin, leukocyte common antigen (CD45), and vimentin. These antibodies are usually negative in neuroblastic tumours. Positive markers are: CD56 (N-CAM), NB84a, (monoclonal neuron specific enolase (NSE), neurofilament triplet protein (NF), synaptophysin, tyrosine hydroxylase, and the protein gene product 9.5). However, it has to be kept in mind that these markers, although often positive, may also be negative in undifferentiated neuroblastomas. In addition, NB84a also reacts with epithelial cell and endothelial cells (see also below). Although GD2 is positive in virtually all cases of neuroblastic tumours and very useful in for example detection of neuroblastic cells in the bone marrow, anti GD2 staining cannot be recommended for use on paraffin sections (due to very high background staining). Moreover, GD2 expression is not restricted to neuroblastic tumours! Lymph nodes. For differential diagnosis see above. In addition, the morphological result can be controlled by immunohistology using anti-CD56 and anti-NSE antibodies. If the NB84a antibody is applied, its reaction with endothelial cells should be kept in mind. Cytologic material. For detection and quantification of tumour cells in bone marrow and fine needle aspirates, anti-GD2 for bone marrow diagnostics, and anti GD2, NB84a, anti CD56 and anti S-100 for material obtained by fine needle aspiration are recommended. For differential diagnosis see above. Determination of the tumour cell content. See Histology/Cytology Report and Biology Guidelines.
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18.8 Exact Determination of the Tumour Cell Content

It is mandatory that the tumour cell content is evaluated in all samples used for molecular-genetic/biological investigations and DNA analyses. GD2, NB84a and common leukocyte antigen as well as the use of S-100 for unequivocal detection of Schwann cells is recommended. If the number of tumour cells in the touch preparations is low and obviously not corresponding to the tumour cell content in the paraffin material the imprints originate from, the touch preparations have to be checked by immunocytology for the presence of tumour/stromal cells. This should be done preferably in combination with FISH for MYCN, 1p36.3 or other chromosomal aberrations (for further details see also Biology Guidelines).
18.9 Pathology Review

The SIOP Europe Neuroblastoma Board and Pathology Committee decided that all tumours are to be reviewed by a central review panel. H&E slides from all available paraffin blocks or slides from 2 representative blocks and 10 unstained sections have to be sent to the National reference pathologist. It is especially important to include the H&E slide from the paraffin block adjacent to the tumour specimens used for molecular-genetic/biological investigations (sample A, B. etc. 1). In addition, 10 unstained sections from the most representative paraffin blocks and a copy of the written report including immunohistologic results should also be forwarded to the National reference pathologist.
BIOLOGY GUIDELINES
18.10 General Remarks and Recommendations for Biology
In neuroblastomas, investigation of the MYCN gene status, of the chromosomal region 1p36.3 and of the DNA content gives critically important information. The recommended methods for the individual parameters are summarised below. Besides the use of moleculargenetic/biological methods, it has to be stressed that classical cytogenetic analyses frequently give excellent results. As already pointed out in the Pathology Guidelines, a reliable interpretation of the molecular-genetic results is possible only if the exact tumour cell content of the specimen used for these investigations is determined. This is possible only if the pathologist evaluates the specimens adjacent to those used for molecular-genetic/biological analyses. An exact determination of the tumour cell content and correct tumour sampling are crucial prerequisites and absolutely necessary for obtaining reliable molecular-genetic/biological results (see also above). The slides used for molecular-genetic/biological analyses and the blots should be stored according to national standards. It is recommended that FISH slides be stored in the dark at 4C.
18.11 Procedures for the Determination of the Tumour Cell Content

In case of resected tumours and open biopsies (see also Pathology Guidelines), FISH/ICM is performed on touch preparations of piece 1 (see Figure 1). In this case, the tumour cell content is determined on the formalin-fixed paraffin-embedded material of piece 1 and is included in the pathology report. No conclusion about the status of genetic markers should be made in case no numeric and no structural chromosome aberrations are found by FISH and/or the ICM indicates a diploid DNA content. First, the pathologist has to indicate whether the cells
analysed are tumour cells or not! Pieces 2 and 3 are snap frozen by the pathologist and are used for immuno-histological investigations and DNA extraction. For interpretation of the SB and PCR results, the knowledge of the exact tumour cell content is especially important. Therefore, the following procedure is recommended: the snap frozen piece is first used for making touch preparations (avoid complete thawing of the piece) for FISH and ICM. Then, frozen sections, ideally cut from two sides, i.e. from the top and from the bottom, are made before extracting DNA for PCR, SB or other investigations. The frozen sections are especially important for determination and documentation of the tumour cell content. Piece 4 in RPMI 1640 is used for cytogenetic studies or for making cell suspension for FCM and cytospin preparations. Therefore, determination of the tumour cell content cannot be carried out using this specific piece. If there is any doubt about the nature of the cells analysed, due to lack of numeric and structural chromosome aberrations, a GD2, NB84a, S-100 and common leukocyte staining is strongly recommended. (to solve this question see also Pathology Guidelines) In case of tru cut biopsies, the snap frozen material received from the pathologist should be handled as indicated above for pieces 2 and 3. Smears and cell suspensions from fine needle aspirations are provided by the cytopathologist who should also indicate the tumour cell content.
18.12 Genetic Parameters to be analysed

18.12.1 MYCN ONCOGENE Methods and general remarks. The MYCN copy number can be determined by Southern Blot (SB) or fluorescence based in situ hybridisation (FISH). Polymerase chain reaction (PCR) is not recommended to be used without a second method. It has to be kept in mind that by using SB as the only method for MYCN evaluation, the chance to miss possible heterogeneities in the MYCN status (see Chapter 1) or to misinterpret low amplification is higher (due to the dilution effect) as compared to FISH analysis which is done on the single cell level. For SB and PCR, the tumour cell content has to be over 60 per cent. For FISH, all slides and all areas of the individual slides have to be screened and analysed carefully. 500 tumour cells is the minimum cell number which should be available for analysis. At least 200 cells should be counted including all cells (different hybridisation patterns and also cell without signals which give valuable information about the hybridisation efficiency). Recommendation of DNA probes. For SB either pNb1 (Dr. Schwab, Heidelberg, FRG) or an equivalent probe and a probe for a single copy gene which has to be located on chromosome 2 (e.g. L2.3) should be used. For FISH either the Oncor (or Vysis) MYCN probe or the pNb9 or pNb101 (Dr. Schwab, Heidelberg, FRG) and a chromosome 2 specific probe either specific to the centromere (D2Z, Oncor) or to chromosomal region 2pter (to avoid misinterpretation by centromeric associations) are recommended. The chromosome 2 specific reference probe must be used in case of more than 3 MYCN signals to be able to discriminate MYCN gains from chromosome 2 polysomies. PCR studies are only accepted when the PCR data are compared with SB or FISH results. MYCN copy number. Irrespective of the method used, the copy number has to be indicated according to the number of chromosomes 2. For MYCN definitions and report of results see Table 1. The terms given in this table should be used for giving the results to the clinicians and for documentation (data base).
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Table 1. Definitions and report of the results for MYCN status determined by Southern blot/Polymerase chain reaction and by fluorescence in situ hybridisation. SB and PCR - MYCN MYCN amplification = over 4-fold increase of the band intensity in relation to the band from the internal reference (on chromosome 2!). MYCN gain (=inconclusive) = 2- to 4-fold increase of the band intensity in relation to the band from the internal standard (on chromosome 2). Needs further clarification by FISH! No MYCN amplification No result (please specify) = Unclear or not interpretable result Sample contains less than 60 % of tumour cells No DNA No tumour Not done
FISH - MYCN MYCN amplification = over 4 fold increase of the MYCN signal number in relation to the number of chromosomes 2. MYCN amplification focal = more or less focal occurrence of cells (at least 50) showing a MYCN amplification surrounded by non-amplified tumour cells MYCN gain (=inconclusive) = 2- to 4- fold excess of MYCN copies in relation to the reference probe on chromosome 2. Needs further clarification! No MYCN amplification No result (please specify) = Unclear or not interpretable result Not enough tumour cells contained in the sample No tumour Not done
18.12.2CHROMOSOME 1P36.3 STATUS Methods and general remarks The integrity of the short arm of chromosome 1 (1p36.3) can be determined by three methods, i.e. PCR, SB and FISH. It has to be borne in mind that investigations carried out with SB and PCR can be used to answer the allelic status of the chromosomal region 1p36.3. In contrast,
analyses done by FISH give information on the chromosomal level, i.e. on the relation between the numbers of centromeres and subtelomeric regions of chromosome 1. If two centromeres to one subtelomeric region is observed, it most likely corresponds to a loss of heterozygosity (LOH) found by PCR or SB. However, every other disproportion of centromeres and subtelomeric regions but with more than one 1p36.3 signals (i.e. a 3 to 2, 4 to 3, 5 to 3 ratio etc.) found by FISH can but does not necessarily reflect the presence of an LOH. Vice versa, lack of an LOH does not necessarily reflect lack of cytogenetic aberrations on chromosome 1p36.3. Therefore, it is strongly recommended to use both kinds of methods (PCR/SB and FISH) for the detection of chromosome 1p36.3 aberrations in order to obtain sufficient information to address all possible changes in this important chromosomal region! For definition and interpretation of a so-called allelic imbalance (PCR) see Table 2. For PCR and SB, the tumour cell content has to be at least 60 per cent For FISH, all slides used and all areas of the individual slides have to be screened and analysed carefully. In general, 500 tumour cells is the minimum cell number which should be available for analysis and at least 200 cells should be counted including all cells (different hybridisation patterns including cells without signals). In case of an unequivocal aberrant result, 50 cells can be regarded as sufficient. Recommendation of DNA probes For PCR e.g. the primers specific for D1S76 and D1S80 or others within the region 1p36.33 are advisable. FISH investigations are recommended to be performed with the probe D1Z2 together with a centromeric probe (D1Z1) or a probe located on the long arm of chromosome 1 (to avoid misinterpretation caused by centromeric associations) in a double colour FISH approach. For SB e.g. the probe CEB15 which is within the chromosomal region 1p36.33 can be recommended. For Chromosome 1p36.3 definitions and report of results see Table 2. The terms given in this table should be used be used for giving the results to the clinicians and for documentation (data base). Recommendation If less than 50% of the tumour cells show deletion or imbalance by FISH, more slides, samples or nuclei isolated from paraffin material shall be analysed; paraffin material should also be used when FISH on touch slides was not successful (e.g. in case of ganglioneuroma). To exclude methodical reasons, the number of cells with less 1p36.3 signals as compared to the number of centromeric signals has to be compared always with the percentage of cells with more 1p36.3 signals. Cells without hybridisation signals have to be counted as well. Report of the molecular-genetic results The molecular-genetic results should be reported in the following manner: A detailed description of the results and the methods used including percentages of tumour cells versus normal cells, hybridisation pattern etc. should be described in a report section. In addition, PCR and FISH results should be discussed together with an interpretation of their meaning. In the conclusion section, the result should be given as briefly and precisely as possible using the terminology given in Tables 1 and 2 and should also contain information about preceding cytotoxic therapy.
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Table 2. Definitions and report of the results for chromosome 1p36.3 status determined by Southern blot/Polymerase chain reaction and by fluorescence in situ hybridisation.
PCR and SB Chromosome 1p36.3 Allelic loss (LOH) = complete or almost complete disappearance of one band Allelic imbalance (=inconclusive) = one band relatively weaker than the other band when compared to the ratio observed with constitutional DNA controls. This can mean either allele disequilibrium (e.g. two paternal and one maternal chromosomes 1) or LOH. In this case, the experiment has to be repeated, the tumour has to be checked by FISH, and the tumour cell content has to be reevaluated! No allelic loss, no allelic imbalance No result (please specify) = Unclear or not interpretable Constitutional homozygosity Sample contains less than 60% of tumour cells No DNA No tumour Not done
FISH Chromosome 1p36.3 Deletion = only one signal for the subtelomeric region of the short arm of chromosome 1 present (ratio 2/1 possibly together with 4/2 in the same tumour, 3/1, 4/1).
FISH imbalance (=inconclusive) = disproportion of the ratio of centromeres of chromosome 1 to the subtelomeric regions of the short arm with more than one subtelomeric regions (ratio 3/2, 4/3, 4/2, 5/3 etc.) Needs further clarification by PCR or SB. Focal deletion/imbalance = more or less focal occurrence of cells showing a deletion/imbalance surrounded by tumour cells with intact chromosome 1p36.3 (see below). No deletion, no FISH imbalance detected by FISH with the probes used. No result (please specify) = Unclear or not interpretable result Not enough tumour cells contained in the sample No tumour Not done
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18.12.3 DNA CONTENT Methods and general remarks. Two methods, i.e. Flow Cytometry (FCM) and Image Cytometry (ICM) can be used for the assessment of the tumour cell DNA content. The number of chromosomes 1 obtained by FISH analysis should not be used for estimation of the DNA content because near-triploid tumours can be disomic or tetrasomic for chromosome 1 and neardiploid tumours can be trisomic for chromosome 1. For FCM, reference cells derived from human tissue (peripheral blood lymphocytes) from normal individuals or the same patient should be used. Report of the FCM/ICM results. The report should indicate the method used and specify the number of tumour cells versus normal cells contained in the sample under investigation. The results on the DNA content of the tumour cells should be given in absolute numbers. 18.12.4 OTHER CHROMOSOMAL REGIONS OF POTENTIAL INTEREST There are a number of reported chromosomal aberrations, such as chromosome 17q gain, 11q loss and 14q loss, that are believed to give further prognostic information. Also telomeric length and telomerase activity have been shown to be associated with a distinct clinical behaviour. 18.12.5 MOLECULAR-GENETIC/-BIOLOGIC INVESTIGATIONS ON TUMOUR MATERIAL OBTAINED
AFTER CYTOTOXIC THERAPY
This kind of tissue often contains only a low number of tumour cells due to therapy induced regressive changes. The possibly low tumour cell content should be especially taken into account with reference to DNA extracting methods. Yet also, preceding cytotoxic treatment can cause major difficulties in the interpretation of the FISH pattern because of the frequently observed polyploidisation occurring after therapy and the often pronounced centromeric associations. These associations of centromeres can either simulate an equal number between the DNA sequence of interest and the reference probe (i.e. the centromeric sequence) instead of an imbalance or a deletion, or they lead to the impression of a gain of the respective DNA sequence instead of a balanced number. The use of non-centromeric reference probes from the concerned chromosome can be of help in critical cases. Sometimes however, it is not possible to give a reliable result. Altogether, test interpretation should be made with caution considering these biological post-treatment changes and the report should always stress that investigations were performed after cytotoxic treatment. 18.12.6 CENTRAL REVIEW It is recommended that the molecular-genetic/biological data are reviewed either by a central review panel or by exchanging tumour material. Alternatively, FISH slides and images from SB and PCR analyses from individual laboratories can be sent to one external laboratory for blind review. Only cases showing divergent interpretations will then be discussed in the central review panel.
BONE MARROW EXAMINATION GUIDELINES

18.13 General Remarks on Bone Marrow Aspirations
The following guidelines have been developed for the purpose of improving initial staging accuracy, treatment response evaluation, and, ultimately, patient care. Bone marrow (BM) aspirates from two sites (left and right) have to be performed. In addition, two trephine biopsies are mandatory. Three sequential aspirations for each puncture site are necessary:
one for bone marrow smears, the second for immunocytology, classical cytogenetics and FISH, and the third which should not contain heparin for PCR or other techniques. The aspirations from the different sites should not be pooled together. Two to four syringes with plugs and 10 to 20 glass slides for the bone marrow smears and one polished cover glass should be prepared. First aspiration and preparation of bone marrow smears Half a milliliter of BM is aspirated into the syringe and immediately dropped on a glass slide. For preparation of the smears see Figure 3. Second aspiration for immunocytology The appropriate amount of anticoagulant [e.g. 200l heparin (5000IE/ml) in 10 ml peripheral blood or 5 ml BM] is aspirated into the syringe. To avoid excessive dilution with peripheral blood, each aspirate should be ideally 2-3 ml. The syringe should be shaken immediately to allow the anticoagulant to mix with the bone marrow. This procedure is repeated for each puncture site. The properly closed syringes should be transferred to the haematology or immunology laboratory at room temperature as fast as possible. Third aspiration for PCR To enable a highly sensitive technique for detecting rare neuroblastoma cells with the PCR technology, an additional BM sample is needed. Aspirate 2 to 3 ml BM in a separate syringe containing the appropriate amount of EDTA or ACD or another anticoagulant specified by the laboratory (e.g. guanidinium thiocyanate containing tubes supplied by Sue Burchill the content of these tubes should be mixed and stored at 80C).
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B C
adjust the angle of the cover slip according to the cell number (usually 30)
Figure 3. Recommendation for making bone marrow smears. Aspirate briefly to obtain 0.2 ml to 0,5 ml bone marrow and put the whole amount on a glass slide (A). Put this slide in an upright position (B) and allow the excess blood to flow down. Dip a polished glass cover slip into the drop of bone marrow (C) and transfer this dipped cover slip to a new glass slide (D). Immediately afterwards pull this small droplet with the cover slip along the slide. Air dry the slides for at least 10 min. Stain slides according to Pappenheim.
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18.14 Handling of the bone marrow cells in the laboratory

18.14.1 SEPARATION OF MNC, PREPARATION AND STORAGE OF CYTOSPIN PREPARATIONS. The mono-nuclear cells (MNC) are separated by a Lymphoprep (Nycomed) or Ficoll density separation (1.077; the instructions of the manufacturer should be followed). The two different sites must be handled separately. It is important to indicate if two or all sites are pooled. After careful taking out the MNC fraction, the cells are washed in PBS or Tris pH 7.0, without FCS and spun down gently (e.g. 10min at 252g). The recovered MNC fraction should be counted and tested for the vitality percentage by the trypan blue dye-exclusion test. For making the cytospin preparations a Hettich centrifuge is usually appropriate. A total number of at least 2x106 MNC should be analysed. Each investigator can choose the method he/she is more comfortable with, as long the cell preparations are not overcrowded and MNCs lie well separated from one another. In fact, cells in clumps can give both false negative and false positive results. The MNC cell preparation can be achieved by wide diameter (17 mm) (Hettich) cytospins containing 0.5x106 MNCs each. MNC sedimentation should be performed on clean glass slides, or, when used for sequential FISH experiments, on sticky slides (Poly-L-Lysine; HistoBond, Marienfeld, FRG). Cell preparations are air-dried for 2 to 24 hours and fixed with 4% buffered formaldehyde for at least 10 min or overnight at 4C (since GD2 is a lipid antigen alcohol fixatives should be avoided). The samples can be immediately stained or stored at 20C or at 80C, for prolonged storage, in air-tight plastic chambers or tightly wrapped in aluminum foil. After removal from the freezer, the slides should be allowed to thaw in closed boxes for at least 2 hours before opening them. Otherwise the development of condensation water impairs or destroys cytomorphology. 18.14.2 GD2 IMMUNOCYTOLOGY. Anti GD2 antibodies provide consistent results in terms of sensitivity and specificity of NB cell detection. GD2 antigen is a disialoganglioside highly and consistently expressed on neuroblastoma cells and not on normal marrow cells. In addition, this method is relatively simple, cheap and widely applicable. However, macrophages with internalised GD2 positive tumour material as well as unspecific adherence of the antibody to the nuclear or cellular membrane can give rise to difficulties. Therefore, the use of further independent or complementary techniques in order to confirm or to disprove doubtful results is recommended (i.e. FISH for NB associated genetic aberrations, RT-PCR for NB specific transcripts). As long as each positive event is directly examined and confirmed under the microscope, fluorescencebased immunocytology or immuno-cytochemistry techniques are equally acceptable, provided a permanent record is possible and the adequate study concept is followed and quality controls are performed. In BM with less than 0.1% tumour cells, at least 2x106 MNC should be analysed. In bone marrow samples with a high tumour cell infiltrate, a smaller number of total cells can be analysed (e.g. 30 fields in infiltrates over 1%). At least three extra unstained cytospin preparations should be stored frozen (ideally at - 70C) for quality control studies. The primary monoclonal antibody employed is left to the preference of each investigator within a list of antibodies: 14.G2a (Ralph Reisfeld, La Jolla), 3F8 (Nai-Kong Cheung, New York),7A4 (Nicole Gross, Lausanne). Either the primary antibody can be used in the purified form or the hybridoma supernatant as long as the lowest concentration which consistently stains the positive control without background staining is employed. The type of secondary antibodies and development systems employed are left to the preference of each investigator as long as the detection system is validated with cell-line scalar dilutions and the proper negative and positive controls are employed.
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Pitfalls. To avoid misinterpretation of positive cells or cellular material in fluorescence based analyses, cellular morphology has to be controlled by phase contrast and DAPI staining. Macrophages and plasma cells or false-positive staining of the nuclear membrane after disruption of the cell membrane can give rise to such sources of errors. When the number of GD2 positive cells is less than 1 in 104 MNCs and morphologic features are not sufficient to characterise the concerned cells as tumour cells, the genetic profile of these cells should be checked by FISH or another technique whether they correspond to the tumour cells (e.g. MYCN amplification, 1p deletion, or other chromosomal aberrations). These sequential analyses can ideally be done using an automated scanning and relocation system, e.g. the RCDetect/Metafer (MetaSystems, Altlussheim, FRG). PCR techniques These methods used in BM samples need further assessment in large clinical trials before they can be recommended as routine methods.
18.14.3 TEST INTERPRETATION, RESULT REPORTING AND QUALITY CONTROL STUDIES For each test, a negative control with a comparable number of MNCs should be examined (this step is not necessary when fluorescence-based detection methods are used). A total of at least 0.5 to 1 x 106 MNCs from normal bone marrow and from the actual sample should be evaluated with a negative control in which the primary antibody is substituted with a buffered saline solution. This test can be omitted when other techniques are used to unambiguously identify disseminated tumour cells (e.g. FISH). For each staining procedure a positive control (prepared with a NB cell line) should be prepared as well. A positive cell should be called as such if it has a well recognisable nucleus and a completely preserved and stained cytoplasm. An interpretable test is one in which the negative control has no significant background staining such as to interfere with positive cell discrimination and/or has no cells that would be scored as positive. In the positive control, cells with brightly and characteristically stained cytoplasm (surface and globular staining pattern) have to be easily recognisable. If these prerequisites are not fulfilled the test should be reported as non-interpretable. In doubtful cases or in cases with a low tumour cell infiltrate, the genetic aberration of the tumour cells should be visualised to ensure that the immunologically stained cell displays the same genetic aberration as the primary tumour. In some instances clumps of amorphous GD2positive material, such as cell membranes or micelles, can be detected. They should be reported as such and quantified as best as possible. For highly positive samples (>1 GD2 positive cell per 200x field) the result can be reported as a percentage after having counted at least 30 fields. The number of positive cells should be reported together with the total number of cells examined. The average cell loss during slide/cytospin preparation and/or staining should be initially verified for each immunocytology method. Slides stained for light microscopy should be stored at room temperature. All slides whether they are positive or negative should be stored. Cell preparations stained for fluorescence microscopy should be stored at 4C. Specific fluorescence signals (due to bound antibody and/or hybridised probes) should be registered and archived either as optically or digitally processed microphotographs.
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Quality Assessment Routine BM cytospin preparations and frozen BM samples are distributed among the participating laboratories for immunocytochemical and immunofluorescence staining. The results of the individual centres are reported at a meeting of all groups. Review Process. Immunocytochemical and immunofluorescence stained slides are reviewed either in an inter-laboratory exchange system or in unclear situations by a central review procedure.
18.14.4 STORAGE OF TUMOUR MATERIAL, SLIDES AND BONE MARROW SAMPLES It is mandatory to store material and slides from each tumour, biopsy and bone marrow and peripheral blood sample. This is important to conduct further/future biological and genetic analyses and to allow review and quality assessment studies. In case of tumour resection and open and tru cut biopsies, the storage of snap frozen material (at 70C or below) is most important. Besides this, it is advisable to store touch preparations and cytospin preparations at 20C and cell suspensions (including DMSO), if available, in liquid nitrogen. Tumour cells obtained by fine needle biopsy can be stored either as cytospin preparations or cell suspensions. The MNC fraction of BM and PB can be stored as cytospin preparation at 20C and cell suspensions (including DMSO) in liquid nitrogen. Furthermore, stained slides, SB and PCR pictures have to be stored adequately for documentation and review purposes.
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19 APPENDIX SCINTIGRAPHY PROTOCOL FOR MIBG SCANNING

19.1 Objectives
To obtain scans of best and constant quality, only nuclear medicine departments with proper equipment and experienced nuclear medicine physicians should perform the examinations. The departments should be able to perform SPECT scanning, if possible with a multiple head camera to reduce the acquisition time. The radioisotope of choice is Iodine-123 (I123) and should be used wherever available, regardless of higher costs. Information about preceding therapeutic procedures like surgery and chemotherapy and the time when they were performed should be available to the nuclear medicine physicians. There are many drugs and compounds which on a theoretical basis could interfere with the concentration of mIBG by neuroblastoma, for example, by interfering with the uptake mechanisms or acting as competitive inhibitors. Therefore the discontinuation of these medicines two weeks before administration of the radio-pharmaceutical should be considered if this is clinically possible. The classes of drugs which have interfered with the uptake of mIBG are tricyclic antidepressants and related drugs, some antihypertensives, phenothiazines, amphetamines and particularly some nasal decongestants and cough preparations.
19.2 Patient Preparation

19.2.1 THYROID BLOCKADE There are quite different guidelines for thyroid blockade and different prescriptions, for example, for Lugols solution within European countries. Thus it would not be easy to achieve a uniform thyroid protocol. However, the different regimens yield comparable results. In the case of I131 mIBG the thyroid has to be blocked at least 24 hours prior to tracer injection. However, in I123 mIBG scintigraphy thyroid blocking should be done as well. Recommended protocol: Beginning on the day before tracer injections until the day after injection, children from one month to three years should receive 32.5mg potassium iodide daily, from three to thirteen years 65 mg, and over this age 130 mg daily. Rapid blockade by intravenous injection of potassium and perchlorate (Irenat) respectively, is an alternative option. 19.2.2 OTHERS To obtain scans without motion artefacts sedation might be necessary in some patients. In small children an intravenous line has to be inserted by the paediatrician prior to tracer injection. Central venous catheters should be avoided if possible .
19.3 Tracer Activity
Considering an activity of 370 Mbq I123 mIBG for adults the EANM paediatric task group generally recommends calculation of the activity for children by the following formula 370 Mbq*body weight (kg) 70
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On the other hand in this patient group the radiation hazard is far less than the risk resulting from false negative or positive scanning results. Longer scanning times provoke motion artefacts. Even rescanning inadequate frames rarely improves the results in our experience. To obtain scintigrams of good quality within a short scanning time a sufficient count rate is necessary. Recommendation Therefore, when using I123 mIBG, in children under 6 kg body weight we recommend an activity of 37 Mbq, in children from 6 to10 kg 50-75 Mbq, from 10 to15 kg 75-100 Mbq, and from 15 to 30 kg 150 Mbq and above 30 kg the formula can be applied.
19.4 Scanning Protocol

As a compromise between best image quality and limitation of scanning time the imaging protocol should not exceed one hour. In the case of a double-head camera in children with a body length under 100 cm the planar whole body scintigraphy can be done within 35 minutes. SPET of the primary tumour region is useful in evaluation after surgery and chemotherapy but might also be important at the initial scintigraphic examination for later comparison. Using a three-head SPET camera the tomography protocol described below takes about 15 minutes for one region. The time for patient positioning and setting of the camera is included in our estimations. The 159 KeV photopeak of I123 has to be centred in a 20% energy window. 19.4.1 PLANAR SCANNING Scanning time: Twenty-four hours after tracer injection; for all frames with equivocal result a second time at 48 hours. A double-headed gamma camera with low energy high resolution parallel hole collimators (LEHR) is recommended. To limit scanning time on all purpose parallel hole low energy collimator (LEAP) can be used in singleheaded gamma cameras. Whole body: anterior and posterior frames of skull/thorax, skull profiles, abdomen/pelvis, and upper extremities; lower extremities in anterior view. Ten minutes for each frame. When time is restricted or in case of a single-head gamma camera 6 minutes/view. The scans are stored in a 2562 pixel matrix.
Collimator:
Views:
Acquisition:
19.4.2 SINGLE PHOTON EMISSION TOMOGRAPHY (SPET) Time: Views: Twenty-four hour after tracer injections. Region of the suspected primary tumour; other regions from skull to pelvis with equivocal planar result. Multiple-head camera system with LEHR collimators. 360o in 6o steps, 30 seconds per step, 642 pixel matrix.
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Collimator: Acquisition:
Processing:
Reconstruction using a ramp filter and 3D post-filter (Butterworth 6th order) or alternatively pre-filtering with a Butterworth filter (6th order) and back-projection with a ramp filter. Reorientation of the images in transaxial and coronal slices.
19.4.3 SCAN ASSESSMENT Each scan should be evaluated directly from the screen by two independent experienced observers. Hard copies of the best possible quality should be archived. Central Review The Nuclear Medicine Subcommittee will organise central review of mIBG scans. MIBG scan results are crucial at the end of induction evaluation, since only patients with an improvement of at least 50% or with a maximum of three residual, but improved mIBG positive spots are eligible for randomisation. Thus it is a prerequisite that the raw data is stored on appropriate media for further evaluation. Ideally the group will build up central reviews via electronic transmission. However, hard copies will have to be used for this purpose till the system is built up.
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Form for Standardised Evaluation of mIBG Scans

Examination Date:. Patient Name:. Trial Reference number:.... Injected activity: Time after injection: 24 hours Other specify: No Site of the primary tumour.Surgery: Yes PRIMARY TUMOUR Is there uptake at the site of the primary tumour? Yes: No: If yes, intensity (*) If yes, homogenous = 1 Heterogenous = 2 SKELETAL LOCALISATIONS
Skull vault Orbit or facial bones (including base of skull) Intensity (*) ----------
If yes, loco-regional extension **
Extension (**) ---------Intensity (*) ----------
Extension (**) ---------Spine Intensity (*) ----------
Extension (**) ---------Ribs and sternum Intensity (*) ----------
Extension (**) ---------Pelvis Intensity (*) ----------
Extension (**) ---------Lower limbs Intensity (*) ----------
Extension (**) ---------Upper limbs Intensity (*) ----------
Extension (**) ----------
SOFT TISSUE LESIONS

Yes: No: If yes, the site of greatest signal intensity -------------------------------------------------------Intensity (*) ---------Extension (**) ---------(*) Intensity (**) Extension 0 = absent 0 = None 1 = possible 1 = A single lesion 2 = Definite but weak 2 = More than one lesion or diffuse 3 = Definite and strong When there is more than one focus in an anatomical location, measure the strongest focus. Quality of scan: Excellent Acceptable Poor
Comments:..
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APPENDIX MONITORING OF RENAL TOXICITIES

20.1 Recommended Procedure for GFR Evaluation
1. Prepare a syringe containing 0.04 MBq of Cr-51-EDTA per kg of patient weight, and using a minimum activity of 0.4 MBq. At the same time prepare an accurately known percentage standard of the EDTA of about 2-4% of the activity to be administered. 2. Administer the EDTA to the patient using the most appropriate iv route, but taking care to thoroughly flush any lines if they are used. 3. Keep the emptied syringe and any other disposable items (e.g. butterfly needle or canal) that have been used in the administration. 4. Take three blood samples of 2-5ml from the patient, using a route of venous access which is different to that used for the administration. The samples should be taken at 1, 2 and 4 hours from administration of the EDTA. The exact timing is not critical but the sampling should not start before 1 hour or extend beyond 4 hours from the time of administration. The time of each sample should be recorded accurately. 5. Spin the blood samples down and transfer 1-2 ml of plasma into tubes suitable for counting on a standard gamma sample counter. Rinse out the contents of the EDTA syringe and any other items saved from the administration of the EDTA into a counting vial. If necessary split this between two or more vials this is the dead space. Ensure all of the plasma samples, the dead space and the standard prepared in (1) are of about the same volume by adding water to any that might need it. 6. Count all the blood samples, the standard and the dead space together with an inactive background sample in an automatic gamma sample counter. Samples should be counted long enough to achieve at least 10,000 counts for each sample and more than 1,000 counts for the background. 7. Calculate the GFR for the patient using a single exponential model. This calculation should yield: GFR, ml/min T1/2 of the fitted line The correlation coefficient for the fitted line The volume of distribution projected to the time of administration 8. In order to ensure data from different centres are compatible data should be pooled centrally, using e-mailed spreadsheets, and in addition to the data listed in (7) ask for: Administered activity (MBq) Percentage standard Standard counts per minute For each sample: Time for administration (minutes) Volume (ml) Counts per minute Background counts per minute
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9. If any centres prefer to use Tc-99m DTPA then they can either (i) use the above procedure ensuring that the standard is diluted sufficiently so that all samples can be counted simultaneously or (ii) count the plasma samples some time after counting the standard and dead space, and this time interval should also be recorded and communicated. Remark: The point in the above list is to avoid prescribing exactly how each stage is done. This is to give basic guidelines on minimum requirements to avoid most frequent errors in evaluating GFR rates by the above method.
Recommendation by M.J. Keir BSc, PhD, MIPEM CONSULTANT Medical Physicist Head of Royal Victoria Infirmary Unit Contact addresses: Royal Victoria Infirmary Queen Victoria Road Newcastle upon Tyne, NE1 4 LP Tel: +44 191282 4038 Fax: +44 191 233 0351 www.rmpd.org.uk
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20.2 Prescription sheet for GFR estimation ESTIMATION OF GLOMERULAR FILTRATION RATE
PATIENTS NAME: ..................................... ADDRESS. ................................................. HOSPITAL NUMBER: ............................. DATE OF BIRTH: .................................... IP / OP Ward: ............................................ HOSPITAL: ...............................................
........................................................................ ........................................................................ Radiopharmaceutical: Cr 51 EDTA Activity of Dose: .......................MBq Volume of Injection: .........................
Date of Test: ..............................................
Instructions To Ward Staff 1. The EDTA should be administered i.v. and the exact time of injection written below. The administration techniques are outlined overleaf. The method of administration (butterfly or multiple lumen central venous catheter) should be recorded below. 2. Replace the blind hub on the empty EDTA syringe and return it to the Medical Physics Department with the blood samples. 3. Blood samples of 5ml each should be taken at 1,2 and 4 hours and placed in heparinised tubes. These times are not critical, but the exact time of sampling should be noted below. All blood samples should come from a site remote from that used to administer the EDTA, but if one lumen of a central venous catheter is used to administer the EDTA, another lumen may be used to obtain samples. Any deviation from this protocol should be noted below Please Record: Time of injection: Weight (kg): Sampling times: Notes: .............................. .............................. 1) butterfly multiple lumen catheter
Height (cm): ............................... 2) ............................ 3) ............................
...........................
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ADMINISTRATION TECHNIQUE In general, always administer using a butterfly needle. i) ii) iii) Prime it with saline which can then be used to check the correct siting of the needle. Administer the EDTA. Use more saline to flush any EDTA remaining in the butterfly into the patient.
FOR PAEDIATRIC ONCOLOGY PATIENTS Administration of EDTA down one lumen of a multiple (double or triple) lumen central venous catheter, with blood sampling from a separate lumen, is permitted as long as the following procedure is followed: 1. Establish that both lumens are flushing and sampling satisfactorily. 2. Inject 5ml 0.9% Saline through bung of one lumen using a blue needle. Other sizes of needle (e.g. green) are not adequate since they are the wrong length. 3. Inject EDTA through bung of same lumen using another blue needle. 4. Inject 10ml 0.9% Saline through bung using another blue needle and follow this with a 5ml Hepsal (or Heplock) flush. 5. Do not remove the bung to attach the syringe directly to the catheter hub during procedures 2,3 or 4 since this does not allow adequate flushing of EDTA. 6. Identify the lumen used for EDTA injection and record this on the front of this form. Do not use again until all sampling complete, unless absolutely necessary for clinical purposes. 7. Sample at 1, 2 and 4 hours from the lumen not used for EDTA injection. 8. Tick the box on the Medical Physics form to indicate that central venous catheter has been used for EDTA administration.
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21 APPENDIX Drug Information

21.1 Carboplatin
Formulation Vials containing 50 mg/5 mls, 150 mg/15 mls or 450 mg/45 mls. Storage At room temperature. Reconstitution Each vial is reconstituted with water for injection BP, glucose injection 5% or sodium injection 0.9% to a final concentration of 10mg carboplatin/ml. Stability When reconstituted with water for injection or glucose 5%, 8 hours at room temperature, 24 hours in refrigerator. After dilution in glucose 5% infusion stable for 24 hours in refrigerator. AdministrationIn this protocol as 1 hour intravenous infusion. Toxicity Myelosuppression, especially thrombocytopenia, nausea and vomiting, low incidence of nephrotoxicity, ototoxicity, neurotoxicity and abnormalities of liver function. May cause urinary loss of serum magnesium, potassium and calcium, so levels of these should be monitored. Toxicity Frequencies
Common Happens to 21 - 100 out of every 100 children Nausea (L), vomiting (L) Myelosuppression Occasional Happens to 5-20 children out of every 100 Electrolyte disturbances (L) Rare Happens to <5 children out of every 100 Metallic taste Peripheral neuropathy, hepatotoxicity (L), renal toxicity (L), ototoxicity (L)
Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 -weeks, prior to the next course
Delayed: Any time later during therapy, excluding the above conditions Late: Any time after completion of treatment 1 Thrombocytopenia is more severe or dose limiting. (L) Toxicity may also occur later.
Secondary leukemia
21.2 Cisplatin
Formulation Amber glass vials containing cisplatin solution 1mg (10, 25, 50 ml). Vials containing lyophilisied cisplatin either 10mg or 50 mg. Storage At room temperature. Reconstitution Powder reconstituted with water for injection to a final concentration of 1mg/ml cisplatin. Must be further diluted before administration. Stability Cisplatin is unstable in aqueous vehicles unless chloride ions are present. Minimum concentration of sodium chloride providing an acceptable level of stability is approximately 0.3%w/v. At adequate chloride concentration, stability is unaffected by presence of glucose. Less than 4% degradation after 24hours at 25C in solutions containing recommended chloride-ion concentration. AdministrationIn this protocol as continuous 24hour intravenous infusion. Toxicity Renal toxicity reduction in glomerular filtration rate and renal tubular loss of electrolytes particularly potassium, magnesium, sodium, and calcium. Ototoxicity, neurotoxicity, peripheral neuropathy, severe nausea and vomiting. Myelosuppression minimal. Rarely ocular toxicity, seizures and anaphylactic-like reactions.
Toxicity Frequencies
Common Happens to 21-100 children out of every 100 Nausea (L), vomiting (L) Occasional Happens to 5-20 children out of every 100 Metallic Taste (L) Electrolyte disturbances (L) Rare Happens to <5 children out of every 100 Anaphylactic reaction Peripheral neuropathy (L), tinnitus (L), seizure (L), liver toxicity (L)
Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 Anorexia (L), weeks, prior to the myelosuppression, next course hypomagnesemia (L), high frequency hearing loss (L), nephrotoxicity (L) Delayed: Any time later during therapy, excluding the above conditions Late: Any time after completion of treatment (L) Toxicity may also occur later.
Hearing loss in the normal hearing range Secondary malignancy
21.3 Etoposide
Formulation Vials containing 100 mg etoposide in 5 ml. Storage At room temperature. Reconstitution Ideally dilute to a concentration of 0.25-0.4 mg/ml in 0.9% sodium chloride or 5% dextrose. Stability Vials are stable for 5 years at room temperature. At concentrations of 0.4 mg/ml in 0.9% saline solutions are stable for 96 hours at room temperature in normal fluorescent lighting, in PVA containers. Solution in PVC infusion bags should be used immediately, to avoid leaching out of potential carcinogenic plasticisers. AdministrationDuring rapid COJEC by intravenous infusion over 4hours - protected from light. During CEM given as 24hours continuous infusions. Caution: Anaphylactic reaction usually manifested as severe hypotension may occur if infusion given too rapidly. Avoid extravasation. Toxicity Bone marrow suppression. Alopecia, headache, fever, hypotension, nausea, vomiting, anaphylactic reactions, second malignancies including leukaemia. Toxicity Frequencies
Common Happens to 21 - 100 children out of every 100 Nausea, vomiting Occasional Happens to 5-20 children out of every 100 Rare Happens to <5 children out of every 100 Hypotension, anaphylaxis, skin rash Peripheral neuropathy, stomatitis
Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 Myelosuppression weeks, prior to next course Delayed: Any time later during therapy, excluding the above conditions Late: Any time after completion of treatment (L) Toxicity may also occur later.
Alopecia (L) , enhanced damage due to radiation, diarrhoea
Secondary malignancy
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21.4 Cyclophosphamide
Formulation 100 mg, 200 mg, 500 mg and 1g vials for reconstitution. Storage At room temperature. Stability Unreconstituted vials stable for 5 years at room temperature. A solution of cyclophosphamide appears to be chemically stable for at least 28 days when stored at 4 C. Reconstituted solution (20 mg/ml) should be used within 3 hours when stored at room temperature, unless prepared under strict aseptic conditions, when it may be used within 8 hours. Administration Cyclophosphamide is given as i.v. bolus followed by post-cyclophosphamide infusion over 24 hours containing mesna. A mesna i.v. bolus is given prior to cyclophosphamide in addition in this protocol. (see individual course details for more information). Toxicity Myelosuppression, nausea, vomiting, alopecia, haemorrhagic cystitis, sterility, second malignancies including leukaemia or bladder cancer. May exacerbate the cardiotoxic effects of anthracycline therapy.
Toxicity Frequencies Common Happens to 21-100 children out of every 100 Anorexia (L), nausea (L), vomiting (L) Occasional Happens to 5-20 children out of every 100 Metallic taste (L), Inappropriate ADH 1 Rare Happens to <5 children out of every 100 Transient blurred vision 1 cardiac toxicity with arrhythmias 1 myocardial necrosis 2 (L)
Immediate: Within 1-2 days of receiving drug
Prompt: Within 2-3 Myelosuppression (L), Hemorrhagic cystitis weeks, prior to the alopecia (L) (L) next course Delayed: Any time Immunosuppression, later during therapy, gonadal excluding the above dysfunction/sterility (L) conditions Late: Any time after completion of treatment Unknown Frequency and Timing: **Fetal and teratogenic toxicities
Pulmonary fibrosis 3 (L)
Secondary malignancy, bladder fibrosis
1 Less -common with lower doses 2 Only with very high doses 3 Risk increased with chest radiation. (L) Toxicity may also occur later. ** Fetal toxicities and teratogenic effects of cyclophosphamide (alone or in combination with other antineoplastic agents) have been noted in humans. Toxicities include: chromosome abnormalities, multiple anomalies, pancytopenia, and low birth weight. Cyclophosphamide is excreted into breast milk. Neutropenia has been reported in breastfed infants. Cyclophosphamide is considered to be contraindicated during breast feeding because of the reported cases of neutropenia and because of the potential adverse effects relating to immune suppression, growth, and carcinogenesis.
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21.5 MESNA (sodium 2-mercaptoethanesulfonate)

Formulation Available in 1000mg/10ml multidose vials which contain 10.4mg/ml of benzyl alcohol* as a preservative, or in 200mg/2ml ampoules without preservatives for neonates and infants or patients with hypersensitivity to benzyl alcohol. Source and Pharmacology: MESNA is a thiol compound with the capacity of inhibiting the urotoxicity of the oxazaphosphorines, ifosfamide and cyclophosphamide. Within 1 hour of administration, MESNA is completely oxidised to DiMESNA, a totally inert compound. After an 800mg dose the t for MESNA and DiMESNA is 0.36 hours and 1.17 hours, respectively. There is little or no tissue penetration. Following glomerular filtration DiMESNA is rapidly reduced in the renal tubules back to MESNA which inactivates acrolein and the oxazaphosphamides, thus preventing bladder toxicity. Young children receiving high doses of benzyl alcohol (> 99 mg/kg/day) may develop the gasping syndrome manifested by gasping, metabolic acidosis and multiple organ system failure. Benzyl alcohol is the preservative in multidose vials of MESNA Storage MESNA is not light-sensitive, but is oxidised to DiMESNA when exposed to oxygen. Non-preserved ampoules should be used immediately after opening, while benzyl alcohol-preserved vials may be stored and used for 8 days. Stability After further dilution for administration, either product is chemically stable for at least 24 hours. Lack of an antimicrobial preservative suggests that the nonpreserved product should be used within 6-8 hours after diluted for administration. For IV administration, dilute to 20 mg/ml with any of the following fluids: 5% dextrose, 5% dextrose in 0.45% sodium chloride, 0.9% sodium chloride or Lactated Ringer's. MESNA may be mixed with ifosfamide or cyclophosphamide. Administration IV. Can be given orally but has a foul taste. Total dose is usually 60% of the oxazaphosphorine dose given in divided doses. Higher doses or continuous infusions are used with high dose ifosfamide or cyclophosphamide, or in patients with a history of hemorrhagic cystitis. Toxicity The package insert for MESNA states that multidose vials contain benzyl alcohol 10.4mg/ml (1%) as a preservative, should not be used in neonates or infants, and should be used with caution in older paediatric patients. A 200mg/2ml ampoule remains available free of charge for paediatric patients less than 2 years old and for patients with hypersensitivity to benzyl alcohol. It may be obtained in the U.S. by calling Bristol- Myers Squibb Oncology at 1-800-437-0994. The medical literature includes reports of gasping syndrome in premature infants receiving saline flushes with benzyl alcohol at benzyl alcohol doses greater than 99 mg/kg/day. There is also a report of metabolic acidosis occurring in a 5 year old girl receiving continuous infusion diazepam which contained 180 mg/kg/day of benzyl alcohol.149 The syndrome includes gasping respiration, severe metabolic acidosis, and multiple organ system failure. It results from inability to adequately conjugate benzoic acid with glycine, a metabolic pathway poorly developed under 8 weeks of age. Even if the amount of benzyl alcohol in MESNA is not enough to cause problems in a patient, a number of other drug products contain benzyl alcohol and therefore could add to the dose a patient is receiving. Your pharmacist can check product contents and calculate the dose of benzyl alcohol any patient is receiving.
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Common Happens to 21 - 100 children out of every 100 Bad taste with oral use Occasional Happens to 5-20 children out of every 100 Nausea, vomiting, stomach pain Rare Happens to <5 children out of every 100 Headache, pain in arms, legs, and joints; fatigue, rash, transient hypotension, allergy Diarrhoea
Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 weeks, prior to the next course Delayed: Any time later during therapy, excluding the above conditions Late: Any time after completion of treatment
21.6 Vincristine
Formulation 1 mg, 2 mg, 5 mg vials with 10 mls diluent. 1 ml, 2 ml, 5 ml vials of solution 1 mg/ml. Also available in pre-filled syringes containing 1 mg in 1 ml, 2 mg in 2 mls unpreserved. Storage At 2-8C in refrigerator. Stability Depends on formulation : Lyophilised powder (0-6C) 3 years. Solution (0-6C) 2 years. Reconstituted injection is stable for 14 days (2-8C). Diluted infusion (in 0.9% saline, 5% dextrose or Ringer's lactate) is stable for 24 hours at 20 g/ml. AdministrationBy bolus intravenous injection. Ensure that needle is well into the vein to avoid extravasation. It is strongly recommended that all vincristine injections are labelled "FOR INTRAVENOUS USE ONLY". Toxicity Local necrosis if extravasated. Jaw pain, paresis, constipation, neurotoxicity and alopecia, paralytic ileus and ADH. Toxicity Frequencies
Common Happens to 21 -100 children out of every 100 Local ulceration if extravasated Hair loss Occasional Happens to 5-20 children out of every 100 Jaw pain Weakness, constipation Rare Happens to <5 children out of every 100
Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 weeks, prior to the next course
Paralytic ileus, ptosis, vocal cord paralysis, myelosuppression, CNS depression, inappropriate ADH, seizure
Delayed: Any time Loss of deep tendon Numbness, tingling and later during therapy, reflexes clumsiness excluding the above conditions Late: Any time after the completion of treatment Unknown Frequency and Timing: **Fetal and teratogenic toxicities
(L) Toxicity may also occur later

** Fetal toxicities and teratogenic effects of vincristine (either alone or in combination with other antineoplastic agents) have been noted in humans. The toxicities include: chromosome abnormalities, malformation, pancytopenia, and low birth weight. 21.7 Busulphan
Formulation 0.5 and 2 mg coated tablets [25 mg tablets are now produced by Novolabs/UK] Storage 0.5 mg tablets - keep dry and store at 2-8C 2 mg tablets - keep dry and store < 25C Stability Three years from manufacture. If 0.5 mg tablets are stored between 8-25C they are stable for 6 months. AdministrationFor oral administration No product licence (in UK) for neuroblastoma Toxicity Myelosuppression, thrombocytopenia, mucositis, nausea, vomiting, diarrhoea, anorexia, hyperpigmentation, interstitial pulmonary fibrosis, veno-occlusive disease. Toxicity Frequencies
Common Happens to 21-100 children out of every 100 Immediate: Within Myelosuppression, 1-2 days of receiving drug thrombocytopenia Prompt: Within 2-3 mucositis, weeks, prior to the veno-occlusive disease next course hyperpigmentation, Delayed: Any time later during therapy, excluding the above conditions Late: Any time after the sterility completion of treatment Occasional Happens to 5-20 children out of every 100 nausea, vomiting, diarrhoea, anorexia Rare Happens to <5 children out of every 100
interstitial pulmonary fibrosis
(L) Toxicity may also occur later.
21.8 Melphalan
Formulation 20 mg vials containing 50 mg anhydrous melphalan hydrochloride with 10 ml buffered diluent. Storage Below 30C protect from light Reconstitution Add 10 ml diluent to 50 mg vial, shake vigorously until dissolution complete. This gives a solution of 5 mg/ml which should be used immediately, or diluted further in 0.9% saline and infused within 2 hours. Dextrose solutions are incompatible with melphalan. AdministrationAs IV bolus into fast running infusion via a central venous line, or as an infusion over 1-2 hours. Toxicity Myelosuppression, irreversible bone marrow aplasia, amenorrhoea, sterility, nausea, vomiting, stomatitis, diarrhoea.
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Common Happens to 21-100 children out of every 100 Immediate: Within Anorexia, ulceration if 1-2 days of receiving extravasated, nausea and drug vomiting Prompt: Within 2-3 Myelosuppression (L), weeks, prior to the mucositis, diarrhoea, next course alopecia Delayed: Any time later during therapy, excluding the above conditions Late: Any time after Completion of treatment (L) Toxicity may also occur later. Occasional Happens to 5-20 children out of every 100 Rare Happens to <5 children out of every 100 Hypotension, diaphoresis, hypersensitivity reaction
Inanition
Pulmonary fibrosis, sterility, secondary malignancy
21.9 Granulocyte Colony Stimulating Factor

(r-metHuG-CSF, G-CSF, filgrastim, Neupogen) NSC #614629 Source and Pharmacology: r-metHuG-CSF (produced in E. coli by recombinant DNA technology) stimulates the production of neutrophils in the bone marrow and selected end-cell activation. The 175 amino acid protein (M.W. of 18,800 daltons) differs from the natural protein in that the N-terminal amino acid is a methionine and it is not o-glycosylated. 3.45 g to 11.5 g of G-CSF (filgrastim) administered subcutaneously resulted in a maximum serum concentration of 4 ng/ml to 49 ng/ml within 2 to 8 hours. The elimination half-life is similar for SQ and IV, approximately 3.5 hours.
Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 weeks, prior to the next course
Common Happens to 21 100 children out of every 100
Occasional Happens to 5-20 children out of every 100 Local irritation at the injection site Medullary bone pain, increased alkaline phosphatase, increased lactate dehydrogenase, increased uric acid, thrombocytopenia
Rare Happens to <5 children out of every 100 Allergic reaction, low grade fever Subclinical splenomegaly, exacerbation of pre-existing skin rashes, alopecia Cutaneous vasculitis
Delayed: Anytime later during therapy, excluding the above conditions Late: Anytime after Completion of treatment
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Formulation and Stability: Supplied as a sterile, clear, colourless and preservative-free solution in pre-filled syringes or vials (1ml or 1.6ml). Vials are preservative free and are intended to be single-use vials; Filgrastim must be stored between 2 and 8 C. Stability has been demonstrated for at least 24 months when stored under these conditions. Do not use if discoloured or if there is particulate matter. G-CSF Administration: Administer once daily, subcutaneously without dilution. For IV use, dilute in D5W to concentrations 15 g/ml; G-CSF (filgrastim) is incompatible with normal saline. At dilutions from 5 g/ml to 14 g/ml, add human serum albumin to a final albumin concentration of 2 mg/ml to protect against absorption of the G-CSF (filgrastim) to container walls (glass or plastic). Filgrastim, when diluted as described above, is compatible with a number of plastics commonly used in the manufacture of syringes, IV bags, infusion sets, and IV pump cassettes. These include polyvinyl chloride, polyolefin, and polypropylene. Diluted filgrastim should be stored at 2 to 8 C and used within 24 hours. Do not shake or freeze. The suggested starting dose is 5 g/kg. Although guidelines are not well documented in the literature, POG protocols typically recommend stopping G-CSF (filgrastim) if the following occurs: ANC > 0.5 x 109/L after the nadir is reached (usually 10- 14 days) or ANC > > 0.5 x 109/L on 2 consecutive days after nadir is reached Generally, the ANC decreases by 50% in 24-48 hours G-CSF (filgrastim) should be stopped 48 hours before restarting chemotherapy.
Supplier: Commercially available. See package insert for further information.
21.10 13-Cis-Retinoic Acid (ISOTRETINOIN , ROACCUTANE)

Source and Pharmacology: The exact mechanism of RA-induced maturation of tumour cells is not known. It has been observed that cyclic AMP-inducing agents have a synergistic effect on RA-induced differentiation of the HL-60 promyelocytic leukemic cell line. The observers have hypothesised that RA may induce increased levels of a cAMP-dependent protein kinase whose activity is potentiated by increased intracellular cAMP levels. The role of cAMP-dependent protein kinases in cellular differentiation has been documented. RA also appears to enhance normal haematopoietic differentiation by increasing the responsiveness of myeloid and erythroid progenitor cells to the action of myeloid colony stimulating activity and erythropoietin, respectively. Metabolism: RA is 99.9% bound in plasma (almost entirely to albumin) and has a half-life of 10-20 hours. The major metabolite is 4-oxoisotretinoin, and excretion is in the urine and feces. A single oral dose of 100mg/m 13-cis retinoic acid will produce peak plasma levels of 1-2mM. The mean peak-time was 3.2 hours after 80mg orally, with a terminal t of 10 to 20 hours. Guidelines for cutting Isotretinoin capsules: Isotretinoin (13-cis retinoic acid) will be provided in 5mg and 20mg capsules depending on the total daily dose required. All capsules are blister packed. The following guidelines have been developed to maximise the amount of drug recovered from the capsule and to minimise the risk of skin contamination especially to women of childbearing age. Gloves must be worn for this procedure. 1. Remove capsule from blister pack and transfer required number of capsules for each dose to the plastic medicine pot.
2. Assemble equipment: 1 pair non-sterile gloves small pair sharp clean scissors (to be used only for this purpose) 1 dessert spoon 1 teaspoon 1 small tray small portion of ice cream/yoghurt kitchen roll kept just for this purpose cytotoxic waste bin 3. Put on gloves. 4. Place dessert spoon on clean surface. 5. Take a capsule between finger and thumb and hold upright firmly. With the scissors snip the tip off the capsule into the tray to avoid any possible harm to the eyes. 6. Carefully squeeze the contents of the capsule onto the dessert spoon. 7. Discard empty capsule in cytotoxic waste bin. 8. Use kitchen roll to wipe any drug from gloves then dispose of kitchen roll immediately in the cytotoxic waste bin. 9. Repeat steps 5 to 8 for each capsule needed. 10. After all the required capsules have been snipped, use the teaspoon to place some soft ice cream or yoghurt onto the dessert spoon. 11. Using the teaspoon mix the ice cream/yoghurt and medicine together. 12. Give medicine to child. 13. Clean surface with kitchen roll and wash all equipment, including scissors, in hot soapy water. 14. Dispose of gloves in cytotoxic waste bin. 15. Wash hands thoroughly. 16. Return cytotoxic waste bin to hospital when full.
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Toxicity: Common Happens to 21-100 children out of every 100 Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 Dry skin (L), dry mucosa weeks, prior to the (L), cheilitis (L) next course Occasional Happens to 5-20 children out of every 100 Nausea and vomiting Rash (L), conjunctivitis (L), musculoskeletal pains (L), fatigue (L), headache (L), serum elevation (L), triglyceride elevation (L), cholesterol elevations (L), transaminase elevations (L) Rare Happens to <5 children out of every 100 Changes in skin pigmentation, nonspecific GI complaints, dizziness, pseudotumour-cerebri, RBC decreases, WBC decreases, retinoic acid syndrome with hyperleukocytosis, respiratory distress, fever, hypotension Skeletal hyperostosis
Delayed: Any time later during therapy, excluding the above conditions Late: Any time after the completion of treatment (L) Toxicity may also occur later.
21.11 Monoclonal Anti-GD2-Antibody (ch 14.18)

Human-mouse chimeric with the human IgG1-Fc part and the specificity for the ganglioside GD2 on human neuroblastoma cells. Produce BioInvent International AB, Lund, Sweden (?). Effect: The complement dependent (CDC) and antibody dependent cellular cytolysis (ADCC) is induced after bondage to neuroblastoma cells. Dosage: 20mg/m x d, d 1-5 as 8hr-infusion. Months 2, 4, 6, 8, 10, 12 after myeloablative therapy. Only for high risk patients. Toxicity 1. During the infusion an intensive visceral pain as well as pain in the extremities is nearly always experienced. This requires a prophylactic therapy with morphine. Generally this pain is quickly reversible after finishing the infusion. 2. Urticarial allergic reactions, which seldom occurs with tachycardia, fits of cough, temperature and CRP increase. Theoretically also anaphylactic reactions can be expected. 3. Reversible paralysation of pupils.
Toxicity Frequencies Common Happens to 21-100 children out of every 100 Visceral pain Occasional Happens to 5-20 children out of every 100 tachycardia, fits of cough, temperature Rare Happens to <5 children out of every 100 Reversible paralysation of pupils.
Immediate: Within 1-2 days of receiving drug Prompt: Within 2-3 weeks, prior to the next course Delayed: Any time later during therapy, excluding the above conditions Late: Any time after the completion of treatment (L) Toxicity may also occur later.
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22 APPENDIX: Pharmacological Guidelines for MAT

22.1 Pharmacodynamics and pharmacogenetics of high-dose busulphan and melphalan
22.1.1 RATIONALE 1. High-dose busulphan and melphalan combination is effective in high-risk neuroblastoma, but induces a significant level of multi-organ toxicity, especially liver toxicity.150;151 It has been suggested that pharmacokinetically guided dose adjustment may improve high-dose chemotherapy regimens, by reducing the inter-patient variability in systemic exposure to toxic alkylating agents.152-154 2. A PK/PD relationship between a high AUC of busulphan after oral administration and risk of severe toxicity has been documented by several teams when busulphan is combined with cyclophosphamide155;156. However, no such correlation has been found when oral busulphan is combined with melphalan in neuroblastoma and Ewing sarcoma. It does suggest that drug interaction may occur between busulphan and melphalan, and/or that exposure to both drugs should be considered to better evaluate PK/PD relationships and to develop appropriate dose adjustment. 3. Pharmacodynamics of high-dose melphalan has been recently reported in children.157 4. Both drugs are alkylating agents, sharing the same metabolic pathway through conjugation to glutathione by glutathione-S-transferases.158-161 Genetic polymorphism have been reported for this metabolic pathway and may be involved in liver toxicity of high-dose busulphan and melphalan.162;163 Genotyping can be easily performed using new molecular technologies. 5. Pharmacokinetics of oral busulphan and melphalan has been extensively studied in children.157;164 Limited sampling strategies using bayesian population methodologies have been defined for both drugs,165-167 and enable prospective, multicentric study with a small number of blood samples needed for each patient. 22.1.2 OBJECTIVES 22.1.2.1 Primary objective To study whether a high AUC of busulphan and/or melphalan is associated with an increased risk of severe liver toxicity in children receiving high-dose oral busulphan and iv-melphalan as a consolidation therapy for high-risk neuroblastoma.
22.1.2.2 Secondary objectives To describe concomitantly the pharmacokinetics of busulphan and melphalan using population PK models and to identify possible drug interactions. To study the PK/PD relationships between exposure to busulphan and melphalan and severe toxicities other than VOD.
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To genotype patients for major detoxifying enzymes (mainly glutathione-transferases) involved in alkylating agents metabolic pathway and to study the risk of severe toxicity with regard to systemic exposure to both drugs, and genotype. To identify the AUCs of busulphan and melphalan that could be prospectively targeted to reduce the risk of severe toxicity in high-dose combined chemotherapy regimens including busulphan (oral or iv) and melphalan.
22.1.3 TYPE OF STUDY This is multi-institution pharmacological study exploring pharmacokinetics, pharmacodynamics and pharmacogenetics of two anticancer drugs using limited blood sampling strategies and NONMEM programmes. This study will be conducted according to Good Laboratory Practice, including interlaboratory cross-validation of busulphan and melphalan assays.
22.1.4 BUSULPHAN BLOOD SAMPLING/ ASSAYS For all blood samples (2 ml), plasma will be separated at 0C and stored at 20C until sample shipment which will be performed every 3 to 6 months for each centre according to their accrual. Busulphan plasma levels will be assayed after the first oral dose and four blood samples will be withdrawn (before administration and at 2, 4 and 6 hours after oral intake). In addition, trough plasma levels will be measured immediately before doses 5, 9 and 13. Busulphan will be assayed by gas-chromatography with two different ways of detection: 1. Gas chromatography with electron capture detector (Sweden). 2. Gas chromatography with Mass spectrometry (France). Briefly, 0.2 ml (heparin) plasma is required for the analysis. Internal standard (1,5bis(methansulfonoxy)pentane) or Deuterium busulphan) is added along with 1 ml sodium iodide (8M) and 0.4 ml n-heptane. A micro magnet is added to the screw cap tube. The reaction is carried on at 70 oC for 45 min under continuous magnet stirring. Then, the organic phase (nheptane) is taken to analysis in the GC system. 22.1.5 MELPHALAN BLOOD SAMPLING/ ASSAYS Melphalan plasma levels will be assayed after the single iv infusion over 15 minutes and 4 blood samples will be withdrawn (before administration and at 5, 23 and 90 minutes Melphalan will be assayed by HPLC with UV detection as previously described.167 22.1.6 PHARMACOKINETIC MODELING Pharmacokinetic parameters will be established according to validated and reported bayesian population (NONMEM program) for busulphan and melphalan157. 22.1.7 GENOTYPING Total blood sample (5 ml) will be collected on EDTA before treatment for genotyping. Gene sequence for GSTs and other detoxifying enzymes will be performed using Taqman technology. Other polymorphisms will be studied later on using micro-arrays.
22.1.8 NUMBER OF PATIENTS 80 - 100 patients over the 4 year accrual of the protocol. 22.1.9 TOTAL AMOUNT OF BLOOD Overall the study requires 5 ml for genotyping and 11 samples (2 ml) over the 5 days of treatment, i.e. a total amount of 27 ml. 22.1.10 REFERENCE LABORATORIES Dr Claude Ardiet, Centre Lon Brard in Lyon, France for melphalan assay. Dr Moustapha Hassan, Huddinge University Hospital in Stockholm, Sweden for busulphan assay in part of the samples. Dr Marc Bonnay, Institut Gustave Roussy, Villejuif for busulphan assay in part of the samples.
Cross-validation study for busulphan assay will be performed prior to initiation of the study. 22.1.11OPERATION OF THE STUDY A Pharmacology committee composed of the study coordinators, a representative of each laboratory of the network, a bio-mathematician and two clinicians involved in the European controlled trial study monitoring committee, will meet twice a year.
22.2 Pharmacodynamics and pharmacogenetics of CEM MAT

22.2.1 THERAPEUTIC DRUG MONITORING OF CARBOPLATIN For those centres able to carry out therapeutic monitoring, real-time measurement of carboplatin pharmacokinetics will be carried out to allow for dose adjustment during the 4 day course of carboplatin therapy. Blood samples for pharmacokinetics will be taken following day 1 administration with samples being sent by overnight courier to the Cancer Research Unit in Newcastle for analysis of free platinum plasma levels on day 2. This will allow dose adjustment of the day 3 and 4 doses of carboplatin where appropriate. Samples to be taken on day 1 prior to administration of carboplatin and 0.5, 1 and 2h after the start of infusion, 5ml of blood should be taken in a heparinised tube and centrifuged at a speed of 1,000 g at 4C. The plasma should be separated immediately, 1ml transferred to an ultrafiltration device (MPS-1, Amicon Centrifree) and centrifuged at 1,000 2,000 g for 15 minutes at 4C. The ultrafiltrate and remaining plasma should be frozen immediately at -20C and transported by overnight courier to Newcastle for analysis (Gareth Veal/Alan Boddy, Tel. +44 0191 222 7134 or +44 0191 222 8233). Each centre participating in pharmacokinetic studies will undertake blood sampling on the remaining 3 days of carboplatin treatment for retrospective pharmacokinetic analysis. This will allow the relationship between intended and administered dose of carboplatin and carboplatin exposure to be investigated. These samples should be processed as above and stored at -20C prior to transport to Newcastle as convenient. Pharmacokinetic samples may also be
taken over the 4 days of treatment for retrospective analysis even if no therapeutic drug monitoring is planned. This will allow possible correlations between carboplatin exposure and response/toxicity to be studied. Carboplatin will be measured in plasma ultrafiltrate samples as free platinum by flameless atomic absorption spectrophotometry (AAS). 22.2.2 MONITORING OF ETOPOSIDE AND MELPHALAN Dosing of etoposide and melphalan in this study are determined by surface area for the majority of children but by body weight for those children whose weight is below a specified cut-off point (12 kg). In addition, dose reductions are also made for patients with a GFR < 100 ml/min/1.73m2. This application of cut-off points for surface area and renal function based dosing is designed to avoid excessive drug toxicities in these groups of patients. However, this general rule may not be applicable to all drugs used in paediatric oncology. Pharmacokinetic sampling of etoposide and melphalan will provide data on the exposure of patients to these two chemotherapeutic agents in each of the dosing groups specified in this study, i.e. dosage based on surface area, dosage adjusted based on renal function and dosage adjusted based on body size. This will enable us to determine whether or not the application of cut-off points, for surface area and renal function based dosing, result in a significant difference in steady state plasma concentrations between these patient populations. 22.2.3 ETOPOSIDE PHARMACOKINETIC SAMPLING AND ANALYSIS Samples to be taken prior to administration of etoposide and 6 hours after the start of infusion with an additional sample taken between 8 and 22 hours after the start of infusion on each of 4 days of treatment. 2ml of blood will be taken in a heparinised tube, centrifuged at a speed of 1,000g at 4C and the plasma separated and frozen at -20C. Measurement of etoposide concentrations in plasma will be undertaken at the Cancer Research Unit, University of Newcastle.
Etoposide plasma concentrations will be determined by LC/MS analysis using techniques previously established in the unit.
22.2.4 MELPHALAN PHARMACOKINETIC SAMPLING AND ANALYSIS Samples to be taken prior to administration of melphalan, and at 5, 23 and 90 minutes after the start of infusion on day 1 of treatment only. 2ml of blood will be taken in a heparinised tube, centrifuged at a speed of 1,000g at 4C and the plasma separated and frozen at -20C. Measurement of melphalan concentrations in plasma will be undertaken at the Cancer Research Unit, University of Newcastle.
Melphalan plasma concentrations will be determined by HPLC analysis using techniques previously established in the unit.
22.2.5 TRANSPORT OF SAMPLES Samples should be sent to Newcastle by overnight courier, packed on dry ice in an insulated container. Plasma samples for pharmacokinetic analysis should be sent together following completion of pharmacokinetic sampling. The Cancer Research Unit should be notified on the day that the samples are sent (contact Gareth Veal or Alan Boddy, Tel. +44 0191 222 7134 or +44 0191 222 8233).
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Carboplatin Sampling Sheet

DAY OF STUDY DATE WEIGHT (kg) NAME S.A (m2) HOSPITAL NUMBER
51
CR EDTA t (min)
CARBOPLATIN DOSE (MG): INFUSION TIME (carboplatin) START: FINISH:
Samples to be taken for carboplatin analysis. Label all tubes with the patient initials, date of study and time of sample or sample number. Sample number 1 2 3 4 Time from start Carboplatin (min) Pretreatment 30 60 120
Time due
Time taken
Samples should reach laboratory for centrifugation within 10 minutes of sampling. 5 ml of blood to be taken in a heparinised tube, centrifuged for 5 min at 1000g and 4C. Remove 1 ml of plasma and freeze remainder at -20C. Transfer the 1 ml of plasma to an Ultrafiltration device (MPS-1, Amicon Centrifree), centrifuge at approximately 1500g for 15 min using a refrigerated centrifuge at 4C and freeze the ultrafiltrate at -20C. Samples to be sent by overnight courier, packed on dry ice in an insulated container. Address for delivery :Gareth Veal / Alan Boddy Cancer Research Unit University of Newcastle Medical School Newcastle upon Tyne NE2 4HH Contact numbers :Gareth Veal : 0191 222 7134 Alan Boddy : 0191 222 8233 Fax : 0191 222 7556 e mail : G.J.Veal@newcastle.ac.uk Alan.Boddy@newcastle.ac.uk
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Etoposide Sampling Sheet

DAY OF STUDY DATE WEIGHT NAME S.A HOSPITAL NUMBER GFR (if available)
ETOPOSIDE DOSE (mg): DAY 1 DAY 2 DAY 3 DAY 4 INFUSION TIME INFUSION TIME INFUSION TIME INFUSION TIME START: START: START: START: FINISH: FINISH: FINISH: FINISH:
Samples to be taken for etoposide analysis Label all tubes with the patient initials, date of study and time of sample or sample number Sample number 1 2 3 4 5 6 7 8 9 Study day 1 1 1 2 2 3 3 4 4 Time from start Etoposide Pretreatment 6h 8-22h 6h 8-22h 6h 8-22h 6h 8-22h Time due Time taken
2 ml of blood to be taken in a heparinised tube and centrifuged for 5 min at 1000g and 4C. Remove plasma and freeze at -20C as soon as possible. COMMENTS:
Samples to be sent packed on dry ice in an insulated container by overnight courier following completion of etoposide treatment. Address for delivery :Gareth Veal / Alan Boddy Cancer Research Unit University of Newcastle Medical School Newcastle upon Tyne NE2 4HH Contact numbers :Gareth Veal : 0191 222 7134 Alan Boddy : 0191 222 8233 Fax : 0191 222 7556 e mail : G.J.Veal@newcastle.ac.uk Alan.Boddy@newcastle.ac.uk
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Melphalan Sampling Sheet

DAY OF STUDY DATE WEIGHT NAME S.A HOSPITAL NUMBER GFR
MELPHALAN DOSE (mg): INFUSION TIME (melphalan) START: FINISH:
Samples to be taken for melphalan analysis. Label all tubes with the patient initials, date of study and time of sample or sample number Sample number 1 2 3 4 Time from start Melphalan Pre-treatment 5 min 23 min 90 min
Time due
Time taken
5 ml of blood to be taken in a heparinised tube and centrifuged for 5 min at 1000g and 4C. Remove plasma and freeze at -20C. COMMENTS:
Address for delivery :Gareth Veal / Alan Boddy Cancer Research Unit University of Newcastle Medical School Newcastle upon Tyne NE2 4HH Contact numbers :Gareth Veal : 0191 222 7134 Alan Boddy : 0191 222 8233 Fax : 0191 222 7556 e mail : G.J.Veal@newcastle.ac.uk Alan.Boddy@newcastle.ac.uk
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23 LEUCAPHERESIS GUIDELINES
23.1 Paediatric apheresis procedure
23.1.1 GENERAL PRINCIPLES IN TECHNIQUE Although operating procedures differ for different apheresis systems, certain principles apply to all types of equipment. Continuous-flow (CF) systems are preferred for paediatric use because they have smaller extra corporal volumes (ECV). In older children with a body weight greater than 30 kg, the technique is very similar to that used in adults. It is in small children that significant modifications of techniques are required to provide safe and effective therapy. The two most important factors for safe apheresis procedures in paediatric patients are the maintenance of both a constant intravascular volume and an adequate red blood cell mass in the circulation. A third factor is prevention of hypocalcaemia that results from chelation of calcium by citrate in the anticoagulant. PBSC harvesting can begin when the peripheral CD34+ count is > 20 cells/l. The paediatric apheresis procedure should be performed by an experienced paediatric team. The team must be familiar with the paediatric basic life support, advanced life support and the typical age dependent problems of children. The parents or a reliable person for the children should be present during the whole procedure. 23.1.2 APHERESIS MACHINE The Cobe Spectra or the Fenwal CS 3000+ is recommended because the continuous flow centrifugation devices are better tolerated than discontinuous flow machines. Equipment should be operated in compliance with the manufacturer's operating guidelines. The Standards of Care protocols should be written and available in the Apheresis Unit. The standard operating procedure will be specific for each machine. Blood Priming Priming of the machine prior to collection should be with ACD and saline according to manufacturer's directions. In haemodynamic instable patients or very small children (BW < 15 kg) the priming should be performed by 5% albumin solution instead of saline solution. For patients less than 25 kg, a secondary prime with IRRADIATED, leukocyte-poor red blood cells should be done. This is described in the standard operating procedures for each machine. The blood prime will be performed with cross-matched, irradiated, filtered red cells. Procedural Support Use of a Cobe in-line blood warmer on the return line will be used for the Cobe machine. A standard blood warmer device can be used with the Fenwal machine. If patients platelet count is <30,000, one may consider to transfuse with platelet prior to apheresis procedure. However, there is evidence in the literature that apheresis procedure could also be performed in children with platelet counts below 20 x 109 /l.168 23.1.3 ANTICOAGULANT Anticoagulant to be used is Acid Citrate Dextrose Formula - A (ACD-A) in a ratio sufficient to prevent extracorporeal clotting. Heparin anticoagulation is not recommended for use in PBSC collections except for patients with an allergy to citrate. Hypocalcemia is a wellrecognised side effect of citrate. To prevent hypocalcaemia a prophylactic calcium fluconate infusion can be used. If patient becomes symptomatic from hypocalcaemia then give oral calcium or alternatively the rate of the calcium gluconate infusion can be increased.
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Whole Blood Flow Rate The following rates are designed to avoid citrate reactions and thus boluses and continuous infusions of calcium can be avoided. <2 years (<15 kg) 15-20 ml/min (initial)* 2-5 years (15-20 kg) 25-40 ml/min >5 years 35-50 ml/min may be increased to 25-30 ml/min by ratio ramping
Collection Goals During each leukapheresis procedure, the volume of whole blood processed should be approximately 240 to 480 ml/kg (3 - 6 blood volumes). The total time necessary for the whole apheresis procedure should not exceed 5 h. Optimal collection goal (total for all collections) is more than 3 x 106 CD 34+ cells/kg for unpurged PBSC with a rescue of 2 x 106 CD 34+ cells/kg. The targeted number of cells can usually be obtained in 1-2 collection days. Patient Monitoring Patients should be observed continuously during the collection. Vital signs should be obtained q 1 hour. Laboratory Studies For patients < 25 kg, a type and cross for PRBC should be performed one day prior to procedure. Pre-apheresis and immediately post-apheresis the following minimal lab values should be obtained: CBC with differential and platelet count, ionised calcium and magnesium. Vascular Access For continuous flow apheresis, two sites of venous access are required. In patients less than 25 kg use the MedComp 8.0 French permanent or temporary catheter as required. For patients greater than 25 kg, the MedComp 8.0 French or other central venous lines can be used. Depending on the situation of the peripheral veins, a Hickman catheter could be used in combination with a peripheral venous access, also in very small children.
23.2 Cryopreservation of PBSC Products

Each collection should be processed and cryopreserved on the day of collection using 10% dimethyl sulfoxide final concentration, controlled-rate freezer, and liquid nitrogen storage. Stem cells should be frozen at a final concentration of 0.5 to 1.2 x 108 nucleated cells/ml. Fluid Management Hydration with D5 0.45 NS +/- KCl or 0.9 NS should begin 2-4 hours prior to the infusion and be continued for at least 4 hours following infusion. Intravenous fluids on the day of PBSC infusion, excluding the volume of cells infused, should total 3000 ml/m/24 hours. Premedication The DMSO cryoprotectant may cause a histamine-like reaction when infused into the patient. Therefore premedication with Antihistamines (i.e. Benadryl) is recommended. Thawing of PBSC PBSC are thawed in a 37oC water bath which is monitored with a mercury thermometer to ensure temperature does not rise above 40oC. Only one bag of PBSC should be thawed at a time. In the event of bag breakage, every effort should be made to maintain sterility and salvage the PBSC component using a syringe with a large bore needle. When the infusion of one bag is completed, the next bag should be thawed. When the final bag of PBSC has been infused, the IV tubing should be flushed with normal saline.
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23.3 PBSC Infusion

Thawed PBSC should be infused as rapidly as tolerated through a central venous catheter. The PBSC product should NEVER be irradiated prior to infusion. Only a 170 micron blood filter should be used during reinfusion. No WBC filter should be used. The unit may be infused by gravity, or the cells may be drawn up into a syringe and pushed by trained personnel. Microaggregate filters and leukodepletion filters MUST NOT be used for infusion of PBSC. If a thawed unit appears clumpy or stringy and these particles cannot be dispersed with gentle kneading, the PBSC product could be infused through a standard 170 micron blood filter. Possible Symptoms during Infusion Precipitating Factor haemolysed red cells cellular clumps and debris cold 10% DMSO microbial contamination plasma proteins Possible Symptoms fever, chills, haemoglobinuria chest pain, hypoxia, hypertension nausea, headache fever, chills, hypotension urticaria
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24 APPENDIX Performance Scales

24.1 Lansky Play-Performance Scale
100 90 80 70 60 50 40 30 20 10 0Fully active, normal. Minor restrictions in physically strenuous activity. Active, but tires more quickly. Both greater restriction of and less time spent in play activity. Up and around, but minimal active play; keeps busy with quieter activities. Gets dressed but lies around much of the day, no active play, able to participate in all quiet play and activities. Mostly in bed; participates in quiet activities. In bed; needs assistance even for quiet play. Often sleeping; play entirely limited to very passive activities. No play; does not get out of bed. Unresponsive.
24.2 Karnofsky Performance Scale

100 90 80 70 60 50 40 30 2010 0Normal, no complaints, no evidence of disease. Able to carry on normal activity, minor signs or symptoms of disease. Normal activity with effort, some signs or symptoms of disease. Cares for self. Unable to carry on normal activity or to do active work. Requires occasional assistance, but is able to care for most of own needs. Requires considerable assistance and frequent medical care. Disabled, requires special care and assistance. Severely disabled, hospitalisation is indicated although death is not imminent. Hospitalisation necessary, very sick, active supportive treatment necessary. Moribund, fatal processes progressing rapidly. Dead.
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25 Appendix Guidelines for Reporting Toxicity/Serious Adverse Events

Any serious adverse event should be reported to the National Data Centre within 24 hours of onset. The Data Centre will notify the Study Co-ordinator by fax within 24 hours. In order to have consistency with the other European neuroblastoma studies CTC toxicity grading will be used in this study. Exceptions will be : 1. Ototoxicity where the Brock grading system will be used 2. Renal toxicity where an isotope method of GFR will be required. 3. For the transplant setting the Bearman classification must be used. All grade 3 or 4 toxicity should be reported as SAE. According to the ICH GCP guidelines (International Committee on Harmonisation Guidelines for Good Clinical Practice) a Serious Adverse Event (SAE) or Serious Adverse Drug Reaction (Serious ADR) is any untoward medical occurrence that at any dose: results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability/incapacity, or is a congenital anomaly/birth defect. (see the ICH Guideline for Clinical Safety Data Management Definitions and Standards for Expedited Reporting: http//:www.ifpma.org/pdfifpma/e2a.pdf) AT THE END OF THE MAT/PBSCR ( CEM OR BUMEL) Haematological toxicity The following are considered serious adverse events and should be notified to the National Data Centre within 24 hours of onset : all neutropenia, (neutrophils <0.5 x 109/l) beyond day 20 post-transplant all thrombocytopenia requiring transfusion support beyond day 30 post-transplant. Extra-haematological toxicity All grade 3 toxicity according to the Bearman classification169 should be notified within 24 hours to the International Data Centre (St. Anna Childrens Hospital/ CCRI; HRNBL1@ccri.univie.ac.at; St.Anna Childrens Hospital/CCRI, Unit for Applied Clinical Research and Statistics, A 1090 Vienna)
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26 APPENDIX: Toxicity Grading

26.1 TOXICITY AFTER HIGH DOSE CHEMOTHERAPY (BEARMAN)169
Grade I Cardiac Mild ECG abnormality, not requiring medical intervention; or noted heart enlargement on CXR with no clinical symptoms Grade II Moderate ECG abnormalities requiring and responding to medical intervention; or requiring continuous monitoring without treatment, or congestive heart failure responsive to digitalis or diuretics. Macroscopic haematuria after 7 days from last chemotherapy dose not caused by infection; or haematuria after 2 days with subjective symptoms of cystitis not caused by infection . Increase in creatinine above twice baseline but not requiring dialysis. CXR with extensive localised infiltrate or moderate interstitial changes combined with dyspnea and not caused by infection or CHF; or decrease of PO2 (>10% from baseline) but not requiring mechanical ventilation or >50% O2 on mask and not caused by infection or CHF. Moderate hepatic dysfunction with bilirubin >6mg% <20mg%, or SGOT increase >5-fold from preconditioning ; or clinical ascites or image documented ascites >100ml, or weight gain >5% from baseline of noncardiac origin. Somnolence with confusion after arousal, or other new objective CNS symptoms with no loss of consciousness not more easily explained by other medication, bleeding, or CNS infection. Pain and/or ulceration requiring a continuous IV narcotic drug (morphine drip). Watery stools >2,000ml every day not related to infection; or macroscopic hemorrhagic stools with no effect on cardiovascular status not caused by infection; or subileus not related to infection. Grade III Severe ECG abnormalities with no or only partial response to medical intervention; or heart failure with no or only minor response to medical intervention; or decrease in voltage by more than 50%. Haemorrhagic cystitis with frank blood, necessitating invasive local intervention with installation of sclerosing agents, nephrostomy or other surgical procedure. Requirement of dialysis. Interstitial changes requiring mechanical ventilatory support or >50% oxygen on mask and not caused by infection or CHF.
Bladder
Macroscopic haematuria after 2 days from last chemotherapy dose with no subjective symptoms of cystitis and not caused by infection. Increase in creatinine up to twice the baseline value (usually the last recorded before start of conditioning). Dyspnea without CXR changes not caused by infection or congestive heart failure; or CXR showing isolated infiltrate or mild interstitial changes without symptoms not caused by infection or congestive heart failure. Mild hepatic dysfunction with 2.0 mg% < bilirubin < 6.0mg%; or weight gain >2.5% and <5% from baseline, of noncardiac origin; or SGOT increase more than 2-fold but less than 5-fold from lowest preconditioning. Somnolence but the patient is easily arousable and orientated after arousal. Pain and/or ulceration not requiring a continuous IV narcotic drug. Watery stools > 500ml but <2,000ml every day not related to infection.
Renal Pulmonary
Liver
Severe hepatic dysfunction with bilirubin >20mg%; or hepatic encephalopathy; or ascites compromising respiratory function. Seizures or coma not explained (documented) by other medication, CNS infection, or bleeding. Severe ulceration and/or mucositis requiring preventive intubation; or resulting in documented aspiration pneumonia with or without intubation. Ileus requiring nasogastric suction and/or surgery and not related to infection; or hemorrhagic entercolitis affecting cardiovascular status and requiring transfusion.
CNS
Stomatitis
Digestive
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26.2 CTC-NCIC CRITERIA

RECOMMENDATIONS FOR GRADING OF TOXIC EFFECTS
Site
H H1 H2 H3 H4 H5 D D1 D2 Haematological Haemoglobin (g/100 ml) 3 Leukocytes (1000/mm ) 3 Granulocytes (1000/mm ) 3 Platelets (100`0/mm ) Haemorrhage Digestive Bilirubin Transaminases (SGOT/SGPT) Alkaline phosphatase Amylase Stomatitis Nausea/vomiting
Grade 0
WNL <4.0 < 2.0 WNL None
Grade 1
> 10.0 3.0-3.9 1.5-1.9 > 75 Petechiae
Grade 2
8.0-9.9 2.0-2.9 1.0-1.4 50-75 Mild blood loss
Grade 3
6.5-7.9 1.0-1.9 0.5-0.9 25-50 Needing blood transfusion or fundal haemorrhages 1.5 - 3 x N 5.1 -20 x N
Grade 4
<6.5 <1.0 < 0.5 < 25 Debilitating blood loss, life threatening > 3x N > 20 x N
WNL 1.26-2.5 x N < 1.25 x N < 1.25 x N WNL No change None 1.26-2.5 x N < 1.5 x N Mild soreness, erythema Nausea
< 1.5 x N 2.6 -5 x N
D3 D4 D5 D6
2.6 -5 x N 1.6-2 x N Painful erythema, oedema, ulcers but can eat solids Transient vomiting < 5 episodes in 24 hrs Tolerable but > 2 days Moderate
5.1 -20 x N 2.1 - 5 x N Ulcerated lesions, requiring liquid diet only Transient vomiting > 5 episodes in 24 hrs Intolerable requiring therapy Severe, abdominal distension 3.1 - 6 x N 4+ or > 10 g/l Gross + clots 156-164/116/124 6.5-6.9/2.1-2.5 3.1-3.3/1.5-1.69 0.6-0.8 50-64 54-40% 54-40% Dyspnoea at normal levels of exertion
> 20 x N >5xN Oral alimentation not possible Intractable vomiting, >10 episodes in 24 hrs Leading to dehydration Ileus >96 hrs; distension & vomiting >6xN Nephrotic syndrome Requires transfusion > 165 < 115 > 7/ < 2 > 3.3/ < 1.5 < 0.6 < 49 < 40% < 40% Dyspnoea at rest
D7 D8 M M M M3 M4 M5 M6 M7 P P1 P2 P3 P4
Diarrhoea Constipation Metabolic/Renal Blood creatinine Proteinuria Haematuria Na + mmol/L K+ mmol/lL Ca + mmol/L Mg++ mmol/l Pulmonary PA O2 DL CO CV Function
None None or no change
Transient < 2 days Mild
WNL No change No change 135-145 3.5-5.4 2.15-2.59 1.5-2.0 > 90 100-75% 100-75% No change
< 1.5 x N 1+ or < 3g/l Microscopic 146-149/130-134 5.5-5.9/3.1-3.4 2.6-2.89/1.9-2.1 1.2-1.4 80-89 74-65% 74-65% Mild symptoms
1.5 - 3 x N 2-3 + or 3-10 g/l Gross no clots 150-155/125/129 6-6.4/2.6-3 2.9-3.09/1.7-1.89 0.9-1.1 65-79 64-55% 64-55% Exertional dyspnoea
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RECOMMENDATIONS FOR GRADING OF TOXIC EFFECTS Grade 2

Mild bronchospasm, urticaria, no parenteral therapy needed Dry desquamation, vesiculation, pruritus
Site
A S I Allergy Skin Infection
Grade 0
No change No change
Grade 1
Oedema, transient rash Macular, papular eruption, erythema, asymptomatic
Grade 3
Bronchospasm, parenteral therapy required General symptomatic macular, papular or vesicular eruption or ulceration Minor infection: 1= culture negative fever without septic shock 2= central catheter related bacteraemia epidermidis, staphylococcus 3= other
Grade 4
Anaphylaxis Exfoliative dermatitis, necrosis requiring surgical intervention Major infection: 1= septicaemia 2= pneumonia 3= urinary infection 4= severe soft tissue infection 5= septic shock Requires monitoring or hypotension or ventricualr tachcardia or fibrillation Severe or refractory congestive cardia failure Acute myocardial infarction < 15% Requiring therapy and hospitalisation. for > 48Hours after stopping agent Hypertensive crisis
C CI C2 C3 C4 C5 C6
Cardiac Rhythm Function Ischaemia Echocardiography (FS) Hypotension Hypertension
None No change None > 30% None or no change None or no change
Asymptomatic, transient ,requiring no therapy Asymptomatic Non specific T wave flattening > 25% and < 30% Changes requiring no therapy Asymptomatic, increase< 20 mmHg or < 150/100 if previous WNL. No treatment required
Recurrent/persistent requiring no therapy Asymptomatic, decline of resting EF > 20% of baseline Asymptomatic St + T wave changes suggesting ischaemia > 20% and < 25% Requiring therapy but no hospitalisation Recurrent/persistent increase > 20 mmHg or > 150/100 if previous WNL. No treatment required
Requires treatment Mild congestive heart failure responsive to therapy Angina without evidence of infarction > 15% and < 20% Requiring therapy and hospitalisation. resolves within 48 hours after stopping the agent Requires therapy
N N1
Neurological Seizures
N2 N3 N4 N5 N6
Cortical Cerebellar Sensory Motor Vision
None or no change None None or no change None or no change
Mild somnolence and agitation Slight incoordination Mild paraesthesia and/or loss of deep tendon reflexes Subjective weakness, no objective findings
Moderate somnolence (< 50% waking hors) or agitation Intention tremor, dysmetria, slurred speech, nystagmus Mild or moderate objective sensory loss; moderate paraesthesia Mild objective weakness, without significant impairment
Severe somnolence, agitation, confusion, disorientation Locomotor ataxia Severe objective sensory loss, or paraesthesia interfering with function Objective weakness with significant impairment of function Symptomatic subtotal loss of vision
Seizures related to: 1=metabolic disorder, 2=sinus thrombosis, 3=fever, 4=other, 5=unknown Coma, encephalopathy, toxic psychosis Cerebellar necrosis
Paralysis Blindness
145
27 APPENDIX PSYCHO-SOCIAL SUPPORT

Qualified psycho-social support for patients and relatives should be an integral part of the treatment strategy. Faced with the diagnosis of cancer implying the risk of death or of permanent disablement, and the need for long-term, aggressive multimodal therapy, patients and relatives need psychological support and crisis management. Moreover, social issues must be dealt with: housing, financial issues, unscheduled leave from work, etc. Patients (and their families) need to continue their normal lives as much as possible, and to allow their minds to turn away from the disease from time to time. Thus, school, structured and spontaneous play, artwork, music therapy, etc. should be available. In the case of paediatric cancer patients, siblings often feel rejected because all of the attention is directed towards the patient, and they can even feel guilty about being healthy. Parents may wonder if they are responsible for their child's disease (wrong food, smoking?). Special attention needs to be paid and support offered to all family members as well as the patient. Close co-operation and regular exchange with the medical staff is of paramount importance in order to optimise both aspects of patient care. Well trained personnel who can offer these services should be permanently available , and need to be integrated into each patient's treatment strategy. The psycho-social support team should include members of the following professions: Clinical psychologist Paediatric nurse Social worker School teacher Nursery school / kindergarten teacher Art / music teacher / therapist Psycho-social support should encompass: Social / psychological family history at first contact (intra-family relationships, coping styles, etc.) Help and guidance with social services, health insurance, social insurance matters, etc. Help and support with practical problems during hospital stays, e.g. housing, transport, organising nannies for siblings, etc. Offering assistance and aid to patients and relatives in stressful or painful situations, e.g. on the way to the operating theatre, diagnostic interventions, etc. Building stable relationships between patient, family, and support team members Crisis intervention (e.g. in case of non-coping, non-compliance, etc.) Support with emotional aspects: coping with disease and therapy Play, artwork, music, ... Visits to the patient's home Psychological and social follow up (coping with memories of the disease and its therapy, rehabilitation, occupational problems, ...) Psychological support in a terminal care situation. The brief description given above can obviously not cover all issues of importance. In every patient, his or her specific situation must be met by adjusted care and support, which requires the permanent involvement of an experienced psycho-social team.
146
28 APPENDIX International Trial Co-ordination

The conduct of this study will be according to the following agreed procedures:
28.1 Status of Study

This is a collaborative study between a number of participating national groups. The coordinators for each group are listed below.
28.2 The Protocol

A common protocol will be used for the International study by all national groups. The finalised master protocol in the English language will be held at the International Main Data Centre. Other national groups will be responsible for producing a literal translation in their own language. Each national co-ordinator will be responsible for the distribution of protocols to centres within their national group via their national data centre as appropriate. Appendices may be added independently by any of the national groups to address local needs, provided they have no bearing on the essential aims of the international protocol. However no change will be allowed to the eligibility criteria or the treatment procedures of the main protocol. Subsequent to finalisation, any amendments to the protocol must be agreed by the SIOP Europe Neuroblastoma Board, the study co-ordinator and the national co-ordinators.
28.3 Study Forms

One common set of forms will be used. The master version (in English) of the study forms will be held at the International Data Centre. Each national co-ordinator will be responsible for distribution of forms to centres within his/her country (using their national data centre as appropriate). Additional forms may be produced independently by any national group for the collection of data additional to that required for the International study. Subsequent to finalisation, amendments to the forms must be agreed by all national groups. The International Data Centre will be responsible for the issue of amended forms.
28.4 Inclusion Procedure for New Patients

New patients will be included online via the internet by each treating physician or through the national co-ordinator. Pre-registration is only possible if the eligibility criteria are met. Registration will be final once the disease description at diagnosis including MycN results is complete.
28.5 Data Collection

Data entry ideally will be done online by each treating physician via internet (adress to be circulated prior to November 1st). If for any reason the local hospital physician prefers to fill in paper forms, the data entry still should be nationally based and done in the data center of the responsible national co-ordinator via internet.
28.6 Randomisation Procedure
Online per internet, available on from November 1st. Internet adress to be added: [
].
147
28.7 Confidentiality of Patient Data

The use of names as patient identifiers on paper forms and on national databases will be according to national practice. An abbreviated patient identifier will be used for data transfer and for the master database.
28.8 Data Quality Control

On receipt of forms at the national data centre, common range and logical checks, agreed by the Study Co-ordinators, will be carried out on data prior to transfer to the master database. Any amendment to the checking programme will require mutual agreement of the Study Coordinators. Data entry verification shall be carried out according to current national practice. Cross checks of data entry will be carried out occasionally, between national centres, on a sample of forms. Data amendments shall only be carried out at the national data centre on the national database. Errors noted on the master database, after receipt of the group database, shall be reported back to the national centre. Data audit of study forms against the patient record forms at the treating institution shall be performed to satisfy national requirements.
28.9 Data Analysis and Monitoring

Data will be released from the International Database to the Trial statistician responsible for interim analysis at given time intervalls. Results of the interim analysis on outcome and toxicity shall be reported to an independent international DMSC as scheduled in the protocol. The DMSC may recommend early stopping, continuation or extension of the study to the TMC. The Study co-ordinators (and national co-ordinators) shall meet as appropriate to consider patient treatment, eligibility and outcome to ensure the smooth running of the study.
28.10 Adverse Events

Any adverse event (death, relapse or Grade 4 non-haematological life threatening toxicity) shall be reported immediately by the treating institution to the national centre and relayed to the Study co-ordinators and the International Data Centre for further reporting according to local practice. The Toxicity criteria used in completion of the Data forms will be the same for all participating groups and appear in the appendix (chapter 26).
28.11 Chemotherapy Review

Chemotherapy forms shall be reviewed by the national group co-ordinator(s) and protocol deviations noted on the database. The review information shall be reported to the Trial coordinators.
28.12 Pathology Review

Pathology review affecting eligibility shall be rapidly reviewed by national group reviewer(s). National group pathology data shall be discussed at the meeting of the international study committee.
148
28.13 Biological Review

Biology will be established within the reference laboratories only. National group reference laboratory biology data will be discussed at the meeting of the international study committee (ENQUA).
28.14 BM /PBSC Harvest Review

Review affecting eligibility shall be rapidly done by the national group reviewer(s). National group BM / PBSC harvests data shall be discussed at the meeting of the international study committee.
28.15 MIBG Review

Review of mIBG scans shall be rapidly reviewed by national group reviewer(s). National group mIBG data shall be discussed at the meeting of the international study committee.
28.16 Radiological Review

The question of the quality of surgery and local radiotherapy on the local relapse rate, the relapse rate and EFS will be addressed. The imaging of the primary tumour site prior to and post surgery, as well as the radiation fields should be centrally reviewed.
28.17 Follow Up Data

All registered patients will be followed up by the national data centres during and after completion of treatment according to the current protocol. .
28.18 Study Approval

The study will be opened consecutively in each of the participating countries.
28.19 Institutional/Local Ethical Approval and Patient Consent

Institutional/local ethical approval will follow accepted national practice. National procedures for patient consent will be used
28.20 Data Monitoring and Safety Committee (DMSC)

An independent DMSC composed of 3 international experts will monitor the progress of the trial on ethical and scientific grounds. The role of the DMSC will be: To review the accrual rate. To examine the interim analysis. Each interim analysis will be reported to the DMSC. These interim analyses will remain confidential. On the basis of these analyses, the DMSC will recommend whether the study can continue, or whether it should be changed or terminated prematurely. To monitor toxicity. Every 6 months the statistician for the trial will circulate a report to the members of the DMSC about toxicity. The DMSC will review these interim toxicity data and any relevant information will be forwarded to each Trial co-ordinator. This biannual procedure should prevent problems of major toxicity persisting.
To examine other trials. The DMSC will review reports of related studies performed by other groups or organisations to determine whether such information materially affects the aims or preliminary findings of the trial. The DMSC will be asked to review any major modification to the study proposed by the TMC prior to its implementation.
28.21 Toxicity Monitoring

Evaluation of non-lethal toxicity is based on CTC -scored reported toxicity of all patients. The evaluation includes myelosuppression, rate of infections, capillary leak syndrome, VOD of the liver, mucosal damage, cardiac, pulmonary, renal, hepatic, central and peripheral nervous toxicity as described in the toxicity report form enclosed in the appendix (chapter 26).
28.22 Treatment Stopping Rules for Individual Patients

If any (non-lethal) adverse event as outlined above occurs in a patient, his or her further treatment according to the study protocol must be discussed with the national study co-ordinator immediately. Consideration will be given to how dangerous continuation of protocol therapy might be, and if there is an alternative treatment option available in the particular situation, or if treatment should be discontinued. As the consequences of either decision may be deleterious to the patient, immediate discussion of the event is mandatory. All deaths on treatment and any unexpected life-threatening toxicity must be reported as SAE to the national study centre immediately (on the next working day)! (For details see chapter 25)
150
29 Appendix - ADDRESS LIST

29.1 SIOP EUROPE NEUROBLASTOMA BOARD
MICHON, JEAN, DR. PRESIDENT Dept. Haematology/ Oncology Institut Curie 26 Rue DUlm F - 75231 Paris Cedex 5 Tel: +33-1-44324558 Fax: +33-1-44324005 Email: jean.michon@curie.net Dept. Haematology/ Oncology Giannina Gaslini Childrens Hospital Largo Gerolamo Gaslini 5 I - 16148 Genova Tel: +39-335-8248264 or 0039-010-5636464 Fax:+39 010 3762322 E mail: brunodebernardi@ospedale-gaslini.ge.it Unidad De Oncologia Pediatrica Hospital Universitario Infantil La Fe Avenida Campanar 21 E - Valencia 46006 Phone: +34-96-3862789 or +34-96-398-7727 Fax: +34-96-3987727 or +34 96 3868 700 Email: castel_vic@gva.es FRANCE
DE BERNARDI, BRUNO, DR. VICEPRESIDENT
ITALY
CASTEL, VICTORIA, DR VICEPRESIDENT
SPAIN
LADENSTEIN, RUTH, ASS.PROF. St Annas Childrens Hospital Kinderspitalgasse 6 DR. A - 1090 Vienna SECRETARY Tel: +43 1 40 170- 475 Fax: +43 1 40170 759 Email:ladenstein@ccri.univie.ac.at PROF. DR ANDY PEARSON FORMER PRESIDENT, ADVISORY Sir James Spence Institute of Child Health Royal Victoria Infirmary Queen Victoria Road UK - Newcastle upon Tyne NE1 4LP Tel: +44 191 202 3024/36 Fax: +44 191 202 3060 E mail: a.d.j.Pearson@ncl.ac.uk
AUSTRIA
UK
151
29.2 DATA MONITORING COMMMITTEE

MATTHAY, KATE K., DR. Dept. of Paediatrics University of California 505 Parnassus Avenue, M 647 USA - San Francisco, CA 94143-0106 Tel: +1-415-476-0603 Fax: +1-415-5024372 Email: katekm@itsa.ucsf.edu Div. of Paediatirc Epidemiology& Clinical Research University of Minnesota PO Box 422 UMHC USA Minneapolis, MN 55455 Tel: +1 612-626-2902 Fax: +1 612-626-4842 Email: robison@epivax.epi.umn.edu Haematology Dept. Princess Margaret Hospital GPOOP Box D 184 AUS- Perth 6001 Tel: +61 8-93408234 Fax: +61 8-93408117 Email: david.baker@health.wa.gov.au AUS USA
USA ROBISON, LESLIE, PROF.
BAKER, DAVID L., DR.
29.3 NATIONAL CO-ORDINATORS

LADENSTEIN, RUTH, ASS.PROF. St Annas Childrens Hospital Kinderspitalgasse 6 DR. A - 1090 Vienna Tel: +43 1 40 170- 475 Fax: +43 1 40170 759 Email:ladenstein@ccri.univie.ac.at LAUREYS, GENEVIEVE, DR. Kliniek voor Kinderziekten (Paediatric Haematology and Oncology) Universitair Ziekenhuis (University Hospital Gent) De Pintelaan 185 B - 9000 Gent Phone: +32-9-2402111 Fax: +32 9 240 3875 Email: genevieve.laureys@rug.ac.be AUSTRIA
BELGIUM
152
SCHRDER, HENRIK, DR.
Department of Paediatrics University Hospital of Aarhus, Skejby Brendstrupgaardsvej 100 DK - 8200 Aarhus Phone: +45-8-9496700 Fax: +45-8-9496023 Email: hsa@sks.aaa.dk Dept Paediatric Oncology Institut Gustave Roussy 39, Rue Camille Desmoulins F - Villejuif Cedex 94805 Phone: +33-1-42114170 Fax: +33 1 4211 5275 Email: valteau@igr.fr Dept. Paediatric Haematology/Oncology Schneider Childrens Medical Center of Israel Kaplan Street 14 IL - Petah Tikva 49202 Phone: +972-3-9253669 Fax: +972-3-9253042 Email: iyaniv@clalit.org.il Istituto Nazionale Tumouri di Milano Unit Operativa Pediatria Via Venezian 1 I - 20133 Milano Phone:+39-02-266462; 0039-02-2390-588/ 592/or 694 Email: luksch@istitutotumouri.mi.it Department of Paediatrics Rikshospitalet Pilestredet 32 N - Oslo 0027 Phone:+47-23-07-46-61 Switchboard:+47-23-07-0000 Fax:+47-23-074557 Email: ingebjorg.storm-mathisen@rikshospitalet.no
DENMARK
VALTEAU-COUANET, DOMINIQUE, DR.
FRANCE
YANIV, ISAAC, DR.
ISRAEL
LUKSCH,ROBERTO, DR
ITALY
STORM-MATHISEN, INGEBJORG, DR.
NORWAY
153
FORJAZ DE LACERDA, ANA MARIA, DR.
Dept. of Paediatrics Instituto Portugues De Oncologia De Francisco Gentil (Portuguese Institute of Oncology) Rua Professor Lima Basto P - Lisboa Codex 1099-023 Phone: +351-21-722-9800 Fax: +351-21-720-0417 Email: hdiap@ipolisboa.min-saude.pt Unidad De Oncologia Pediatrica Hospital Universitario Infantil La Fe Avenida Campanar 21 E - Valencia 46006 Phone: +34-96-3862789 or +34-96-398-7727 Fax: +34-96-3987727 or +34 96 3868 700 Email: castel_vic@gva.es Childhood Cancer Research Unit, Barncancerforskningsenheten Astrid Lindgren Children's Hospital, Q6:05 Karolinska Hospital SE - Stockholm 17176 Phone: +46-8-51773534 (Pager:0740-113185) Fax: +46-8-5177 3475 Email: Per.Kogner@ks.se Dept. of Paediatrics University Hospital (CHUV) Rue du Bugnon CH-1011 Lausanne Phone: +41-21-3143567 Fax: +41-21-3143332 Email: Maja.Nenadov-Beck@chuv.hospvd.ch Dept. of Paediatric Oncology Great Ormond Street Hospital GB London WC1N 3JN Phone: +44 207 829 8832; Switchboard:+44-207 405 9200 Fax: +44-207-813-8588 Email: brockp@gosh.nhs.uk
PORTUGAL
CASTEL, VICTORIA, DR
SPAIN
KOGNER, PER, DR.
SWEDEN
NENADOV-BECK, MAYA, DR.
SWITZERLAND
BROCK, PENELOPE, DR.
UK
154
SUBCOMMITTEES 29.4 BIOLOGY SC

AMBROS, INGE, DR. CCRI Childrens Cancer Research Institute St Anna Childrens' Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43 1 40170-421 Fax: +43-1 4087230 Email: ambros@ccri.univie.ac.at CCRI Childrens Cancer Research Institute St Anna Childrens' Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43 1 40170-411 Fax: +43-1 4087230 Email: ambros@ccri.univie.ac.at Labor de Gntique Hpital Erasme Route deLennik, 808 B 1070 Bruxelles Phone: +32-2-555 41 66 Fax: +32-2-555 42 12 Email: pheimann@ulb.ac.be Centre for Medical Genetics-OK5 University of Ghent De Pintelaan 185 B - 9000 Ghent Phone: +32-9-240 2451 Fax: +32-9-240 49 70 Email: franki.speleman@rug.ac.be Centre for Medical Genetics-OK5 University of Ghent De Pintelaan 185 B - 9000 Ghent Phone: +32-9-240 2451 Fax: +32-9-240 49 70 Email: nadine.vanroy@rug.ac.be AUSTRIA
AMBROS, PETER, DR.
AUSTRIA
HEIMANN, PIERRE, DR.
BELGIUM
SPELEMAN, FRANK, DR.
BELGIUM
VAN ROY, NADINE, DR.
BELGIUM
155
BENARD, JEAN, DR.
Laboratoire de Pharmacologie Clinique et Moleculaire Institut Gustave Roussy 39, rue Camille Desmoulins F - Villejuif-Cedex Phone: +33-142114818 Fax: +33-1 42115280 Email: benard@igr.fr Immunologie-Centre Leon Berard 28 rue Laennec F - 69373 Lyon Cedex 08 Phone: +33-4-78782828 Fax: +33-4-78782717 Email: combaret@lyon.fnclcc.fr Laboratoire de Genetique des Tumeurs Pavillon Trouilhet Rossignol, Institut Curie 26 rue dUlm F - 75231 Paris Cedex 05 Phone: +33-1-4432-4216 Email: jerome.couturier@curie.net Laboratoire de Genetique des Tumeurs Pavillon Trouillet Rossignol, Institut Curie 26 rue dUlm F - 75231 Paris Cedex 05 Phone: +33-1-4234-6678 Fax: +33-1-4234-6630 Email: olivier.delattre@curie.fr Laboratory of Population Genetics National Institute for Cancer Research/CBA (IST) Advance Biotechnology Center I - Largo Benzi, 10 16132 Genova Phone: +39-010-5737-463/ or-490/ or-430 Fax: +39-010-5737-463 Email: tonini@cba.unige.it Institute of Human Genetics University of Amsterdam, Academic Medical Centre Meibergdreef 15 NL-1105 Amsterdam Phone: +31-20-5665111 Fax: +31-20-6918626 Email: h.n.caron@amc.uva.nl
FRANCE
COMBARET, VALERIE, DR.
FRANCE
COUTURIER, JEROME, DR
FRANCE
DELATTRE, OLIVIER, DR
FRANCE
TONINI, GIAN PAOLO, DR
ITALY
CARON, HUIB, DR.
THE NETHERLANDS
156
BOAVIDA, MARIA GUIDA, DR
Instituto Ricardo Jorge/National Institute of Health Dept. of Human Genetics Avenida Padre Cruz P - 1699 Lisboa-Codex Phone: +351-1-7519324 Fax: +351-1-7590441 Email: m.guida.boavida@insa.min-saude.pt Departamento de patologa Facultat de medicina Avda Blasco Ibaez 17 E - 46010 Valencia Phone: +34-96-386 41 46 Fax: +34-96-386 41 73 Email: rnoguera@uv.es Woman and Child Health, Childhood Cancer Research Unit Dept. of Pediatrics Karolinska Hospital S-10401 Stockholm Phone: +46-8-517-73-534 Fax: +46-8-517-73-475 Email: Per.Kogner@lab.ks.se Department of Clinical Genetics Gahlgrenska University Hospital/East Dept. of Clinical Genetics S-41685 Gothenburg Phone: +46-31-434-803 Fax: +46-31-842 160 Email: Tommy.Martinsson@clingen.gu.se Onco-Hematology Laboratory Pediatrics Department University Hospital CHUV CH - 1011 Lausanne Phone: +41-21-314-3622 Fax: +41-21-314-3558 Email: nicole.gross@chuv.hospvd.ch Division of Human Genetics University of Newcastle upon Tyne 19/20 Claremont Place GB - Newcastle upon Tyne NE2 4AA Phone: +44-191 222 7143 Fax: +44 191 222 6662 Email: Nicholas.Bown@newcastle.ac.uk
PORTUGAL
NOGUERA, ROSA, DR
SPAIN
KOGNER, PER, DR
SWEDEN
MARTINSSON,TOMMY, DR.
SWEDEN
GROSS, NICOLE, DR
SWITZERLAND
BOWN, NICHOLAS, MR.
UK
157
LUNEC, JOHN, DR
Cancer Research Unit University School University of Newcastle upon Tyne UK - Newcastle upon Tyne NE2 4HH Phone: +44-191-222-8057 Fax: +44-191-222-7556 Email: john.lunec@newcastle.ac.at
UK
29.5 BONE MARROW STUDIES SC

AMBROS, PETER, DR. CCRI Childrens Cancer Research Institute St Anna Childrens' Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43 1 40170-411 Fax: +43-1 4087230 Email: ambros@ccri.univie.ac.at Department of Pediatrics and Genetics Laboratorium of Haematology, 1P8, West Ghent University Hospital De Pintelaan 185 B - 9000-Ghent Phone:+32-9-240-31-67 Fax:+32-9-240-49-85 Email: jan.philippe@rug.ac.be Department of Pediatrics and Genetics Laboratorium of Haematology, 1P8, West Ghent University Hospital De Pintelaan 185 B - 9000-Ghent Phone:+32-9-240-34-31 Fax:+32-9-240-49-85 Email: katrien.swerts@rug.ac.be Service dhmatologie biologique Dpartement de biologie clinique Institut Gustave Roussy, (12me etage) 39 rue Camille Desmoulins F - 94805 Villejuif Cedex Phone: +33 1 4211-4233 Fax: +33 1 4211-5268 Email: abenna@igr.fr AUSTRIA
PHILIPP, JAN
BELGIUM
SWERTS, KATRIEN
BELGIUM
BENNACEUR, ANNE-LISE, DR.
FRANCE
158
SCHUMACHER, ROSWITHA, DR. University of Kln, Dept. of Paediatrics Klinik und Poliklinik fr Kinderheilkunde der Universitt zu Kln Joseph-Stelzmann-Strae 9 D - 50924 Kln Phone: +49 221 4784390 Fax:+49 221 4784689 Email:roswitha.schumacherkuckelkorn@medizin.uni-koeln.de CORRIAS, MARIA VALERIA, DR. Laboratorio di Oncologia Instituto G. Gaslini largo Gaslini 5 I - 16147 Genova Fax: +39-010-3779820 Email:mariavaleriacorrias@ospedalegaslini.ge.it FAULKNER, LAWRENCE, DR. Servizio Emotrasfusionale e di Terapie Cellulari Azienda Ospedale Meyer Via Luca Giordano 13 I - 50132 Firenze Phone: +39-0555662437 or +39-055-5662448 Fax: +39-335-5621861 Email: lfaulkn@tin.it Dept. of Pathology Rikshospitalet Pilestredet 32 N - 0027 Oslo Phone:+47 23 07 14 00, +47 23 07 40 76 (office) Fax: +47 228 68 596 Email: klaus.beiske@labmed.uio.no Unidad de Oncologia Pediatrica Hospital Infantil La Fe Avda. de Campanar 21 E - 46009 Valencia Phone: +34 963 987727, +34 963 862789 Fax: +34 963 987727, +34 963 868700 Email: fernandez_jma@gva.es
GERMANY
ITALY
ITALY
BEISKE, KLAUS, DR.
NORWAY
NAVARRO, JOSE M FERNNDEZ, DR.
SPAIN
159
GROSS, NICOLE, DR.
Onco-Haematology Unit, Paediatric Department University Hospital (CHUV) rue du Bugnon CH 1011 Lausanne Email: Nicole.gross@chuv.hospvd.ch ICRF Cancer Medicine Research Unit St. James University Hospital GB - LS9 7TF Leeds Phone:+44-113-206 5873 Fax:+44-113-242 9886 Email:S.A.Burchill@cancermed.leeds.ac.uk
SWITZERLAND
BURCHILL, SUE, DR.
UK
29.6 IMMUNOTHERAPY SC
FELZMANN, THOMAS, DR. CCRI Childrens Cancer Research Institute St Anna Childrens' Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43 1 40170-406 Fax: +43-1 4087230 Email: felzmann@ccri.univie.ac.at CCRI Childrens Cancer Research Institute St. Anna Childrens Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43-1-40170-475 Fax: +43-1-40170-430 Email: ladenstein@ccri.univie.ac.at Department of Paediatrics Institut Gustave Roussy 39, rue Camille Desmoulins F - 94805 Villejuif Phone: +33-1-42114170 Fax: +33 1 4211 5275 Email: valteau@igr.fr Dept. of General Paediatrics Charite Children's Hospital Augustenburgerplatz 1 D - 13353 Berlin Phone: +49-30-45066422 Fax: +49-30-450-66916 Email: holger.lode@charite.de AUSTRIA
LADENSTEIN, RUTH, ASS.PROF., DR.
AUSTRIA
VALTEAU-COUANET, DOMINIQUE, DR.
FRANCE
LODE, HOLGER, DR.
GERMANY
160
PISTOIA, VITO, DR.
Giannina Gaslini Children's Hospital Laboratory of Oncology Largo Gaslini 5 I - 16145 Genova Phone: +39 010 56 36 342 Email: vitopistoia@ospedale-gaslini.ge.it Unidad de Oncologia Pediatrica Hospital Infantil La Fe Avda. De Campanar 21 E - 46009 Valencia Phone: +34 963 987727, +34 963 862 789 Fax: +34 963 987 727, +34 963 868 700 Email: canyete_ade@gva.es Queen Silvia's Children's Hospital Sv/ostra SE 41685 Goteborg Phone: +31 3434100 Fax: +31 215486 Email: jonas.abrahamsson@pediat.gu.se
ITALY
CAETE, ADELA, DR.
SPAIN
ABRAHAMSSON, JONAS, DR.
SWEDEN
29.7 NUCLEAR MEDICINE AND PHYSICS

BECHERER, ALEXANDER, DR. University Clinic for Nuclear Medicine Whringer Grtel 18-20 A - 1090 Vienna Phone: +43-1-40400-5550 Fax: +43-1- 40400-5552 Email: alexander.becherer@univie.ac.at Department of Nuclear Medicine University Hospital Ghent De Pintelaan 185 B - 9000 Ghent Phone: +32-9-2403028 Fax: +32-9-240-3807 Email: Boudewijn.Brans@rug.ac.be Institut Claudius Regaud 20-24 rue du Pont Saint Pierre F - 31052 Toulouse Phone: +33 5 61 42 42 11 Fax: +33 5 61 42 46 35 Email: Boneu@icr.fnclcc.fr AUSTRIA
BOUDEWIJN, BRANS, DR.
BELGIUM
BONEU, ANDRE, DR.
FRANCE
161
GIAMARILLE, FRANCESCO, DR. Sevice de Mdecine Nuclaire Centre Lon Brard 28 rue Laennec F - 69373 Lyon cedex 08 Phone: +33 4 78 78 26 82 Fax: +33 4 78 78 29 06 Email: giammari@lyon.fnclcc.fr HAHN, KLAUS, PROF.DR.MED. Dept. of Nuclear Medicine Univ. Clinic Munich Ziemssenstr.1 D - 80336 Munich Phone: +49 89 5160-2423 Fax: +49 89 5160-4555 Email: hahn@nuk.med.uni-muenchen.de Dept. of Nuclear Medicine Univ. Clinic Munich Ziemssenstr.1 D - 80336 Munich Email: Ute.Porn@nuk.med.uni-muenchen.de Dip. Medicina Nucleare Istituto Nazionale Tumouri di Milano Nuclear Medicine Division Via Venezian 1 I - 20133 Milano Phone: +39-02-23902512 Fax: +39 023 367874 Email: castellani@istitutotumouri.mi.it Istituto di Medicina Nucleare Ospedale Policlinico Gemelli Univ. Catt. del Sacro Cuore Largo Gemelli 8 I - 00168 Roma Phone: +39-06-30154978 Fax: +39-06-3058185 Email: v.rufini@tiscalinet.it Service of Nuclear Medicine Galliera Hospital Mura delle Capuccine 14 I - 16128 Genoa Phone: +39-010-5634538 Fax: +39-010-5632356 Email: g.villavecchia@galliera.it
FRANCE
GERMANY
PORN, UTE, DR.
GERMANY
CASTELLANI, MARIA RITA, DR.
ITALY
RUFINI, VITTORIA, DR.
ITALY
VILLAVECCHIA, GIAN PIERO, DR.
ITALY
162
COLARINHA, PAULA, DR.
Instituto Portugues De Oncologia De Francisco Gentil (Portuguese Institute of Oncology) Rua Professor Lima Basto P - 1099-023 Lisboa Codex Email: paula@esoterica.pt Medicina Nuclear Hospital Universitario de Getafe Crta.Toledo Km.12,500 E - 28905 Madrid Phone: 34 916833661 Fax: 34-916839748 Email: mmitjavila@mundivia.es Department of Radiology Karolinska Hospital SE - 17176 Stockholm Phone: +46 8 517 73581 Fax: +46-8517-74939 Email: hans.jacobsson@ks.se Chef du Service de Mdecine Nuclaire Centre Hospitalier Universitaire Vaudois rue du Bougnon CH - 1011 Lausanne Phone: +41 21 314 43 46/47 Fax: +41 21 314 43 49 Email: Angelika.BischofDelaloye@chuv.hospvd.ch Sevice de Mdecine Nuclaire BH-07 Centre Hospitalier Universitaire Vaudois rue du Bougnon CH - 1011 Lausanne Phone: +41 21 131 44353 Fax: +41 21 314 43 43 Email: Ariane.Boubaker@chuv.hospvd.ch Institute of Human Genetics University of Amsterdam Academic Medical Centre Meibergdreef 15 NL-1105 Amsterdam Phone: +31-20-5665111 Fax: +31-20-6918626 Email: h.n.caron@amc.uva.nl
PORTUGAL
MITJAVILA, MERCEDES, DRA.
SPAIN
JACOBSSON, HANS, PROF. DR.
SWEDEN
BISCHOF-DELALOYE, ANGELIKA, DR.
SWITZERLAND
BOUBAKER, ARIANNE, DR.
SWITZERLAND
CARON, HUIB, DR.
THE NETHERLANDS
163
HOEFNAGEL, CORNELIS A., DR
Dept. Of Nuclear Medicine The Netherlands Cancer Institute Plesmanlaan 121 NL - 1066 CX Amsterdam Phone: +31 20 512 2286 Fax: +31 20 512 2290 Email: hoef@nki.nl Joint Department of Physics Royal Marsden NHS Trust & Institute of Cancer Research Downs Road GB SM2 5PT Sutton, Surrey Phone: +44 (0)20 8661 3485 Fax: +44 (0)20 8643 3812 Email: glenn@icr.ac.uk Dept. of Paediatric Oncology Royal Marsden Hospital Downs Road GB - SM2 5PT Sutton, Surrey Phone: +44-20-86613329 Fax: +44-20-86613617 Email: stmell@aol.com
THE NETHERLANDS
FLUX, GLENN, PHD MIPEM SRCS
UK
MELLER, SIMON, DR.
UK
29.8 PATHOLOGY SC
AMANN, GABRIELE, DR. Univ. Clinic of Pathology Whringer Grtel 18-20 A - 1090 Vienna Phone: +43-1-40400-3654 Fax: +43-1-4053402 Email: gabriele.amann@akh-wien.ac.at CCRI Childrens Cancer Research Institute St. Anna Children's Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43-1-40170-421 Fax: +43-1 4087230 Email: ambros@ccri.univie.ac.at AUSTRIA
AMBROS, INGE, DR.
AUSTRIA
164
HEIMANN, PIERRE, DR.
Labor de Gntique Hpital Erasme Route deLennik, 808 B 1070 Bruxelles Phone: +32-2-555 41 66 Fax: +32-2-555 42 12 Email: pheimann@ulb.ac.be Dept. of Pathology University Hospital Leuven Minderbroedersstraat 12 B 3000 Leuven Phone: +32-16-336647 Fax: +32-16-336548 Email: raf.sciot@uz.kuleuven.ac.be The Institute of Pathology Odense University Hospital DK - 5230 Odense C Phone: +45-65414-824 Fax: +45-65912-943 Email: gitte.kerndrup@ouh.fins_amt.dk Service d'Anatomie et de Cytologie Pathologiques, hpital Robert Debr EA3102 Universit Paris 7 48, Boulevard Srurier F - 75019 Paris Phone: +33-1-40 03 23 70 Fax: +33-1-40 03 47 13 Email: michel.peuchmaur@rdb.ap-hop-paris.fr Institute of Pathology Via Gabelli 61 I 35100 Padova Phone: +39-424-888778 Fax: +39 049 827 2263 Email: eda@uxl.unipd.it Giannina Gaslini Children's Hospital Largo Gaslini 5 I - 16148 Genova Phone: +39-010-5636210 Fax: +39-010-3776590 Email: claudio.gambini@ospedale-gaslini.ge.it
BELGIUM
SCIOT, RAF, MD
BELGIUM
KERNDRUP, GITTE, DR.
DENMARK
PEUCHMAUR, MICHEL, PROF.
FRANCE
D'AMORE, EMANUELE S.G., DR.
ITALY
GAMBINI, CLAUDIO, DR.
ITALY
165
BEISKE, KLAUS, DR.
Dept. of Pathology Rikshospitalet Sognsvannsveien 20 N - 0027 Oslo Phone: +47-23-071400 or office: +47-23074076 Fax: +47-23-071410 Email: klaus.beiske@labmed.uio.no Instituto Portugues De Oncologia De Francisco Gentil (Portuguese Institute of Oncology) Rua Professor Lima Basto P - 1099-023 Lisboa Codex Email: emenndonca@netcabo.pt Instituto Portugues De Oncologia De Francisco Gentil (Portuguese Institute of Oncology) Rua Professor Lima Basto P - 1099-023 Lisboa Codex Email: psoliveira@mail.telepac.pt Departamento de patologa Facultad de medicina Avda. Blasco Ibaez 17 E - 46010 Valencia Phone: +34 96 3864 146 Fax: +34 96 3864 173 Email: Samuel.navarro@uv.es Dept. of Paediatric Pathology Karolinska Hospital PO Box 100 SE - 17176 Stockholm Phone: +46-87296162 Fax: + 46 8 729 6165 Email: Bengt.Sandstedt@pat.ds.sll.se University Hospital (CHUV) rue du Bugnon CH - 1011 Lausanne Phone: +41 21 314 7162 Fax: +41 21 314 7115 Email: Kathleen.MeagherVillemure@chuv.hospvd.ch
NORWAY
MENDONA, EVELINE, DR.
PORTUGAL
OLIVEIRA, PEDRO, DR.
PORTUGAL
NAVARRO, SAMUEL, DR.
SPAIN
SANDSTEDT, BENGT, DR.
SWEDEN
MEAGHER-VILLEMURE, KATHLEEN, DR.
SWITZERLAND
166
CULLINANE, CATHERINE, DR, FCRPATH
Pathology Dpt. St. James's University Hospital Beckett Street UK - Leeds LS9 7TF Phone: +44 113 2065163 Email: ccullinane@doctors.org.uk
UK
29.9 RADIOLOGY SC
HRMANN, MARCUS, DR. Dept. of Paediatrics University Clinic Whringer Grtel 18-20 A 1090 Vienna Phone: +43 1 40400-7620/ or-4891 Email: marcus.hoermann@akh-wien.ac.at Imagerie mdicale Cliniques universitaires St. Luc Avenue Hippocrate 10/2972 B 1200 Bruxelles Phone: +32-2-7642972 Fax: +32-2-7705574 Email: philippe.clapuyt@rdgn.ucl.ac.be Dept. of Radiology Institut Gustave Roussy 39 rue Camille Desmoulins F - 94805 Villejuif Phone: +33-1-42114872 Fax: +33-1-42115279 Email: valteau@igr.fr Institut fr Radiologie; Abteilung Kinderradiologie (Paediatric Radiology) Universitt Kln Joseph-Stelzmann.Str. 9 D - 50933 Cologne Phone: +49 221 478 4228 Fax: +49 221 478 3347 AUSTRIA
CLAPUYT, PHILIPPE, DR.
BELGIUM
COUANET, DOMINIQUE, DR.
FRANCE
BENZ-BOHM, GABRIELE, PROF.DR.
GERMANY
GIAN MICHELE MAGNANO, DR. Department of Radiology Giannina Gaslini Childrens Hospital Largo Gaslini 5 I - 16148 Genova - Quarto Phone: +39-010-5636 531 Fax: +39-010-385599 Email: gianmichelemagnano@ospedale-gaslini.ge.it
ITALY
167
SILVA, JOO PAULO CONCEIAO E, DR.
Instituto Portugues De Oncologia De Francisco Gentil (Portuguese Institute of Oncology) Rua Professor Lima Basto P - 1099-023 Lisboa Codex Email: conceicaosilvaj@netc.pt Servicio de Radiologia Infantil Hospital Infantil La Fe Avda de Campanar 21 E - 46009 Valencia Phone: +34 963868772 Fax: +34 963868773 Email: mpal@servitel.es Department of Radiology Karolinska Hospital SE - 17176 Stockholm Phone: +46 8 517 73581 Fax: +46-8517-74939 Email: hans.jacobsson@ks.se Department of Radiology Royal Marsden Hospital Downs Road GB - SM2 5PT Sutton, Surrey Phone: +44-208-661-3222 Email: david.macvicar@rmh.nthames.nhs.uk
PORTUGAL
MURO, M. DOLORES, DR.
SPAIN
JACOBSSON, HANS, PROF. DR.
SWEDEN
MACVICAR, DAVID, DR
UK
29.10 RADIOTHERAPY
PTTER, RICHARD, PROF. DR. Univ.Klinik f. Strahlentherapie und Strahlenbiologie Whringer Grtel 18-20 A - 1180 Vienna Phone: +43-1-40400 2694 Fax: +43-1-40400 2693 Email: Richard.Poetter@univie.ac.at Univ.Klinik f. Strahlentherapie und Strahlenbiologie Whringer Grtel 18-20 A - 1180 Vienna Phone: +43-1-40400 2665 Fax: +43-1-40400 2693 Email: karin.dieckmann@str.akh.magwien.gv.at AUSTRIA
DIECKMANN, KARIN, DR.
AUSTRIA
168
RUTTEN, ISABELLE, DR
Service de Radiothrapie Domaine Universitaire du Sart Tilman, Bat. B35

B 4000 LIEGE
BELGIUM
Email: isabelle.rutten@chrcitadelle.be HABRAND, JEAN LOUIS, PROF. Service de Radiothrapie Institut Gustave Roussy 39, rue C. Desmoulins F - 94800 Villejuif Phone : +33 1 42 11 49 95 Email : habrand@igr.fr Service de Radiothrapie B Institut Curie 26 rue dUlm F 7548 Paris Phone: +33-1 44324636 Fax: +33-144-324616 Email: sylvie.helfre@curie.net FRANCE
HELFRE, SYLVIE, DR
FRANCE
BENZ-BOHM, GABRIELE, PROF. Institut fr Radiologie Abteilung Kinderradiologie DR. (Paediatric Radiology) Universitt Kln Joseph-Stelzmann.Str. 9 D - 50933 Kln Phone: +49 221 478 4228 Fax: +49 221 478 3347 SALVINA, BARRA, DR. Service of Radiotherapy National Cancer Research Institute Viale Benedetto XV I - 16132 Genova Phone: +39 010 5600014 Fax: +39 010 5600014 Email: Barra@hp380.ist.unige.it Istituto Nazionale Tumouri Dip.di Radioterapia Via Venezian 1 I - 20133 Milano Tel: +39-02-23902477 Fax: +39-02-2665605, +39-010-3367874 Email: gandola@istitutotumouri.mi.it
GERMANY
ITALY
LORENZA GANDOLA, DR.SSA
ITALY
169
OLDENBURGER, FOPPE, DR.
Academic Medical Center Dept. of Radiotherapy Meibergdreef 9 NL - 1105 AZ Amsterdam Phone : +31 20 5664231 Fax : +31 20 6091278 Email : F.Oldenburger@amc.uva.nl Servicio de Radioterapia Hospital Universitario La Fe Avda Campanar 21 E - 46009 Valencia
Phone:+34 963862700 Ext.:50445 Fax: +34-963-868-789
THE NETHERLANDS
BADAL, DRA. DOLORES
SPAIN
Email: Ibadal@ctv.es GAZE, MARK, DR. The Meyerstein Institute of Oncology The Middlesex Hospital Mortimer Street UK - London W1N 8AA Phone: +44-207-380-9301 Fax: +44-207-436-0160 Email: mark@gaze.demon.co.uk UK
29.11 STATISTICAL SC
PTSCHGER, ULRIKE, M.SC. CCRI Childrens Cancer Research Institute St. Anna Childrens Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43-1-40170-477 Fax: +43-1-40170-430 Email: poetschger@ccri.univie.ac.at Institut Curie 26 rue DUlm F - 75231 PARIS CEDEX 5 Phone: +33 1 4432-4665 Fax: +33-1-44-32-40-78 Email: veronique.mosseri@curie.net Unit of Clinical Epidemiology and Trials Advanced Biotechnology Center National Institute for Cancer Research I - 16136 GENOVA Phone: +39-010-5737477 Fax: +39-010-354103 Email: Bruzzi@ermes.cba.unige.it AUSTRIA
MOSSERI, VERONIQUE, DR.
FRANCE
BRUZZI, PAOLO, DR.
ITALY
170
IMESON, JOHN, MR.
Department of Epidemiology and Public Health UKCCSG Data Centre University of Leicester, 3rd Floor Hearts of Oak House 9 Princess Road West GB - Leicester LE1 6TH Phone: +44-116-249-4460 Email: ime@leicester.ac.uk Visiting Professor of Clinical Trial Methodology UKCCSG Data Centre University of Leicester, 3rd Floor Hearts of Oak House 9 Princess Road West GB - Leicester LE1 6TH Phone: +44-116-249-4460 Email: David@machin-home.freeserve.co.uk
UK
MACHIN, DAVID, PROF.
UK
29.12 STEM CELL SC

WITT, VOLKER, DR. St. Anna Childrens Hospital Kinderspitalgasse 6 A - 1090 Vienna Phone: +43 1 40170-752 Fax: +43-1-40170-752 Email: witt@stanna.at BTC Oost-Vlaanderen Ottergemsesteenweg 413 B-9000 Gent Phone: +32-9-244 56 56 Fax: +32-9-244 56 64 Email: bart.vandekerckhove@bl.rodekruis.be Cellular Therapy Unit Institut Gustave Roussy 11 rue Camille Desmoulins F - 94805 Villejuif Cedex Phone: 33142115148 Fax: 33142115303 Email: boccacio@igr.fr AUSTRIA
VANDEKERCKHOVE, BART DR.
BELGIUM
BOCCACCIO, CATHERINE, DR.
FRANCE
171
DALLORSO, SANDRO, DR.
Unita' Trapianto di Midollo Osseo Institute Gaslini largo Gaslini 5 I - 16147 Genova Phone: +39 010 5636 507 Fax: +39 010 3777 133 Email: s.dallorso@libero.it BMT Unit Instituto Portugues de Oncologia Centro de Lisboa Rua Prof. Lima Basto 1099 P 23 Lisboa Fax: +351 21 722985 Email: flcosta@ipolisboa.min-saude.pt Banco de Sangre Hospital Universitario La Fe Avda. Campanar No. 21 E - 46009 VALENCIA Phone: +34-963862745 Fax: +34 963868789 Email: delarubia_jav@gva.es Queen Silvia's Children's Hospital Sv/ostra SE 41685 Goteborg Phone: +31 3434100 Fax: +31 215486 Email: jonas.abrahamsson@pediat.gu.se Consultant Paediatric Oncologist Regional Paediatric Oncology Unit St James's University Hospital GB - Leeds LS9 7TF Phone: +44 - 113 206 4984 Fax: +44- 113 2470248 Email: susan.picton@gw.sjsuh.northy.nhs.uk
ITALY
LEAL DA COSTA, FERNANDO, DR.
PORTUGAL
DE LA RUBIA, JAVIER, DR.
SPAIN
ABRAHAMSSON, JONAS, DR.
SWEDEN
PICTON, SUE, DR.
UK
29.13 SURGERY
HORCHER, ERNST, PROF. Dept. For Paediatric Surgery Univ. Clinic for Surgery Whringer Grtel 18-20 A - 1090 Vienna Phone: +43 1-40400 6838 Fax:+43 1-40400 6898 Email: Ernst.Horcher@akh-wien.ac.at AUSTRIA
172
DE WEVER, IVO, PROF. DR.
Head of Department Surgical Oncology University Hospital Gasthuisberg Herestraat 49 B - 3000 Leuven Phone: +32 16 34 6831 Fax: +32 16 34 68 34 Email: Ivo.Dewever@uz.kuleuven.ac.be CHR Citadelle Boulevard du XIIme de Ligne, 1 B - 4000 Liege Phone: +32 4 225 6283 Fax: +32 42238712 Email: martine.demarche@chrcitadelle.be Department of Surgical Gastroenterology A Odense University Hospital DK- 5000 Odense C Phone: +45 6541 2121 or 0045 6541 2239 Fax:+45 6591 9872 Email: lars.rasmussen@ouh.fyns-amt.dk Hpital d'enfants de la Timone F - 13385 Marseille Cedex 5 Phone: +33 4 91 386 682 Fax: +33 491 384714
BELGIUM
DEMARCHE, MARTINE, DR.
BELGIUM
RASMUSSEN, LARS, DR
DENMARK
GUYS, JEAN MICHEL, PROF
FRANCE
CECCHETTO, GIOVANNI, PROF. Paediatric Surgery Divisione Chirurgia Pediatrica Istituto di Chirurgia Pediatrica dell'universit di Padova Via Giustiniani 3 I - 35128 Padova Phone: +39 049 8213 681 Fax: +39 049 8213689 Email: cecchett@child.pedi.unipd.it GRANATA, CLAUDIO, DR. Dept. of Paediatric Surgery Giannina Gaslini Institute Largo Gaslini 5 I 16145 Genoa Phone: +39 010 5636 217 / 392 Fax: +39 010 377 6590 Email: cgranata@panet.it
ITALY
ITALY
173
MONCLAIR, TOM, DR.
Department of Surgery Rikshospitalet Pilestredet 32 N - 0027 Oslo Phone: +47 23 07 09 59 Fax: +47 23 07 09 61 Instituto Portugues De Oncologia De Francisco Gentil (Portuguese Institute of Oncology) Rua Professor Lima Basto P - 1099-023 Lisboa Codex Instituto Portugues De Oncologia De Francisco Gentil (Portuguese Institute of Oncology) Rua Professor Lima Basto P - 1099-023 Lisboa Codex Email: agentilmartins@netc.pt Dept. of Paediatric Surgery St. Georges Hospital Blackshaw Road GB - London SW17 O2T Phone: +44 20 87252926 Fax: +44-20-8725711 Email: kholmes@doctors.org.uk
NORWAY
SOUSINHA, MRIO, DR.
PORTUGAL
MARTINS, ANTNIO GENTIL, DR.
PORTUGAL
HOLMES, KEITH, DR.
UK
174
30 APPENDIX: DECLARATION OF HELSINKI
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI

Ethical Principles for Medical Research Involving Human Subjects
Adopted by the 18th WMA General Assembly Helsinki, Finland, June 1964 and amended by the 29th WMA General Assembly, Tokyo, Japan, October 1975 35th WMA General Assembly, Venice, Italy, October 1983 41st WMA General Assembly, Hong Kong, September 1989 48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 52nd WMA General Assembly, Edinburgh, Scotland, October 2000
30.1 Introduction
The World Medical Association has developed the Declaration of Helsinki as a statement of ethical principles to provide guidance to physicians and other participants in medical research involving human subjects. Medical research involving human subjects includes research on identifiable human material or identifiable data. It is the duty of the physician to promote and safeguard the health of the people. The physician's knowledge and conscience are dedicated to the fulfillment of this duty. The Declaration of Geneva of the World Medical Association binds the physician with the words, "The health of my patient will be my first consideration," and the International Code of Medical Ethics declares that, "A physician shall act only in the patient's interest when providing medical care which might have the effect of weakening the physical and mental condition of the patient." Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects. In medical research on human subjects, considerations related to the well-being of the human subject should take precedence over the interests of science and society. The primary purpose of medical research involving human subjects is to improve prophylactic, diagnostic and therapeutic procedures and the understanding of the aetiology and pathogenesis of disease. Even the best proven prophylactic, diagnostic, and therapeutic methods must continuously be challenged through research for their effectiveness, efficiency, accessibility and quality. In current medical practice and in medical research, most prophylactic, diagnostic and therapeutic procedures involve risks and burdens. Medical research is subject to ethical standards that promote respect for all human beings and protect their health and rights. Some research populations are vulnerable and need special protection. The particular needs of the economically and medically disadvantaged must be recognized. Special attention is also required for those who cannot give or refuse consent for themselves, for those who may be subject to giving consent under duress, for those who will not benefit personally from the research and for those for whom the research is combined with care.
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Research Investigators should be aware of the ethical, legal and regulatory requirements for research on human subjects in their own countries as well as applicable international requirements. No national ethical, legal or regulatory requirement should be allowed to reduce or eliminate any of the protections for human subjects set forth in this Declaration.
30.2 Basic Principles for All Medical Research

It is the duty of the physician in medical research to protect the life, health, privacy, and dignity of the human subject. Medical research involving human subjects must conform to generally accepted scientific principles, be based on a thorough knowledge of the scientific literature, other relevant sources of information, and on adequate laboratory and, where appropriate, animal experimentation. Appropriate caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected. The design and performance of each experimental procedure involving human subjects should be clearly formulated in an experimental protocol. This protocol should be submitted for consideration, comment, guidance, and where appropriate, approval to a specially appointed ethical review committee, which must be independent of the investigator, the sponsor or any other kind of undue influence. This independent committee should be in conformity with the laws and regulations of the country in which the research experiment is performed. The committee has the right to monitor ongoing trials. The researcher has the obligation to provide monitoring information to the committee, especially any serious adverse events. The researcher should also submit to the committee, for review, information regarding funding, sponsors, institutional affiliations, other potential conflicts of interest and incentives for subjects. The research protocol should always contain a statement of the ethical considerations involved and should indicate that there is compliance with the principles enunciated in this Declaration. Medical research involving human subjects should be conducted only by scientifically qualified persons and under the supervision of a clinically competent medical person. The responsibility for the human subject must always rest with a medically qualified person and never rest on the subject of the research, even though the subject has given consent. Every medical research project involving human subjects should be preceded by careful assessment of predictable risks and burdens in comparison with foreseeable benefits to the subject or to others. This does not preclude the participation of healthy volunteers in medical research. The design of all studies should be publicly available. Physicians should abstain from engaging in research projects involving human subjects unless they are confident that the risks involved have been adequately assessed and can be satisfactorily managed. Physicians should cease any investigation if the risks are found to outweigh the potential benefits or if there is conclusive proof of positive and beneficial results. Medical research involving human subjects should only be conducted if the importance of the objective outweighs the inherent risks and burdens to the subject. This is especially important when the human subjects are healthy volunteers.
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Medical research is only justified if there is a reasonable likelihood that the populations in which the research is carried out stand to benefit from the results of the research. The subjects must be volunteers and informed participants in the research project. The right of research subjects to safeguard their integrity must always be respected. Every precaution should be taken to respect the privacy of the subject, the confidentiality of the patient's information and to minimize the impact of the study on the subject's physical and mental integrity and on the personality of the subject. In any research on human beings, each potential subject must be adequately informed of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliations of the researcher, the anticipated benefits and potential risks of the study and the discomfort it may entail. The subject should be informed of the right to abstain from participation in the study or to withdraw consent to participate at any time without reprisal. After ensuring that the subject has understood the information, the physician should then obtain the subject's freely-given informed consent, preferably in writing. If the consent cannot be obtained in writing, the non-written consent must be formally documented and witnessed. When obtaining informed consent for the research project the physician should be particularly cautious if the subject is in a dependent relationship with the physician or may consent under duress. In that case the informed consent should be obtained by a well-informed physician who is not engaged in the investigation and who is completely independent of this relationship. For a research subject who is legally incompetent, physically or mentally incapable of giving consent or is a legally incompetent minor, the investigator must obtain informed consent from the legally authorized representative in accordance with applicable law. These groups should not be included in research unless the research is necessary to promote the health of the population represented and this research cannot instead be performed on legally competent persons. When a subject deemed legally incompetent, such as a minor child, is able to give assent to decisions about participation in research, the investigator must obtain that assent in addition to the consent of the legally authorized representative. Research on individuals from whom it is not possible to obtain consent, including proxy or advance consent, should be done only if the physical/mental condition that prevents obtaining informed consent is a necessary characteristic of the research population. The specific reasons for involving research subjects with a condition that renders them unable to give informed consent should be stated in the experimental protocol for consideration and approval of the review committee. The protocol should state that consent to remain in the research should be obtained as soon as possible from the individual or a legally authorized surrogate. Both authors and publishers have ethical obligations. In publication of the results of research, the investigators are obliged to preserve the accuracy of the results. Negative as well as positive results should be published or otherwise publicly available. Sources of funding, institutional affiliations and any possible conflicts of interest should be declared in the publication. Reports of experimentation not in accordance with the principles laid down in this Declaration should not be accepted for publication.
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30.3 Additional Principles for Medical Research Combined with Medical Care
The physician may combine medical research with medical care, only to the extent that the research is justified by its potential prophylactic, diagnostic or therapeutic value. When medical research is combined with medical care, additional standards apply to protect the patients who are research subjects. The benefits, risks, burdens and effectiveness of a new method should be tested against those of the best current prophylactic, diagnostic, and therapeutic methods. This does not exclude the use of placebo, or no treatment, in studies where no proven prophylactic, diagnostic or therapeutic method exists. At the conclusion of the study, every patient entered into the study should be assured of access to the best proven prophylactic, diagnostic and therapeutic methods identified by the study. The physician should fully inform the patient which aspects of the care are related to the research. The refusal of a patient to participate in a study must never interfere with the patient-physician relationship. In the treatment of a patient, where proven prophylactic, diagnostic and therapeutic methods do not exist or have been ineffective, the physician, with informed consent from the patient, must be free to use unproven or new prophylactic, diagnostic and therapeutic measures, if in the physician's judgement it offers hope of saving life, re-establishing health or alleviating suffering. Where possible, these measures should be made the object of research, designed to evaluate their safety and efficacy. In all cases, new information should be recorded and, where appropriate, published. The other relevant guidelines of this Declaration should be followed.
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31 APPENDIX: SAMPLE INFORMATION SHEETS / CONSENT FORMS

As there are a number of different countries participating in this study, and therefore a number of different languages involved, it is left to each National co-ordinator to write the information sheets for parents and informed consent form as appropriate. The following parent information sheet and informed consent document has been prepared for use within this protocol, and may be adapted by other participants.
31.1 Sample Information Sheet/ Consent Form

Dear Parent You have been told that your child has been diagnosed as having a disease called neuroblastoma. Neuroblastoma is a rare malignant disease (Cancer) and in children over the age of one year is often very difficult to treat. However, some children are cured, but others despite very intensive treatment do not do well. Children with high-risk neuroblastoma often respond to standard treatment at first, but there is a high risk that the cancer will come back. We invite you to agree to your childs participation in the research study named the High-Risk Neuroblastoma Study1/ESIOP which aims to increase the number of children with high-risk neuroblastoma who can be cured. This research study has been reviewed and approved by the Institutional Review Board (IRB) [name of the institution], which is an independent board composed of [name of institution] physicians and staff members, and representatives of the community. The IRB has reviewed this study, evaluated the potential benefits and risks and has granted approval for the inclusion of participants. The hospital maintains a Multiple Project Assurance of Compliance, a document that explains how the hospital provides for protection of human subjects. You/your child will receive a copy of this assurance if so requested. This study is a clinical trial (a research study involving human patients). Clinical trials include only patients who choose to take part. You are being asked to allow your child to take part in this study because he/she has been diagnosed with high-risk neuroblastoma. Please take your time to make your decision. Before you can decide whether or not to volunteer/allow your child to volunteer for this study, you must understand the purpose, how it may help you/your child, any risks to you/your child, and what is expected of you/your child. This process is called informed consent. You are asked to read this information sheet and discuss your decision with friends and family. This information sheet gives you information about the study which will be discussed with you/your child. Once you understand (and possibly your child understands) the study, and if you agree to participate/to allow your child to participate, you will be asked to sign the Informed Consent Form, assuming that your child will not be able to sign. (However, your childs assent will be required if the child is seven years of age or older.) Before you learn about the study, it is important to know the following: You/your childs participation in the study is entirely voluntary. You may decide not to participate/allow your child to participate or to withdraw/withdraw your child from the study at any time without penalty. If the study is changed in any way which could affect your/your childs participation, you will be told about the changes and may be asked to sign a new informed consent form.
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WHAT IS THIS STUDY ABOUT? Neuroblastoma is a rare disease and doctors will not see enough cases to be able to plan treatments based just on their own experience, or even that seen in all the centres in their own country. For this reason doctors in a number of European countries have worked together to plan this Clinical Trial in order to study and treat in a planned and systematic way children with high-risk Neuroblastoma and to collect information on a large group of patients. Doctors specialised in treating this disease have combined their knowledge and experience to create a treatment protocol based on what they all believe to be the best available treatment. Experts from many centres in Europe have contributed to the design of this treatment program which is believed to be as effective (or more effective) than treatment used in other major centres throughout the world. These specialists of the field are working together within the International Society of Pediatric Oncology (SIOP) Europe Neuroblastoma (NBL) Group. The SIOP Europe NBL Group represents more than 10 European countries including more than 200 hospitals that treat children with cancer. It is common medical practice world-wide to treat children with neuroblastoma on research studies like this one. What is high-risk neuroblastoma? Neuroblastoma is a solid, cancerous tumour that shows up as a lump or mass in the belly or around the spinal cord in the chest, neck, or pelvis. This treatment protocol is called High-Risk Neuroblastoma Study and aims to improve survival in children recognised to be at particularly high risk. This may be due to disease that has spread throughout the body, in particular bone marrow, bones, lymph nodes, liver or more rarely brain or lungs. In addition over recent years it has become apparent that certain biological features (that it is now possible to test for) may provide additional information about the anticipated prognosis. To date the most important biological feature is something called MycN. MycN amplification has been recognised to be related to a poor survival chance even if the disease is still localised. All these patients groups mentioned above are assumed to need very intensive treatment to have a chance of surviving this disease. How can a malignant disease (cancer) be treated ? There are different methods of attacking and ultimately destroying or overcoming tumour cells: one is CHEMOTHERAPY, using a group of drugs which interact with tumour cells and destroy them. Since tumour cells usually show rapid growth these drugs mainly affect the tumour cells, some however interact with normal tissues and cause what is known as side effects of chemotherapy, i.e. loss of hair, poor blood counts, pronounced immunosuppression, elevated risk of infection and mucositis. All treatments of high-risk tumours are associated with side effects which may be very severe and even life-threatening. There are justified risks in view of the life-threatening nature of the cancer which would otherwise cause the childs death within a short period of time. Drugs may be used at different dose intensity which affects the type of supportive care associated with their administration. At an appropriate time surgeons will try to remove (excise) the main tumour bulk aiming at a COMPLETE SURGICAL EXCISION of the primary tumour. RADIOTHERAPY also has the capacity to kill residual tumour cells, causing damage in cells with a high turn over rate (i.e. tumour cells). Newer approaches to treatment include induction of differentiation of the tumour cells, in other words to turn aggressive immature tumour cells into non-aggressive mature cells. This effect on cells is seen after treatment with certain vitamins, in particular Vitamin A (Retinoid acid) and is called DIFFERENTIATION TREATMENT. A recent study demonstrated the beneficial effect on patient survival when this was added. IMMUNOTHERAPY is another approach that may help to improve survival after the disease has come under control and this study will try to investigate one type of immunotherapy (see below).
What is a randomisation? For some treatment options, developed and accumulated over the years in the various countries world-wide, it is not known today which one is superior in its capacity to improve survival on the one hand and which is characterised by a better toxicity profile on the other hand. In order to compare two types of treatment patients need to be randomised. Random assignment means that the treatment to which your child is assigned is based on chance. It is like flipping a coin, except that the assignment is done by a computer. Neither you nor the researcher chooses which group your child will be in. Your child will have an equal chance of being placed in either group. Parents need to understand and agree to this philosophy prior to their child entering the study and have to allow the randomisation process by signing a consent form. Parents should remember that if doctors knew which of the treatment was better, they would offer it to your child. Parents are not therefore being asked to choose the treatment offered. Doctors will however inform you of the different toxicities associated with both approaches. The parts of the study which include a randomisation should not be regarded as experimental since proposed treatments have been explored and used previously in national treatment protocols. Doctors hope that by studying treatments in a randomised way they will understand the disease better, and will then be able to provide the best treatment available, based on sound evidence of which is best able to cure the disease. Which treatment options are randomised within this study? R0: A Supportive Care Question This randomisation asks the question whether the addition of the human growth factor G-CSF (filgrastim) can reduce the number of days with fever during the induction period. The induction regime has previously been used in Europe for 8 years without any growth factor support. There are some concerns however that the bone marrow stem cell pool may become exhausted if stimulated over a prolonged period of time and thus the quality of the stem cell harvest may be impaired. G-CSF should not be used in an uncontrolled fashion outside the randomisation unless indicated for lifethreatening infection. R1: Comparison of two very high dose chemotherapy regimens This randomisation compares two different intense, high dose chemotherapy regimens. After exposure to such myeloablative therapy (MAT), there is a need to repopulate the bone marrow with its normal cell population. This is done by giving the patients back their previously harvested own stem cells. Two MAT regimens will be compared within this study, both of them are expected to be superior to the other by the groups having developed them (USA and France). According to previous experience both should have the capacity of improving survival, but show a different toxicity profile. The profiles however appear similar in terms of overall risk. R2: Explores the potential benefit of using immunotherapy in addition to differentiation treatment with cis-retinoic acid. This randomisation will investigate whether the addition of immunotherapy in the form of a neuroblastoma specific, humanised mouse antibody in addition to differentiation treatment could help to further improve survival. Both will be given in a treatment phase where usually only minimal residual disease, which is currently very difficult to detect, is suspected in your childs body. This immunotherapy consists of a monoclonal antibody directed against neuroblastoma cells and is an investigational drug not available outside of the randomisation process. Parents may withdraw their agreement to randomisation at any given time point. Your doctors will accept such a decision and continue to treat your child with the treatment arm that is referred to
as standard in this study. Also in the case that parents do not agree to the randomisation process at all, the child will receive what is called the standard treatment. Refusal to participate in the randomisation or a decision to withdraw will not affect your childs treatment or relationship with the doctor or team. What is standard treatment? Standard treatment: Induction treatment without G-CSF Surgery (non-randomised treatment element) MAT treatment with Busulphan and Melphalan (BUMEL) followed by stem cell reinfusion Local radiotherapy (non-randomised treatment element) Differentiation treatment with 13 cis-retinoic acid without additional immunotherapy
SPECIFIC TREATMENT PLAN OF THE HR-NBL1/ESIOP STUDY The current protocol consists of 5 distinct treatment periods. These treatment phases are outlined in the attached, Outline of Treatment Plan for High Risk Neuroblastoma. 1) INDUCTION TREATMENT (COJEC): tries to reduce the tumour cell load in the body and at the primary tumour site. To reach this aim chemotherapy is given every 10 days disregarding blood cell counts over a period of 70 days. This creates a need for your child to be in hospital most of the time, but will be discharged in-between chemotherapy cycles whenever their general well being is sufficient and there is no fever or infection requiring hospital care. There will be an investigational question during this period to find out if adding growth factor support could be beneficial for your child by lowering chemotherapy associated toxicities. Patients will be randomly assigned to receive or not to receive the growth factor G-CSF (filgrastim). 2) INTERIM PHASE WITHOUT CHEMOTHERAPY: Time to evaluate disease response carefully with a variety of diagnostic measures and to aim for complete surgical excision (removal) of the main tumour at the so called primary tumour site. During this period doctors will also try to harvest stem cells (Stem cells are the cells that create new blood cells, such as red blood cells, white blood cells, and platelets). These very young cells are usually found in the bone marrow and have the capacity to develop into all the necessary blood cells and replace those which have been destroyed by very high doses of chemotherapy (myeloablative therapy: MAT). These stem cells are harvested from your childs peripheral blood and frozen for use after high-dose therapy. 3) MYELOABLATIVE TREATMENT (MAT) is a very intense way of administering chemotherapy. High doses of chemotherapy are given to attack residual tumour cells more intensively; this treatment is given over about a week followed by a recovery period in hospital of about 2 additional weeks. Once your child has received MAT his/her own stem cells are thawed and reinfused via a central venous line. Those cells have the capacity to re-establish the whole bone marrow population and to allow for normal peripheral blood values within a time frame of 10 to 20 days. During this period your child will be cared for in the hospital. This part of the study is randomised because two kinds of intensive chemotherapy regimens (myeloablative regimens/MAT) will be compared. Patients will be randomly assigned to one of the two MAT regimens.
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4) RADIOTHERAPY (lasts 2 to 3 weeks), is to be initiated in all children once he/she has recovered from the MAT procedure and will involve the primary tumour site. This should improve the local control of the disease, since the primary tumour site is an area where previously re-growth of the tumour has been observed with a fairly high frequency (reported in up to 40% of affected children) even after complete surgical excision. 5) DIFFERENTIATION TREATMENT with 13 cis-retinoic acid (Vitamin A) (6 months of oral intake) is supposed to change the behaviour of neuroblastoma cells into one characteristic of normal cells. 6) IMMUNOTHERAPY is based in this study on an investigational drug, a neuroblastoma specific humanised mouse antibody. This is an experimental part to explore if the addition of this immunotherapy to differentiation treatment may further improve survival in children with high risk neuroblastoma. WHY IS THIS STUDY BEING DONE? The primary goals of this study are to find out if there is any difference in the number of children cured after one of the two MAT regimens and peripheral stem cell reinfusion. If immunotherapy with chimeric 14.18 anti-GD2 antibody, following MAT, in addition to differentiation therapy with 13-cis retinoic acid, adds to the cure rate observed in patients with high risk neuroblastoma. The secondary goals of this study are To find out if the biological features of neuroblastoma tumours can be used to predict survival. To confirm that metastatic response rates of tumours after this intense, rapid induction schedule as used in this study are better than metastatic response rates previously documented Europewide. To compare the toxicity and, in particular, episodes of febrile neutropenia associated with induction therapy with a rapid schedule platinum regimen (COJEC) when administered with and without G-CSF (filgrastim). To improve the rate of local control with the help of more aggressive local surgery aiming at complete primary tumour resection and the addition of local radiotherapy in all patients. To evaluate if elective haemopoietic support with G-CSF (filgrastim) during induction therapy influences the success of peripheral blood stem cell harvesting (PBSCH) after induction therapy. To determine the efficacy of and to compare the acute and long term toxicities of busulphan and melphalan (BUMEL) and carboplatin, etoposide and melphalan (CEM) used as MAT. To study the long-term side effects of treatment for neuroblastoma on health and quality of life. A side effect is an unintended result of a treatment unrelated to the reason the treatment is being given. HOW MANY PEOPLE WILL TAKE PART IN THIS STUDY? It is expected that about 1000 patients from more than 10 European countries will be treated on this study over a 5-year period. WHAT WILL HAPPEN TO MY CHILD ON THIS STUDY? If you decide to allow your child to participate in the first randomisation (R0), your child will be randomly assigned at the time of registration to receive G-CSF or not during COJEC induction treatment.
If your child is eligible for the high dose chemotherapy myeloablative treatment programme (MAT) your doctors will explain to you the details of the MAT procedure and ask you again for your consent. You will be given a separate informed consent document to read and sign (R1). After successful completion of this part of the treatment and after local radiotherapy the third randomisation is due which will randomly assign your child to receive immunotherapy or not in addition to differentiation treatment. Your doctors will inform you again in detail about the treatment options and ask your consent again to participate in the third randomisation. You will be given a separate informed consent document to read and sign (R2). It may be that your child is ineligible for participation in the myeloablative treatment programme (MAT). Your child could be ineligible for MAT because of factors such as, infection, problems with the stem cell collection, or poor response to the initial treatment given on the study. Patients who are ineligible for MAT and stem cell transplant will be reassigned to receive either additional conventional chemotherapy courses within different controlled studies to improve the disease response status or to other regimens to get control of infection or to conventional bone marrow harvest. Your doctors will explain to you the best possible way of continuing your childs treatment. Methods for Giving Drugs Various methods will be used to give drugs to your child. Some drugs will be given in tablet form by mouth. Other drugs will be given using a needle subcutaneously (inserted under the skin) or directly into a central venous line Central Line For drugs to be given intra-venously, your childs doctor will be likely to recommend that your child has a central venous line placed. A central line is a special type of tubing inserted into a large vein in the chest by a surgeon during a short operation. Your child will be anaesthetised for this procedure and receive pain medication afterwards to keep him/her comfortable. The central line is used to administer chemotherapy drugs and to withdraw small amounts of blood for testing during treatment. The risks associated with central lines will be explained to you. . If your child is to have a central line inserted, you will be given a separate informed consent document to read and sign. Schedules of how and when the drugs will be given during each phase of therapy is in the attached Flowsheet of Therapy for High Risk Neuroblastoma. Induction Phase All patients will begin treatment with the induction phase of chemotherapy, which is divided into 8 chemotherapy cycles every 10 days. There are three different drug combinations in use during the eight cycles. Cycle A uses the anti-cancer drugs vincristine, etoposide and carboplatin, cycle B uses vincristine and cisplatin and cycle C cyclophosphamide, etoposide and vincristine. Additional drugs given during these cycles are MESNA and drugs reducing nausea and vomiting (antiemetics). MESNA is given to help protect the bladder from the potentially damaging effects of cyclophosphamide. G-CSF (granulocyte colony stimulating factor) stimulates the production of white blood cells (infection fighting cells) and will be given in a randomised fashion to properly evaluate the risks and benefits of its use. The balance of benefits and risks is not yet known, which is the reason why it should only be used in a controlled fashion within this trial. It is likely that your child will still need blood transfusions (either red packed blood cells or platelets) and feeding through a naso-gastric tube during this induction period. Stem Cell Harvest After induction is completed and the disease has responded sufficiently, according to given criteria, your childs stem cells will be collected using a procedure called leukapheresis. Either a
special catheter will be placed into a large vein or the pre-existing central line (if in place and appropriate) will be used. One side of the catheter will collect circulating blood into a machine that filters out the stem cells. The filtered blood will be returned to your childs body through the other side of the catheter or a different venous line. Each leukapheresis procedure takes up to 6 hours. The procedure may need to be done several times to collect a sufficient number of stem cells. The physician may decide that the collection of stem cells should come from the bone marrow instead of the peripheral blood, if the collection from the peripheral blood is not satisfactory. This alternative will be discussed in more detail if it affects your child. Your childs collected stem cells (either from the peripheral blood or from the bone marrow) will be frozen and stored at your childs treating institution. Surgery After the end of COJEC induction, your child will have surgery to remove as much of the primary tumour as possible. Myeloablative Chemotherapy and Stem Cell Transplant If your child is to undergo stem cell transplant, he/she will begin consolidation chemotherapy after the end of induction therapy. Most of the children will have already undergone surgery , but this is not a prerequisite prior to MAT. The consolidation phase of therapy is one course of treatment with high doses of anti-cancer drugs. The type of drugs depend on the schedule assigned by randomisation and will be called either CEM consisting of carboplatin, etoposide, and melphalan or BUMEL consisting of busulphan and melphalan. After these anti-cancer drugs are given, your child will have a rest period during which no drugs are given. Your childs stored stem cells will then be given to him/her through the central line , like a transfusion. Five days after the stem cell transplant, your child will be given daily G-CSF injections whether or not your child was randomised to receive G-CSF during the induction treatment. This is because there is evidence the G-CSF is beneficial after transplant. Your child will continue to receive G-CSF for 2-3 weeks, or until blood tests show that the cell count in your childs blood is adequate. During this phase of therapy, your child will be hospitalised. Your child will also require blood transfusions and probably feeding through a naso-gastric tube. It may take only 10 days, but may extend to two or more weeks for the transplanted stem cells to grow and begin to make enough white blood cells so that your child can go home. When your child is medically stable, he/she will be discharged from the hospital. Your child may still need blood transfusions and feeding through a naso-gastric tube for another one or two months. Radiation Therapy Approximately two months after transplant, your child will need to have radiation treatment. Radiation will be given once a day on 5 week days (rest at week-ends) until a total dose of 21 Gy has been administered. Thus radiation will be given on 14 working days and will not exceed a total period of 21 days.
13-CIS-RETINOIC ACID THERAPY
Approximately 3 months following stem cell transplant or immediately after radiation therapy, your child will receive six monthly treatments with 13-cis-retinoic acid. 13-cis-retinoic acid is a drug closely related to vitamin A and has been shown to help stop the multiplication of any remaining neuroblastoma cells in your childs body. Your child will take this drug twice a day by mouth for 14 days and then not take it for the following 14 days. This 28-day cycle will be repeated 6 times. Entry into the last treatment phase or immunotherapy If your child was eligible for stem cell transplant, your child's physician will approach you about the option of entering your child into the last randomisation. If you allow your child to be entered for
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randomisation into the last treatment phase, he/she will be randomised to receive or not 5 additional courses of immunotherapy each given over a week during the rest periods of the 13-cis-retinoic acid. The randomisation will be explained to you in more detail after the MAT procedure before radiotherapy. If you decide to allow your child to be randomised, you will be asked to sign a separate informed consent document. HOW LONG WILL MY CHILD BE ON THIS STUDY? The treatment portion of the study will last about 12 months. However, the researchers would like to continue to observe your child periodically for many years following the study. The researchers may decide to remove your child from the treatment part of the study if your childs cancer gets worse, or your child experiences effects from the treatment that are considered too severe. You can remove your child from the treatment part of the study at any time. However, if you consider removing your child from the study, we encourage you to talk to your childs regular physician and to the research physician before making a final decision. WHAT ARE THE RISKS OF THE STUDY? While on the study, your child is at risk for the side effects listed below. Your child will not necessarily suffer all of the listed side-effects. None of these side-effects would be accepted in a healthy child, but the risk to encounter some of them during treatment is only accepted in view of your childs life-threatening disease. Your child will be closely monitored for the risk of these side effects, and most of the side-effects can be managed effectively. There may also be other side effects that cannot be predicted. Higher dose therapy, such as the treatment your child will receive on this study, is associated with a range of increased side effects compared to standard therapy. Some of the increased side effects may occur during therapy (such as low blood counts, allergic reaction, increased risk of infection, reduced kidney function, impaired hearing, poor nutritional status). Other side effects can occur later for example those relating to fertility. Some side effects can become life threatening, or even fatal. It is also rare, but possible that your child could develop another cancer as a result of treatment on this study. You should discuss these potential risks with the researcher and/or your regular doctor. Other drugs will be given to make certain side effects less serious and/or less uncomfortable. Patients are watched carefully and where possible treatment is stopped if serious side effects develop. Reproductive risks: It is unknown what effect(s) these treatments may have on an unborn child. For this reason, if your child is of child-bearing age, your child will be asked to practice an effective method of birth control while participating in this study. The use of chemotherapy may cause a fertility impairment, in particular high doses as used during the MAT phase. In particular Busulphan is known to cause sterility in the majority of patients, melphalan is thought to reduce fertility particularly in boys. Side effects of chemotherapy drugs: See attached toxicity sheet, which outline the risks associated with each specific drug given on this study. Side effects of radiation therapy: Side effects will vary depending on the site being irradiated. Some side effects may not show up until months or years after treatment. Ask your childs doctor about the specific side effects associated with your childs particular radiation treatment. General side effects include: Nausea and vomiting, sleepiness, temporary weakness, sores in the mouth or intestines if this is the area in the treatment area, redness or soreness of the skin in the area of treatment, decrease in growth of muscle and bone in the treatment area, increased risk of developing a second cancer.
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Side effects of peripheral blood stem cell collection include: Nausea and vomiting, dizziness and/or fainting, seizures (rare), blood loss, infection, muscle cramps, tingling of the lips, changes in heart rhythm (rare and easily treated), skin rash, hives, flushing (redness and warmness of the skin), increased bleeding (caused by a drug given to prevent clotting of the blood during collection). Side effects of bone marrow stem cell collection include: Pain and swelling at the collection site, infection. Side effects of consolidation chemotherapy and stem cell transplant: The high doses of chemotherapy used during preparation for the stem cell transplant cause: Severe lowering of the number of blood cells, damage to normal tissue, severe impairment of your childs ability to fight infection. Due to the severe lowering of blood cells, transfusions are required and your child is at risk of potentially life-threatening infections. The general side effects related to the stem cell transplant are: severe mouth sores, nausea and vomiting, diarrhoea, abdominal pain, skin damage with redness and peeling, irritation of the liver. If there are tumour cells in the harvested stem cells, it is possible that they may cause tumour re-growth in your child when the stem cells are given back to your child. Please note that not all of these side effects will be experienced by all children; they will be closely monitored and can be managed effectively. WILL MY CHILD BENEFIT FROM THIS STUDY? While the hope is that more children with neuroblastoma are cured on this study than on previous studies, there may not be any direct medical benefit to your child if he/she participates in the study. It is hoped that the information learned from this study may however help future patients with high-risk neuroblastoma.
ARE THERE OTHER TREATMENT OPTIONS? There are other options, which will vary from country to country, and are mainly used in nonEuropean countries. WILL MY CHILDS RECORDS BE CONFIDENTIAL? You may read your childs medical record. Efforts will be made to keep your childs personal information confidential. Unfortunately, absolute confidentiality cannot be guaranteed. Information about your child may be disclosed if required by law. Your childs name will not be used when the research results are published. Organisations that may inspect/or copy your childs research records for quality assurance and data analysis include: The SIOP NBL GROUP The Data Monitoring Committee The Institutional Review Board of your childs hospital
WILL I HAVE TO PAY FOR THIS TREATMENT? Taking part in this study may lead to added costs for your insurance company. Please ask about any expected added costs or insurance problems. In the case of injury or illness resulting from this study, emergency medical treatment will always be provided. No funds have been set aside to compensate you in the event of injury. You or your insurance company or your social security will be charged for continuing medical care and/or hospitalisation. However, additional insurance is provided for the
investigational drug, which is the immunotherapy by the neuroblastoma specific antibody ch 14.18 anti-GD2 for side effects unforeseeable today .No-one will receive payment for taking part in this study. WHAT ARE MY CHILDS RIGHTS AS A STUDY PARTICIPANT? Taking part in this study is voluntary. You may choose not to allow your child to take part in this study. If you allow your child to participate, you may remove your child from the study at any time. If you remove your child from the study, physicians and hospital personnel will still take care of your child. You also have the right to know about new information that may affect your childs health, welfare, or your willingness to let him/her participate in the study. You will be provided with this information as soon as it becomes available. Whether you participate or not, your child will continue to get the best medical care your hospital can provide. WHAT IF I HAVE QUESTIONS OR PROBLEMS? For questions about the study or an injury related to the research, please call ____________________ ___________________ NAME at TELEPHONE NUMBER For questions about your rights as a study participant, please call ______________________________________________________ NAME OF INSTITUTIONAL REVIEW BOARD REPRESENTATIVE* ___________________ at TELEPHONE NUMBER *The Institutional Review Board is a group of people who review the research study to protect your rights. WHAT HAPPENS TO THE INFORMATION COLLECTED ON MY CHILD? If you are willing to allow your child to participate in this study, information about your child's tumour and his/her response to the treatment given will be collected, and stored on a computerised database (held in your own country and also in the main data centre). The data will be analysed along with the information from other similar children from all over Europe, so that we can answer some of the questions we have about this disease. We hope in this way that we will be able to more accurately plan treatment for children in the future. All information relating to your child will be treated in the strictest confidence. Any publication or presentation relating to the results of the study will not include any information that enables your child to be identified. If you do decide to allow your child to be entered into the study you may decide at any time to change this decision, and this will not affect your childs treatment in any way. Please ask the doctors caring for your child for any further information. You will be given a copy of this consent form.
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31.2 Trial consent form
High Risk Neuroblastoma 1 study /ESIOP
TRIAL CONSENT FORM

The parent(s) should complete the whole of this sheet himself/herself, (themselves) Please delete as necessary I have been given a copy of and read the information sheet . I have had an opportunity to ask questions and discuss this study . I have received satisfactory answers to all of my questions ? I have understood that if I agree to my child taking part in the study he/she will be treated following the protocol guidelines. For each randomisation a separate consent will be sought at the different time points. Who have you spoken to ? Dr/Mr/Ms ..................................... I have understood that I can withdraw my child from the study : at any time without having to give a reason and without affecting the future medical care of my child I agree to allow my child to take part in this study ? Name of Patient Name of Parent/Guardian Signed Date Name of Physician Signature Date ........................................... ........................................... ........................................... ........................................... ............................................ ............................................ ............................................ YES / NO YES / NO YES / NO
YES / NO
YES / NO YES / NO
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31.3 Supportive Care Randomisation on the Use of G-CSF: R0

INFORMATION SHEET WHAT IS SUPPORTIVE CARE? Supportive care is the treatment that your child is given to try and help him cope with the effects of the cancer s/he has and the side effects of the treatment that s/he is being given to cure him. RANDOMISATION When a group of doctors get together to try and study the effects of a particular treatment on a group of children with a specific disease one of the best ways of looking at the results is to compare a new treatment with a standard treatment which has already shown itself to be effective. If the comparison is to be correct the same number of children of the same age and sex with similar disease characteristics need to be treated in each way. In order to do this the children are randomly assigned to one or other treatment by a central data base which allocates the treatment arm in such a way that the same number of children of the same age and sex and disease characteristics receive each treatment. This is called a randomised study and often the two different treatments are referred to as arms, treatment arm A and treatment arm B.
WHAT IS G-CSF? G-CSF stands for Granulocyte-Colony Stimulating Factor. G-CSF is drug which when entering the body acts on the bone marrow cells. The cells which divide in the bone marrow and have the capacity to mature into different types of blood cells are called stem cells. The pool of cells which are in our bone marrow and have this capacity is called the stem cell pool. G-CSF acts on the stem cell pool which makes white blood cells driving cells to divide and mature. G-CSF thus increases the amount of circulating mature white blood cells (granulocytes, or neutrophils) playing a major role in the control of infections (infection fighting cells). When the number of neutrophils in the body is low and your child develops a fever this is described as febrile neutropenia.
WHAT IS KNOWN ABOUT G-CSF? Administration of G-CSF (filgrastim) before any symptoms of infection have occurred is called prophylactic administration. This is expected to shorten the period of neutropenia. Randomised trials in adults have revealed that G-CSF reduces the incidence of chemotherapy-associated febrile neutropenia in patients at especially high risk of infection . In children, clinical studies suggest that, although not all patients benefit from universal use of G-CSF with chemotherapy, prophylaxis with G-CSF does reduce the incidence of febrile neutropenia and confirmed infections in some patients receiving high intensity or dose intensive chemotherapy for high-risk, advanced stage cancer.
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WHEN WILL IT BE USED?

THE INTENSE, RAPID INDUCTION TREATMENT CALLED (COJEC) IN THE HIGH NEUROBLASTOMA 1/ESIOP STUDY is the first treatment step aiming to reduce the tumour cell
RISK load in the body and at the primary tumour site. To achieve this 8 cycles of chemotherapy will be given every 10 days regardless of the blood cell count over a period of 70 days. There is a risk that your child will be in hospital most of the time, however he or she will be discharged in between chemotherapy cycles whenever their general well being is sufficient and they have no fever or infection requiring hospital care. It is likely that your child will need blood transfusions (either red packed blood cells or platelets) at regular intervals and might require feeding through a naso-gastric tube during this period. At the end of this period stem cells will be collected from your childs peripheral blood to be used later to help your child recover his or her blood count after receiving high-dose therapy. This induction chemotherapy was used in the European Neuroblastoma Study Group (ENSG) 5 study over a period of 10 years. During the induction period this Clinical Trial aims to clarify the potential benefits and or risks of using the growth factor G-CSF (granulocyte colony stimulating factor). If you agree, your child will be randomly assigned to receive or not to receive the growth factor GCSF (filgrastim) to clarify two Major Scientific Questions of R0 Randomisation: 1) Can G-CSF (filgrastim) significantly reduce the number of febrile neutropenic episodes. 2) Is there an influence on feasibility and quality of peripheral stem cell harvest when G-CSF (filgrastim) is applied over an extended period such as COJEC induction. The use of G-CSF (filgrastim) during the COJEC Schedule will require close monitoring of patient data, and strict application of the rules of administration as given below. The G-CSF trial will be stopped in the case of undesired side effects. Concerns have been expressed about the possible difficulty of harvesting peripheral stem cells after prolonged use of G-CSF. However there is previous experience from the US that even after the use of G-CSF during COJEC induction, stem cells may be collected successfully for the high-dose procedure. If necessary, stem cells may also be collected with a bone marrow collection. This procedures will be explained to you separately if relevant for your child. HOW WILL G-CSF BE GIVEN ? The randomised use of G-CSF (filgrastim) has to follow the given rules within this protocol Patients randomised to receive G-CSF (filgrastim) will be given a single daily subcutaneous (inserted underneath the skin) injection of 5 g/kg/day G-CSF (filgrastim) beginning 24 hours after the last chemotherapy dose G-CSF (filgrastim) has to be stopped at least 24 hours prior to next chemotherapy administration in between COJEC courses. G-CSF (filgrastim) is never to be administered on the days of chemotherapy, nor on the day before chemotherapy, nor the day following cisplatin. The administration of G-CSF (filgrastim) should be discontinued if the absolute neutrophil count (ANC) is more than10x109/l. After last Cycle C, when stem cells are to be harvested, G-CSF (filgrastim) should be continued until harvest is completed according to the guidelines for G-CSF (filgrastim) dosing during peripheral blood stem cell harvest.
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WHAT ARE THE EXPECTED SIDE EFFECTS? G-CSF is given as a subcutaneous infusion. It is very unusual but there can be a local reaction with redness at the injection site. It is advisable to change the site of injection every day. Patients receiving G-CSF can sometimes complain of flu like symptoms particularly with muscle or joint pain. These symptoms can be relieved with paracetamol. The concern about reducing the number of stem cells at the time of harvest has already been discussed. The balance between the benefits and risks of G-CSF during rapid COJEC is not known to date, which is the reason why it should only be used in a controlled fashion within this trial.
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31.4 Consent Form for Supportive Care Randomisation R0
High Risk Neuroblastoma 1 Study /ESIOP CONSENT FORM For Supportive Care Randomisation R0
R0
The parent(s) should complete the whole of this sheet himself/herself, (themselves) Please delete as necessary I have been given a copy of and read the information sheet . I have had an opportunity to ask questions and discuss this study . I have received satisfactory answers to all of my questions ? I have understood that if I agree my child will be randomised for this supportive care question and will receive G-CSF as indicated by the randomisation result. Who have you spoken to ? Dr/Mr/Ms ..................................... I have understood that I can withdraw my child from the study : at any time without having to give a reason and without affecting the future medical care of my child I agree to allow my child to take part in this study ? Name of Patient Name of Parent/Guardian Signed Date Name of Physician Signature Date ........................................... ........................................... ........................................... ........................................... ............................................ ............................................ ............................................ YES / NO YES / NO YES / NO
YES / NO
YES / NO YES / NO
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31.5 Randomisation R1 concerning the choice of MAT regimen

You have been told that your child has high risk neuroblastoma which is a life threatening disease. Intensive treatments as proposed below are intended to offer a realistic chance to your child overcoming this disease. Any toxicities encountered due to the treatment are only justified in view of the severity of your childs disease. The reason for randomisation has been explained to you previously at diagnosis. You have now reached the time point to reconfirm your willingness to participate in the R1 randomisation which will assign your child by chance to one of the two proposed MAT regimens. WHAT IS MYELOABLATIVE THERAPY (MAT) FOR? A first response to treatment should be reached after your childs disease has been attacked by induction chemotherapy. Further reduction of the tumour cell load is achieved by surgery aiming to remove as much tumour as possible from the primary site. In neuroblastoma complete removal (resection) is often difficult to achieve. Now a very intense, high dose, chemotherapy is proposed, using multiple agents, to increase the effectiveness of tumour cell elimination. Residual tumour cells are known to build up progressive resistance against conventional dose chemotherapy. After induction treatment they are either still detectable by diagnostic means or no longer detectable by these means. However, the necessity of further intensive treatment was discovered due to the high rate of recurring disease previously observed even in situations where a child was supposed to have reached complete remission (no further detectable disease by diagnostic means). Residual tumour cells are thought to hide somewhere in the body and await good conditions to start to re-grow. In the MAT approach either drugs (carboplatin and etoposide) already used during induction are taken, but at a higher dose, or drugs that are not used initially, because they produce severe bone marrow toxicity (busulphan and/or melphalan). When these drugs are used at high doses, stem cells in the bone marrow get severely affected (myeloablation) or even completely destroyed. This explains the necessity to preserve the childs own stem cells (stem cell harvest procedure) prior to such treatment so that they can be given back after MAT. This is called stem cell reinfusion or rescue.
WHAT TYPE OF MAT IS PROPOSED? IS THERE EXPERIENCE WITH IT ? Two different MAT regimens are proposed in this trial, namely BUMEL and CEM. For both regimens investigators report better survival chances in comparison with other MAT regimens reported previously. However, the BUMEL and CEM regimens have never been compared in the same trial demonstrating either that one may be better than the other or showing equal efficacy against high-risk neuroblastoma but with different toxicity profiles. 1. The BUMEL regimen was developed in Europe. A single institution experience on more than 200 high-risk neuroblastoma patients undergoing MAT claims a significantly improved outcome for those that have received the BUMEL regimen. Overall more than 250 children with neuroblastoma have been treated in Europe with this combination, however at different doses and some with additional drugs. The BUMEL MAT regimen consists of the administration of busulphan divided in 16 oral doses on four consecutive days followed by melphalan. Melphalan is administered as a 15minute short infusion at least 24h after the last busulphan dose. Peripheral blood stem cells are reinfused 24 hours after the melphalan administration.
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2. The CEM MAT-regimen was developed in the US using dose escalation studies. The doses as proposed in this trial have been used in about a hundred children previously in the US. The same CEM regimen is currently used in the US for high-risk neuroblastoma. WHAT ARE THE SCIENTIFIC AIMS OF R1 RANDOMISATION? The scientific aims are to test if the use of MAT with BUMEL or CEM will ultimately result in better survival rates, to determine and compare the acute and long term toxicities of BUMEL and CEM used as MAT. Finally it is sought to monitor drug levels of MAT and to relate them to patients outcome and toxicity. HOW WILL MY CHILD BENEFIT? The potential benefit to be gained from participation in this research study is improved control of your childs disease. Either MAT regimen is supposed to improve survival chances over what was previously experienced within the framework of this high-risk study. Information will also be gained that will be useful to researchers studying this disease. WHO IS ALLOWED ON STUDY? Irrespective of the amount of post-operative residual primary tumour, children having achieved a tumour load reduction in metastatic sites of at least 50% and who have no further bone marrow involvement on will be randomised to receive either BUMEL or CEM as MAT regimen by day 110. Patients not fulfilling these response criteria will not be eligible for randomisation and are off the HR-NBL1/ESIOP study. However, these patients will be offered different treatment. ARE THERE PREREQUISITES TO MAT? Yes there are. 1. Stem Cells need to be collected and stored Before considering such intensive treatment as MAT the patient needs to have his stem cells safely backed up, i.e. frozen for later use. These stem cells will allow repopulation of the bone marrow and reconstitution of normal function of blood components: red blood cells for oxygen transport, white blood cells with their multiple functions in both immune and infection defence and finally platelets with their role in blood clotting protection in case of bleeding . 2. The eligibilty criteria for MAT need to be met Your childs disease must have responded sufficiently before he/she is eligible for MAT therapy. Sufficient stem cells harvested. Your child must have recovered from any prior infections. Your child must have recovered from surgery. WHEN WILL MAT BE GIVEN? Randomisation should be done by day 90. MAT is scheduled for day 110 from diagnosis if the eligibility criteria are met and patient randomised. HOW AND WHEN WILL THE STEM CELLS BE GIVEN BACK? Stem cells will be infused via the central venous line after the MAT regimen after a specified rest period from completion of chemotherapy within 1 hours of thawing. During the infusion the child will be closely monitored
ARE THERE SIDE EFFECTS OF MAT TO BE EXPECTED? The side effects of consolidation chemotherapy and stem cell transplant: The high doses of chemotherapy used during preparation for the stem cell transplant cause: Severe lowering of the number of blood cells Damage to normal tissue Severe impairment of your childs ability to fight infection. Due to the severe lowering of blood cells, transfusions are required and your child is at risk of potentially life-threatening infections. The general side effects related to the stem cell transplant ar severe mouth sores, nausea and vomiting, diarrhoea, abdominal pain, skin damage with redness and peeling, irritation of the liver, hepatic veno-occlusive disease (HVOD). Please note that not all of these side effects will be experienced by all children; they will be closely monitored and can be managed effectively. WHAT WILL HAPPEN TO THE INFORMATION COLLECTED ON MY CHILD? If you are willing to allow your child to participate in this randomisation, information about your child's tumour and his/her response to the treatment given will be collected, and stored on a computerised database (held in your own country and also in the main data centre). The data will be analysed along with the information from other similar children from all over Europe, so that we can answer some of the questions we have about this disease. We hope in this way that we will be able to more accurately plan treatment for children in the future. All information relating to your child will be treated in the strictest confidence. Any publication or presentation relating to the results of the study will not include any information that enables your child to be identified. If you do decide to allow your child to be entered into the study you may decide at any time to change this decision, your child will then be treated in a standard way. Please do not hesitate to ask the doctors caring for your child for any further information.
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31.6 Consent Form for Randomisation R1
High Risk Neuroblastoma 1 Study /ESIOP CONSENT FORM For the Randomisation R1
R1
The parent(s) should complete the whole of this sheet himself/herself, (themselves) Please delete as necessary I have been given a copy of and read the information sheet . I have had an opportunity to ask questions and discuss this study . I have received satisfactory answers to all of my questions ? I have understood that if I agree my child will be randomised for this MAT question and will receive MAT as indicated by the randomisation result. YES / NO YES / NO YES / NO
YES / NO
Who have you spoken to ? Dr/Mr/Ms ..................................... I have understood that I can withdraw my child from the study : at any time without having to give a reason and without affecting the future medical care of my child I agree to allow my child to take part in this study ? Name of Patient Name of Parent/Guardian Signed Date Name of Physician Signature Date ........................................... ........................................... ........................................... ........................................... ............................................ ............................................ ............................................
YES / NO YES / NO
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31.7 Immunotherapy Randomisation R2

INFORMATION SHEET This randomisation will investigate if the addition of immunotherapy in the form of a neuroblastoma specific, humanised mouse antibody could help to further improve the survival chances of your child. This treatment will be given in addition to the differentiation treatment with cis-retinoic acid (13cis-RA) in a treatment phase where usually only minimal residual disease is suspected in your childs body. This means disease which is no longer detectable with methods available to date but which could be responsible for producing recurrent disease later .- This immunotherapy consists of an antibody (adheres to a specific neuroblastoma cell surface marker, like a lock and key mechanism) directed against neuroblastoma cells and is an investigational drug not available outside of this randomisation process. HOW IS THE IMMUNOTHERAPY EXPECTED TO WORK? One concept in the treatment of neuroblastoma is the application of monoclonal antibodies directed against neuroblastoma cells. The antibody, recognises (a surface antigen) which is expressed by virtually all neuroblastoma cells. The antibody is called chimeric, because it was genetically engineered to consist of 30% mouse-protein and of 70% human protein. After binding to neuroblastoma cells, the antibody induces killing of tumour cells. This antibody may have a role in the treatment of neuroblastoma by applying a different concept of anti-tumour action to the standard treatments used to date. HAS THIS ANTIBODY ALREADY BEEN USED? Yes, early studies have been done in the US and in Europe mainly in patients with very advanced disease having already experienced recurrence of their disease. Clinical responses were observed in these patients. WHAT IS THE SCIENTIFIC AIM OF THE R2 RANDOMISATION? Today it is not clear if additional immunotherapy with this antibody increases survival. The current study will recruit enough patients to address this question. The antibody will be given in addition to 13-cis RA after high-dose therapy and local irradiation to those patients elected by the randomisation process. The aim is to combine two independent mechanisms of anti-neuroblastoma activity realised by the two additional therapies, which target the tumour from two different angles. One is indirect via activation of the immune system, the other is direct by induction of neuroblastoma cell death. WHO IS ALLOWED ONTO THIS PART OF THE STUDY? The monoclonal antibody ch 14.18 will be given only to patients fulfilling R2 criteria and being randomised to the immunotherapy arm. Prior to the start of the antibody treatment, patients have to be in good health without clinical or laboratory signs of infection.
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IS MY CHILD ALLOWED TO RECEIVE IMMUNOTHERAPY OUTSIDE THE RANDOMISATION? No, this chimeric 14.18 anti-GD2 antibody will not be available outside the randomisation process, i.e. you cannot opt for your child to receive the antibody. WHEN WILL IMMUNOTHERAPY BE GIVEN? Patients will be randomised (R2) to either additional immunotherapy or no additional immunotherapy after completion of the evaluation following high-dose therapy. Those randomised to additional immunotherapy will receive the antibody treatment. The first course will start three weeks after initiation of retinoid treatment. HOW IS THIS ANTIBODY GIVEN? The antibody is given by slow intravenous infusion through the central line. Your child will be admitted to hospital the day or the evening prior to the antibody infusion. Before the infusion starts your child will be given pre-medication. This will consist of anti-allergic medication, and a continuous pain killing morphine infusion. The morphine co-medication is mandatory during the antibody infusion . ARE THERE ANY SIDE EFFECTS? Yes, this antibody has side effects (SE). Based on previous experience they may be well controlled with adequate supportive care and necessitate hospitalisation of about a week each time. Frequency of side effects (SE) encountered so far: Frequent SE (up to 60% chance): fever Common SE (up to 30 % chance): pain in spite of standard morphine medication, anaphylactic reactions (urticaria, signs of inflammation in the blood (raised CRP, cough) less common (up to 10%): hypotonia in 12%, elevations in liver function tests taken by blood sampling, reduced general condition, oedema rare (up to 1%): serum sickness, seizures with fever, oxygen requirements, reversible pupil palsy. In order to lower the overall risk of SE, the antibody will not be administered during an infectious condition. Pain During the infusion intensive pain in the abdomen and pain in the extremities has to be anticipated. Therefore morphine or opioid treatment is a necessity. The aim is to achieve absence of pain . The morphine dose may need to be adapted, the individual dose required by a particular child to achieve complete absence of pain varies widely. It is important to know that pain symptoms are usually quickly reversible once the antibody infusion is stopped. If your child experiences pain, the antibody infusion will be stopped for 30 minutes, and the pain medication will be adapted to your childs needs. Usually the morphine dose can be drastically reduced during the interval in between antibody infusions. However, acute side effects of high morphine medication such as constipation and difficulty urinating are encountered frequently and require adequate care (laxatives and/ or placement of a urinary catheter). When reducing the dose overnight during the course children usually come off the morphine treatment without withdrawal symptoms.
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Anaphylactic Reactions Allergic reactions are seen quite frequently with associated symptoms such as coughing attacks and fever. This explains the need for the close surveillance of your child during the antibody infusion. If an allergic reaction appears, the antibody infusion will be stopped for at least 30minutes and adequate treatment will be given according to the severity of symptoms. If the reaction resolves easily with adequate medication, the antibody infusion will be continued. If the reaction is severe, the infusion will be stopped for the day. However it will be restarted again the next day with appropriate premedication and a reduction of the antibody dose to 50% for one day, trying to increase to 75% the day thereafter if the 50% regime was well tolerated. If you are willing to allow your child to participate in this randomisation, information about your child's tumour and his/her response to the treatment given will be collected, and stored on a computerised database (held in your own country and also in the main data centre). The data will be analysed along with the information from other similar children from all over Europe, so that we can answer some of the questions we have about this disease. We hope in this way that we will be able to more accurately plan treatment for children in the future. All information relating to your child will be treated in the strictest confidence. Any publication or presentation relating to the results of the study will not include any information that enables your child to be identified. If you do decide to allow your child to be entered into the study you may decide at any time to change this decision, and this will not affect your childs treatment in any way. Please do not hesitate to ask the doctors caring for your child for any further information which you might require.
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31.8 Consent Form for Randomisation R2
High Risk Neuroblastoma 1 Study /ESIOP CONSENT FORM for the Randomisation R2 of the Chimeric 14.18 anti-GD2 antibody
R2
The parent(s) should complete the whole of this sheet himself/herself, (themselves) Please delete as necessary I have been given a copy of and read the information sheet . I have had an opportunity to ask questions and discuss this study . I have received satisfactory answers to all of my questions. I have understood that if I agree my child will be randomised and will receive the chimeric 14.18 anti-GD2 antibody only if indicated by the randomisation result. Who have you spoken to ? Dr/Mr/Ms ..................................... I have understood that I can withdraw my child from the study : at any time without having to give a reason and without affecting the future medical care of your child I agree to allow my child to take part in this randomisation ? Name of Patient Name of Parent/Guardian Signed Date Name of Physician Signature Date ........................................... ........................................... ........................................... ........................................... ............................................ ............................................ ............................................ YES / NO YES / NO YES / NO
YES / NO
YES / NO YES / NO
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32 Reference list
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24. Corbett R, Pinkerton R, Pritchard J et al. Pilot study of high-dose vincristine, etoposide, carboplatin and melphalan with autologous bone marrow rescue in advanced neuroblastoma. Eur.J.Cancer 1992; 28A:1324-28. 25. Hartmann O, Benhamou E, Beaujean F et al. Repeated high-dose chemotherapy followed by purged autologous bone marrow transplantation as consolidation therapy in metastatic neuroblastoma. J.Clin.Oncol. 1987; 5:1205-11. 26. McElwain TJ, Hedley DW, Gordon MY et al. High dose melphalan and non-cryopreserved autologous bone marrow treatment of malignant melanoma and neuroblastoma. Exp.Hematol 1979; 7 Suppl 5:360-71:360-71. 27. Philip T, Bernard JL, Zucker JM et al. High-dose chemoradiotherapy with bone marrow transplantation as consolidation treatment in neuroblastoma: an unselected group of stage IV patients over 1 year of age. J.Clin.Oncol. 1987; 5:266-71. 28. Pritchard J, McElwain TJ, Graham-Pole J. High-dose melphalan with autologous marrow for treatment of advanced neuroblastoma. Br.J.Cancer 1982; 45:86-94. 29. Evans AE, August CS, Kamani N et al. Bone marrow transplantation for high risk neuroblastoma at the Children's Hospital of Philadelphia: an update. Med.Pediatr.Oncol. 1994; 23:323-27. 30. Kletzel M, Becton DL, Berry DH. Single institution experience with high-dose cyclophosphamide, continuous infusion vincristine, escalating doses of VP-16-213, and total body irradiation with unpurged bone marrow rescue in children with neuroblastoma. Med.Pediatr.Oncol. 1992; 20:64-67. 31. Kushner BH, O'Reilly RJ, Mandell LR et al. Myeloablative combination chemotherapy without total body irradiation for neuroblastoma. J.Clin.Oncol. 1991; 9:274-79. 32. Pole JG, Casper J, Elfenbein G et al. High-dose chemoradiotherapy supported by marrow infusions for advanced neuroblastoma: a Pediatric Oncology Group study [published erratum appears in J Clin Oncol 1991 Jun;9(6):1094]. J.Clin.Oncol. 1991; 9:152-58. 33. Pritchard J, Germond S, Jones D et al. Is high dose melphalan of value in treatment of advanced neuroblastoma? J.Clin.Oncol.Proceedings of the American Society of Clinical Oncology 1986; 5. 34. Dini G, Lanino E, Garaventa A et al. Myeloablative therapy and unpurged autologous bone marrow transplantation for poor-prognosis neuroblastoma: report of 34 cases. J.Clin.Oncol. 1991; 9:962-69.
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35. Garaventa A, Rondelli R, Lanino E et al. Myeloablative therapy and bone marrow rescue in advanced neuroblastoma. Report from the Italian Bone Marrow Transplant Registry. Italian Association of Pediatric Hematology-Oncology, BMT Group. Bone Marrow Transplant. 1996; 18:125-30. 36. Hartmann O, Kalifa C, Benhamou E et al. Treatment of advanced neuroblastoma with high-dose melphalan and autologous bone marrow transplantation. Cancer Chemother.Pharmacol. 1986; 16:165-69. 37. Mugishima H, Harada K, Suzuki T et al. Comprehensive treatment of advanced neuroblastoma involving autologous bone marrow transplant. Acta Paediatr.Jpn. 1995; 37:493-99. 38. Philip T, Zucker JM, Bernard JL et al. Improved survival at 2 and 5 years in the LMCE1 unselected group of 72 children with stage IV neuroblastoma older than 1 year of age at diagnosis: is cure possible in a small subgroup? J.Clin.Oncol. 1991; 9:1037-44. 39. Zucker JM, Philip T, Bernard JL et al. Single or double consolidation treatment according to remission status after initial therapy in metastatic neuroblastoma: first results of LMCE 3 study in 40 patients. Prog.Clin.Biol.Res. 1991; 366:543-51:543-51. 40. Kremens B, Klingebiel T, Herrmann F et al. High-dose consolidation with local radiation and bone marrow rescue in patients with advanced neuroblastoma. Med.Pediatr.Oncol. 1994; 23:470-75. 41. Ladenstein R, Lasset C, Hartmann O et al. Comparison of auto versus allografting as consolidation of primary treatments in advanced neuroblastoma over one year of age at diagnosis: report from the European Group for Bone Marrow Transplantation. Bone Marrow Transplant. 1994; 14:37-46. 42. McCowage GB, Vowels MR, Shaw PJ et al. Autologous bone marrow transplantation for advanced neuroblastoma using teniposide, doxorubicin, melphalan, cisplatin, and total-body irradiation. J.Clin.Oncol. 1995; 13:2789-95. 43. Kamani N, August CS, Bunin N et al. A study of thiotepa, etoposide and fractionated total body irradiation as a preparative regimen prior to bone marrow transplantation for poor prognosis patients with neuroblastoma. Bone Marrow Transplant. 1996; 17:911-16. 44. Matthay KK, O'Leary MC, Ramsay NK et al. Role of myeloablative therapy in improved outcome for high risk neuroblastoma: review of recent Children's Cancer Group results. Eur.J.Cancer 1995; 31A:572-75.
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HIGH RISK NEUROBLASTOMA STUDY 1 OF SIOP-EUROPE

SIOP EUROPE NEUROBLASTOMA BOARD

PAEDIATRIC ONCOLOGY NATIONAL CO-ORDINATORS

SIOP EUROPE NEUROBLASTOMA SUB-COMMITTEES

Final version of HR-NBL 1/ESIOP 14.03.06

Final version of HR-NBL 1/ESIOP 14.03.06

Final version of HR-NBL 1/ESIOP 14.03.06

INTERNATIONAL DATA CENTRE

Final version of HR-NBL 1/ESIOP 14.03.06

TRIAL DESIGN......................................................................................................................................................... 11 2.1 2.2 2.3 2.4 2.5 2.6 2.7

BACKGROUND AND RATIONALE ..................................................................................................................... 14 3.1 3.2

ELIGIBILITY AND PATIENT ENTRY CRITERIA........................................................................................... 26 4.1 4.2 4.3 4.4

SURGERY .................................................................................................................................................................. 35 6.1 6.2 6.3 6.4 6.5 6.6 6.7

PATHOLOGY............................................................................................................................................................ 39 7.1 7.2 7.3 7.4

BIOLOGICAL STUDIES ......................................................................................................................................... 41 8.1 8.2 8.3

Final version of HR-NBL 1/ESIOP 14.03.06

STATISTICS AND BIOMETRICAL METHODOLOGY ............................................................................... 86

Final version of HR-NBL 1/ESIOP 14.03.06

APPENDIX SCINTIGRAPHY PROTOCOL FOR MIBG SCANNING..................................................... 113

APPENDIX: PHARMACOLOGICAL GUIDELINES FOR MAT............................................................... 131

Final version of HR-NBL 1/ESIOP 14.03.06

APPENDIX - ADDRESS LIST....................................................................................................................... 151

APPENDIX: DECLARATION OF HELSINKI............................................................................................... 175

Final version of HR-NBL 1/ESIOP 14.03.06

Slettet: Slettet: Sideskift

Final version of HR-NBL 1/ESIOP 14.03.06

2.1 Induction Chemotherapy (Supportive Care Randomisation R0)

days days days days days days days days

750mg/m 175mg/m 1.5mg/m 80mg/m 1050mg/m 0

Final version of HR-NBL 1/ESIOP 14.03.06

2.2 PBSC Harvest

Final version of HR-NBL 1/ESIOP 14.03.06

2.6 Differentiation Therapy

W: weeks related to start of 13-cis RA treatment

2.7 Novel Immunotherapy Approach (Second Randomisation - R2)

GD2 RA W15 rest GD2 W16

GD2 RA W19 rest GD2 W20

W: weeks related to start of 13-cis RA treatment

Final version of HR-NBL 1/ESIOP 14.03.06

3 Background and Rationale

Final version of HR-NBL 1/ESIOP 14.03.06

FIRST TREATMENT MODALITY: COJEC AS INDUCTION REGIMEN

18% 17% 16%

ENSG 5 OVERALL SURVIVAL BY TREATMENT GROUPS

Final version of HR-NBL 1/ESIOP 14.03.06

2.1 1.2 3.6 1.1 3 89 12

COMPARISON OF EVENT FREE SURVIVAL: LMCE3 AND RAPID COJEC

Final version of HR-NBL 1/ESIOP 14.03.06

SECOND TREATMENT MODALITY: SURGERY

THIRD TREATMENT MODALITY: COMPARISON OF TWO MAT REGIMENS BUMEL VS CEM

Final version of HR-NBL 1/ESIOP 14.03.06

Institut Gustave Roussy Data (Olivier Hartmann et al)

Final version of HR-NBL 1/ESIOP 14.03.06

FOURTH TREATMENT MODALITY: RADIOTHERAPY

Final version of HR-NBL 1/ESIOP 14.03.06

FIFTH THERAPEUTIC CHOICE: DIFFERENTIATION THERAPY WITH 13-CIS-RETINOIC ACID

Final version of HR-NBL 1/ESIOP 14.03.06

Eligibility and Patient Entry Criteria

4.1 Eligibility Criteria for the Study

4.2 Eligibility Criteria for the R0 Randomisation

Final version of HR-NBL 1/ESIOP 14.03.06

4.3 Eligibility Criteria for the R1 Randomisation

4.4 Eligibility Criteria for the R2 Randomisation

Final version of HR-NBL 1/ESIOP 14.03.06