Clinical Trial Protocol EWOG-MDS 2006...2007/07/24 · PO box 43515, 2504 AM The Hague Tel. +31...

SKION-Versie 2.0 (juli 2007) 1

Clinical Trial Protocol EWOG-MDS 2006

Prospective non–randomized multi-center study for epidemiology and characterization of Myelodysplastic Syndromes (MDS) and Juvenile

Myelomonocytic Leukemia (JMML) in childhood

SKION Versie 2.0 (juli 2007)

Implementatiedatum: 01-01-2007

Leyweg 299, 2545 CJ The Hague PO box 43515, 2504 AM The Hague Tel. +31 (0)70 367 45 45 Fax +31 (0)70 367 08 68 E-mail: [email protected]

mailto:[email protected]


STICHTING KINDERONCOLOGIE NEDERLAND

Clinical Trial Protocol EWOG-MDS 2006

Prospective non–randomized multi-center study for epidemiology and characterization of Myelodysplastic Syndromes (MDS) and Juvenile Myelomonocytic Leukemia (JMML) in

childhood

BESTUUR Prof. Dr. R.M. Egeler, voorzitter Prof. Dr. R. Pieters, secretaris Prof. Dr. H.N. Caron, penningmeester Dr. M.B. Bierings Prof. Dr. P.M. Hoogerbrugge Prof. Dr. W.A. Kamps Prof. Dr. G.J.L. Kaspers

CENTRAAL BUREAU Dr. J.G. de Ridder-Sluiter, directeur

LABORATORIUM Dr. E.R. van Wering, hoofd laboratorium A. Abdulovski A.A. Choluj E.J. Dam-Boorsma L.J. Goudriaan J.W. Koning-Goedheer J. Koningen E. Laene-Bruyn W.L. de Lannoy-Houtschild B.E.M. van der Linden-Schrever Drs. A.J. van der Sluijs-Gelling J.M. van Wijngaarde-Schmitz

SECRETARIAAT J.M.F. Bouwman J. Pauptit I.A. van Rijn I. van der Veen M.C.J. Yap

DATAMANAGEMENT Ir. C. Korbijn, hoofd datamanagement A. van Sonsbeek-Spierings Dr. H.A. de Groot-Kruseman M.M.Scheffers-van Schie Drs. M.M.J.W.C. Verheijen J. Zijdenbos

APPLICATIE BEHEER M.L. Tros-Batist Ing. J. Godlieb

KWALITEITSMEDEWERKER E.M. Bom

FINANCIËN H. Blokdijk-van der Veen N.B. Zwinkels-Paalvast


Ziektecommissie Myeloïde Maligniteiten Dr. S.S.N. de Graaf, voorzitter Dr. E.S.J.M. de Bont Dr. G.J.L. Kaspers Protocolcommissie MDS 2004 Dr. M.M. van den Heuvel-Eibrink, voorzitter Dr. E.Th. Korthof Dr. P.P.T. Brons Dr. E.R. van Wering Drs. A.C.H. de Vries (fellow) Mevr. A. van Sonsbeek-Spierings (DM)


Introductie Geachte collegae, Voorliggend protocol is de basis voor van een aantal diagnostiek en therapie protocollen die internationaal zijn ontwikkeld voor kinderen met MDS en JMML. Het voorliggende zgn. “master” protocol betreft diagnostiek en registratie en gaat uit van de vernieuwde klassificatie van MDS en JMML. In het komende jaar zullen de 4 therapie protocollen volgen te weten: 1. EWOG-MDS RC-06 voor RC patiënten met hypoplastisch beenmerg en trisomie 8 zonder MSD, 2. EWOG-MDS RC RIC voor alle andere RC patiënten m.u.v. die met monosomie 7 en complex karyotype, 3. EWOG-MDS SCT MDS-06 voor advanced MDS, RC met -7 en complex karyotype en 4. EWOG-MDS SCT JMML 06 voor kinderen met JMML. Op de volgende pagina treft u dit schematisch aan. Er is bewust voor gekozen om het “master” protocol reeds te gaan gebruiken vooruitlopend op de behandelingsprotocollen, aangezien de klassificatie aanzienlijk gewijzigd is ten opzichte van het het voorgaande protocol. Voor de diagnostische procedure verwijs ik u naar pagina 60, sectie 7.3.1 met speciale aandacht voor de Nederlandse bijlage vanaf pagina 107. Omdat er bij aanvang van de ziekte bij RC een overlap is met andere acquired beenmerg falen syndromen zoals bv. Aplastische Anemie maar ook met virusgeassocieerd beenmerg falen wijs ik tevens op appendix 9 Diagnostiek Beenmerg Falen. Het doel hiervan is om bij de SKION te registreren patiënten te filteren op bv. virusgeassocieerde pancytopenieën en aangeboren vormen van beenmergfalen die pas later in het leven aan het licht komen. Tevens wijs ik u op appendix 10 (PNH bij kinderen). In Nederland zullen we patiënten met MDS waarbij evidente PNH klonen gevonden worden registreren in de internationale IPIG registry. Alleen indien er sprake is van de hemolytische variant is therapie met Eculizumab zinvol, hiernaar wordt verwezen in de bijlage. In de hoop dat dit protocol zal bijdragen aan het verbeteren van de overlevingskansen voor kinderen met MDS/JMML in Nederland. Met vriendelijke groet, namens de SKIONPC- MDS M.M. van den Heuvel-Eibrink


Overview of studies

Diagnostic procedures, standards of care, follow-up, registration

EWOG-MDS 2006 (“master protocol“)

EWOG-MDS RC 06

RC

Diagnostics for all RC,Standards on IST

EWOG-MDS SCT RC RIC 06

Chimerism

RAEB/RAEB-T

EWOG-MDS SCT MDS 06

Chimerism

JMML

EWOG-MDS SCT JMML 06

Minimal residual disease

SKION- Versie 2.0 (juli 2007) 6

Clinical Trial Protocol

EWOG-MDS 2006

Prospective non–randomized multi-center study for

epidemiology and characterization of

Myelodysplastic Syndromes (MDS) and

Juvenile Myelomonocytic Leukemia (JMML) in childhood

Final Version

15.03.06

Coordinating Investigator: Regional Coordinator

Name and address Prof. Dr. Charlotte Niemeyer Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 79106 Freiburg Germany

Name and address

Sponsor: University Hospital Freiburg Hugstetterstr. 49 79096 Freiburg Germany

This document may not be published, disclosed, reproduced or passed on without the written permission of the Principal Investigators or Sponsor. These limitations relate to all confidential information and data which will be obtained in the future.


List of Abbreviations AA Aplastic Anemia ANC Absolute Neutrophil Count AP Alkaline Phosphatase Ara-C Cytosine Arabinosid BM Bone Marrow BUN Blood Urea Nitrogen CBC Complete Blood Count CBMF Congenital Bone Marrow Failure CI Cumulative Incidence CMML Chronic Myelomonocytic Leukemia CNS Central Nervous System CRF Case Report Form CSC Coordinating Study Center CT Computerized Tomography CTC Common Toxicity Criteria DBA Diamond Blackfan Anemia DC Dyskeratosis Congenita DLI Donor Lymphocyte Infusion DMC Data Monitoring Committee EC European Community EFS Event Free Survival EORTC European Organization for Research and Treatment of Cancer FA Fanconi Anemia FAB French American British Group GCP Good Clinical Practice GGT Gamma Glutamic Transferase GLP Good Laboratory Practice GM-CSF Granulocyte-Macrophage Colony Stimulating Factor GMP Good Manufacturing Practice GvHD Graft versus Host Disease GvL Graft versus Leukemia HSCT Hematopoietic Stem Cell Transplantation IEC Independent Ethics Committee ICH International Conference on Harmonization IP Investigational Product IPSS International Prognostic Scoring System jCML Juvenile Chronic Myeloid Leukemia JMML Juvenile Myelomonocytic Leukemia LDH Lactate Dehydrogenase MDR-AML Myelodysplasia Related Acute Myeloid Leukemia MDS Myelodysplastic Syndrome MFD Matched Family Donor MNC Mononuclear Cells NF1 Neurofibromatosis 1 NIH National Institute of Health (US) NMDP National Marrow Donor Program (US) n.s. Non significant PB Peripheral Blood PI Principal Investigator


PNH Paroxysmal Nocturnal Hemoglobinuria QOL Quality Of Life RA Refractory Anemia RAEB Refractory Anemia with Excess Blasts RAEB-T Refractory Anemia with Excess Blasts in Transformation RC Refractory Cytopenia RBC Red Blood Cell Count SAA Severe Aplastic Anemia SAE Serious Adverse Event SAP Statistical Analysis Plan SCN Severe Congenital Neutropenia SDS Shwachman Diamond Syndrome SDV Source Data Verification SGOT Serum Glutamic Oxaloacetic Transaminase SGPT Serum Glutamic Pyruvic Transaminase UD Unrelated Donor WBC White Blood Cell Count WHO World Health Organization


Table of Contents Introductie .................................................................................................................................. 4 Overview of studies ................................................................................................................................. 5 List of Abbreviations............................................................................................................................... 7 Synopsis .................................................................................................................................12 Flow Chart JMMLDiagnostic Approach and Follow-Up .............................................................14 Flow Chart Refractory Cytopenia Diagnostic Approach and Follow-Up ......................................15 Flow Chart High grade MDS Diagnostic Approach and Follow-Up............................................16 1. Responsibilities ...............................................................................................................................17 2. Introduction .................................................................................................................................27

2.1 Significance and Background.................................................................................................27 2.2 Classification ........................................................................................................................27 2.3 Epidemiology ........................................................................................................................29 2.4 Juvenile Myelomonocytic Leukemia (JMML) ........................................................................29 2.5 Myelodysplastic Syndrome (MDS) ........................................................................................38

2.5.1 Morphology ............................................................................................................ 38 2.5.2 Cytogenetics and Molecular Genetics ..................................................................... 41 2.5.3 Refractory Cytopenia .............................................................................................. 43 2.5.4 High Grade MDS.................................................................................................... 47 2.5.5 MDS after Chemo- or Radiation Therapy............................................................... 49 2.5.6 MDS after Bone Marrow Failure Disorders............................................................ 50

3. Study Objectives .............................................................................................................................56 3.1 Rationale of the study............................................................................................................56 3.2 Primary Objectives................................................................................................................56 3.3 Secondary Objectives ............................................................................................................56

4. Investigational Plan.........................................................................................................................57 4.1 Study Design.........................................................................................................................57 4.2 Participating centers..............................................................................................................57 4.3 Number of patients................................................................................................................57 4.4 Central and Reference Laboratories .......................................................................................57

5. Study Population.............................................................................................................................58 5.1 Study Population...................................................................................................................58 5.2 Inclusion Criteria ..................................................................................................................58 5.3 Exclusion Criteria .................................................................................................................58

6. Enrollment and Patient Registration.................................................................................................59 6.1 Time of enrollment ................................................................................................................59 6.2 Minimal requirements for enrollment .....................................................................................59 6.3 Mode of enrollment ...............................................................................................................59

7. Methodology .................................................................................................................................60 7.1 Study Schedule .....................................................................................................................60 7.2 Written Informed Consent .....................................................................................................60 7.3 Patient Evaluation .................................................................................................................60

7.3.1 Initial Diagnostic Procedures .................................................................................. 60 7.3.3 Laboratory Tests/Special Examinations During Study Period ................................ 62

7.4 Duration of Study Participation.............................................................................................63 7.5 Use of Patients’ Material.......................................................................................................63 7.6 Asservation of Patients’ Material...........................................................................................63

8. Data Handling and Reporting ..........................................................................................................64 8.1 Reporting and Recording of Data...........................................................................................64 8.2 Data Management and Handling............................................................................................64

9. Quality assurance............................................................................................................................65 9.1 Data Supervision Board ........................................................................................................65


9.2 Steering Committee...............................................................................................................65 9.3 Authorized Supervision .........................................................................................................65 9.4 Data Verification...................................................................................................................65 9.5 Auditing Procedures ..............................................................................................................65

10. Statistics .................................................................................................................................67 10.1 Trial design...........................................................................................................................67 10.2 Patients included in the analyses ............................................................................................67 10.3 Statistical analysis.................................................................................................................67

11. Conditions for protocol amendments................................................................................................68 11.1 Changes in Protocol ..............................................................................................................68

12. Ethical and Legal Considerations.....................................................................................................69 12.1 Patient Information and Informed Consent .............................................................................69

12.1.1 Patient Withdrawal ................................................................................................. 69 12.1.2 Premature discontinuation of the study by the investigator ..................................... 69

12.2 Disclosure and Confidentiality...............................................................................................69 12.3 Independent Ethics Committee (IEC) / Institutional Review Board .........................................70 12.4 General Disclosure Duty .......................................................................................................70 12.5 Insurance ..............................................................................................................................70

13. Study Documents and Archiving of Records ....................................................................................71 13.1 Investigator’s File .................................................................................................................71 13.2 Documentation of Patient Data ..............................................................................................71

13.2.1 Case Report Form (CRF) ....................................................................................... 71 13.2.2 Documentation of data in the patient’s file ............................................................. 71 13.2.3 Patient Identification List ....................................................................................... 71

13.3 Archiving of Records ............................................................................................................71 14. Administrative Considerations.........................................................................................................72

14.1 Financing..............................................................................................................................72 14.2 Final Report..........................................................................................................................72 14.3 Publication of Study Results..................................................................................................72

15. References .................................................................................................................................73 16 Protocol Approval...........................................................................................................................84 17 Investigator Statement.....................................................................................................................85 Appendix 1 List of participating study centers..............................................................................86 Appendix 2 Declaration of Helsinki .............................................................................................87 Appendix 3 CH-GCP-Guidelines .................................................................................................92 Appendix 4 CRFs........................................................................................................................93 Appendix 5 Invoice Forms...........................................................................................................94 Appendix 6 Patient /Parent Information/ Informed Consent ..........................................................95 Appendix 7 Approval by Ethics Committee................................................................................105 18 Nederlandse bijlagen .....................................................................................................................107

18.1 Bijlage I: Diagnostiek fase van JMML/ MDS ......................................................................107 18.1.1 Anamnese ............................................................................................................. 107 18.I.2 Onderzoek bij diagnose......................................................................................... 107

18.2 Laboratoriumonderzoek (SKION) .......................................................................................107 18.2.1 Cytologische diagnostiek (uitstrijkpreparaten)...................................................... 108 18.2.2 Biopsie ten behoeve van ( SKION review) ........................................................... 108 18.2.3 Immunophenotypering and Celbank (Hemoblok – SKION).................................. 108

18.3 Insturen materiaal naar SKION bij Beenmergfalen (appendix 9 vanaf stap2), diagnose en follow-up JMML/MDS...........................................................................................................................110 18.4 Verzenden per koerier naar het SKION laboratorium ...........................................................111 18.5 Cytogenetisch onderzoek .....................................................................................................112 18.6 Patiënten informatie formulieren..........................................................................................113 18.7 Gegevens verzameling per Case Record Form......................................................................124 18.8 Ondersteunende maatregelen bij protocol EWOG-MDS-2006 ..............................................125


Appendix 8 MEC-Verklaring.....................................................................................................127 Appendix 9 SKION protocol diagnostiek beenmergfalen ............................................................129 Appendix 10 Registratie, diagnostiek en behandelingsrichtlijn voor kinderen met PNH klonen in Nederland. ...............................................................................................................................142 Appendix 11 SKION add-on study : Determination of phenotypical and biological characteristics of mesenchymal stromal cells....................................................................................................................147 Appendix 12 Invoice Form ..................................................................................................................192


Synopsis

Title of the international study: PROSPECTIVE NON–RANDOMIZED MULTI-CENTER STUDY FOR EPIDEMIOLOGY AND CHARACTERIZATION OF MDS AND JMML IN CHILDHOOD Protocol No.: EWOG-MDS 2006 Objectives: To assess the epidemiology and to characterize subtypes of MDS and JMML in childhood. Design: Prospective, non-randomized, multi-center study

Planned Study Duration: The total study duration is of 5 years. The study ends 12 months after enrollment of the last patient (total study end). Study duration for each patient is a minimum of 12 months (from inclusion) to a maximum of 5 years. Study Population: A total of 260 patients are expected to be enrolled. Inclusion Criteria:

• Written informed consent by the caretakers and whenever possible the patient’s assent. • Confirmed diagnosis of MDS or JMML (morphology, cytogenetics) • Myeloid leukemia of Down syndrome (patients aged > 6 years). • Age less than 18 years

Exclusion Criteria: • Denied informed consent and/or assent by caretakers/patient. • Myeloid leukemia of Down syndrome (patients < 6 years). • Participation in another study within the last 4 weeks (except for therapy optimizing studies

in cancer or bone marrow failure disorders and studies in diagnostics). Endpoints Primary:

• To evaluate the frequency of the different subtypes of MDS in childhood and adolescence by a standardized diagnostic approach

• To evaluate the frequency of cytogenetic and molecular abnormalities, using array-CGH to evaluate the frequency of subtle chromosomal imbalances, using mFISH to identify unknown chromosomal aberrations

Secondary:

• To assess survival for children and adolescents with MDS and JMML • To evaluate relapse rate, morbidity and mortality in children with MDS and JMML treated

by HSCT Methodology: All patients undergo a standardized initial diagnostic approach. The follow-up covers 12 month periods for the whole study duration. Statistical Methods: The final analysis will be performed six months after the end of the study. One interim analysis two years after start is planned. Survival times will be calculated according to the Kaplan-Meier method and comparisons between probabilities in different patient groups will be performed using the log-rank test.


Timetable: Start of Study: Begin 2006 Enrollment: 48 months End of Study: Fourth quarter 2010 Data available: Begin 2011 Study report: Second quarter 2011 Coordinating Investigator Prof. Dr. Charlotte Niemeyer Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 D – 79106 Freiburg Germany Sponsor University Hospital Freiburg Hugstetterstr. 49 D – 79096 Freiburg Germany


Flow Chart JMMLDiagnostic Approach and Follow-Up

Diagnostic Procedures

Follow-up Every 12 months

HSCT Data

After HSCT

Day 100

After HSCT Yearly

Inclusion/ Exclusion Criteria X Physical examination + vital signs Including signs of NF1

X X

Labs (Hematology, Serum Chemistry) X X X Viral Serology X Transfusion history X X X Concomitant therapy X X X Buccal swab or skin biopsy** X (Freiburg) Cytogenetics X X* X Mutational study (PTPN11, RAS)** X (Freiburg) GM-CSF hypersensitivity in vitro (optional) X Bone marrow aspirate/ (biopsy optional) X X* X Survival X X X X Lansky score X X Secondary malignancy X X Allograft data (graft, conditioning, transplantation, engraftment)

X

GvHD X X Chimerism X X* Relapse/ DLI X X Complications (infections, non-infectious) X X

* if available ** Appendix 5 See also the diagnostic work-up at page 107 and further


Flow Chart Refractory Cytopenia Diagnostic Approach and Follow-Up



HSCT Data

After HSCT Day 100

After HSCT Yearly

Inclusion/ Exclusion Criteria X Physical examination + vital signs Including signs of associated abnormalities

X X

Labs (Hematology, Serum Chemistry) X X X Viral serology X Exclusion of Fanconi anemia X Stool elastase or serum typsinogen, serum isoamylase

X

PNH clone: ITCRV β analysis** X (Rotterdam)

Transfusion history X X X Concomitant Therapy X X X Cytogenetics X X* X Bone marrow aspirate/biopsy X X* X Second bone marrow aspirate/biopsy # X Survival X X X X Lansky score X X Secondary malignancy X X Allograft data (graft, conditioning, transplantation, engraftment)

X

GvHD X X Chimerism X X* Relapse/ DLI X X Complications (infections, non-infectious) X X * if available ** invoice form appendix 12 # recommended within 3 months See also the diagnostic work-up at page 107 and further (including appendix 9)


Flow Chart High grade MDS Diagnostic Approach and Follow-Up



HSCT Data

After HSCT Day 100

After HSCT Yearly

Inclusion/ Exclusion Criteria X Physical examination + vital signs Including signs of associated abnormalities

X X

Labs (Hematology, Serum Chemistry) X X X Viral Serology X Exclusion of Fanconi anemia X Transfusion history X X X Concomitant Therapy X X X Cytogenetics X X* X Bone marrow aspirate/ biopsy X X* X Second bone marrow aspirate/biopsy X Diagnostic lumbar puncture X Survival X X X X Lansky score X X Secondary malignancy X X Allograft data (graft, conditioning, transplantation, engraftment)

X

GvHD X X Chimerism X X* Relapse/ DLI X X Complications (infections, non-infectious) X X * if available See also the diagnostic work-up at page 107 and further


1. Responsibilities Coordinating Investigator: Charlotte Niemeyer, M.D. Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 D – 79106 Freiburg Germany Data Management: Alexandra Fischer, R.N. Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 D – 79106 Freiburg Germany Statistics: Peter Noellke, MPH Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 D – 79106 Freiburg Germany Data Supervision Board Independent Members: Maria Grazia Valsecchi, Ph.D.

Dip. di Medicina Clinica, Prevenzione e Biotecnologie Sanitarie University of Milano – Bicocca Via Cadore 48 I - 20052 Monza Italy Martin Zimmermann, Ph.D. Kinderklinik der Medizinischen Hochschule Hannover Carl-Neuberg-Str. 1 D - 30625 Hannover Germany

Sponsor: University Hospital Freiburg Hugstetterstr. 49 D – 79096 Freiburg Germany


Steering Committee:

Monika Trebo, M.D. St. Anna Kinderspital Kinderspitalgasse 6 A - 1090 Wien Austria

Jan Starý, M.D. Dept. of Pediatric Hematology and Oncology University Hospital Motol Vúvalu 84 150 06 Prague 5 Czech Republic

Charlotte Niemeyer, M.D. Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 D – 79106 Freiburg Germany

Marco Zecca, M.D. Pediatric Hematology / Oncology IRCCS Policlinico San Matteo P. le Golgi 2 27100 Pavia Italy

Marry van den Heuvel-Eibrink, M.D. Sophia Children´s Hospital Room Sp 2429 Dr. Molewaterplein 60 3015 GJ Rotterdam The Netherlands [email protected] T: 010-4636691 F: 010-4636801

Dorota Wójcik, M.D. Ph.D. Department of Pediatric Hematology/Oncology and Bone Marrow Transplantation BMT CIC 817 44 Bujwida St. 50-345 Wroclaw Poland

Henrik Hasle, M.D. Department of Pediatrics Aarhus University Hospital Skejby 8200 Aarhus N Denmark

Eva Bergsträßer, M.D. Department of Oncology University Children’s Hospital Steinwiesstr.75 CH - 8032 Zürich Switzerland



Austria: Regional Coordinator: Monika Trebo, M.D St. Anna Kinderspital Kinderspitalgasse 6 A - 1090 Wien Austria Cytomorphology: N.N. Pathology: Irith Baumann, M.D.

Institut für Pathologie im Klinikum Bayreuth Preuschwitzer Str. 101 95445 Bayreuth Germany

Oncogenetics: Oskar Haas, M.D. St. Anna Kinderspital Kinderspitalgasse 6 A - 1090 Wien, Austria


Czech Republic Regional Coordinator: Jan Starý, M.D. Dept. of Pediatric Hematology and Oncology University Hospital Motol Vúvalu 84 150 06 Prague 5 Czech Republic Cytomorphology: Jan Starý, M.D. Dept. of Pediatric Hematology and Oncology University Hospital Motol Vúvalu 84 150 06 Prague 5 Czech Republic Pathology: Gitte Birk Kerndrup, M.D.

Institute of Pathology Odense University Hospital 5000 Odense C, Denmark

Oncogenetics: Kyra Michalova, M.D.

Center of Tumor Cytogenetics General Faculty Hospital Charles University Unemocnice 1 12808 Prague, Czech Republic


Germany Regional Coordinator: Charlotte Niemeyer, M.D. Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 D – 79106 Freiburg Germany Cytomorphology: Charlotte Niemeyer, M.D. Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology University of Freiburg Mathildenstr 1 D – 79106 Freiburg Germany Pathology: Irith Baumann, M.D.


Oncogenetics: Brigitte Schlegelberger, M.D.

Department of Pathology Medizinische Hochschule Hannover Carl-Neuberg-Str. 1 D - 30625 Hannover Germany


Italy Regional Coordinator: Marco Zecca, M.D.

Pediatric Hematology / Oncology IRCCS Policlinico San Matteo P. le Golgi 2 27100 Pavia Italy

Cytomorphology: Susanna Fenu, M.D.

UOD di Ematologia Lab. di Citomorfologia Az. Osp. “San Giovanni – Addolorata”

Via dell’ Amba Aradam 9 00184 Roma Italy

Laura Sainati, M.D.

Clinica Oncoematologica Pediatrica Universitá di Padova Via Gustiniani 3 35128 Padova Italy

Pathology: Prof. Brunangelo Falini

Laboratorio di Emopatologia Policlinico di Monteluce Via Brunamonti 51 06122 Perugia Italy

Oncogenetics: Laura Sainati, M.D. Clinica Oncoematologica Pediatrica Universitá di Padova Via Gustiniani 3 35128 Padova Italy


The Netherlands Regional Coordinator: Marry van den Heuvel-Eibrink, M.D. Sophia Children´s Hospital Room Sp 2429 Dr. Molewaterplein 60 3015 GJ Rotterdam The Netherlands [email protected] T: 010-4636691 F: 010-4636801 Cytomorphology: Elisabeth van Wering, M.D.

Dutch Childhood Oncology Group P.O. Box 43515 2504 AM The Hague The Netherlands

Pathology: Jan van den Tweel, M.D. University Medical Center Utrecht Department of Pathology H 04123 Heidelberglaan 100 P.O. Box 85500 3508 GA Utrecht The Netherlands

Oncogenetics: Berna Beverloo, Ph.D.

Dept. Clinical Genetics Erasmus University Rotterdam P.O. Box 1738 3000 DR Rotterdam The Netherlands



Poland Regional Coordinator: Dorota Wójcik, M.D. Ph.D.

Department of Pediatric Hematology/Oncology and Bone Marrow Transplantation BMT CIC 817 44 Bujwida St. 50-345 Wroclaw Poland

Cytomorphology: Dorota Wójcik, M.D. Ph.D.

Department of Pediatric Hematology/Oncology and Bone Marrow Transplantation BMT CIC 817 44 Bujwida St. 50-345 Wroclaw Poland

Pathology N.N. Oncogenetics Olga Haus, M.D.

Dept. of Clinical Genetics CM UMK Curie-Sklodowska 9 85-094 Bydgoszcz Poland


The Nordic Countries (Denmark, Finland, Iceland, Norway, Sweden): Regional Coordinator: Henrik Hasle, M.D.

Department of Pediatrics Aarhus University Hospital Skejby 8200 Aarhus N Denmark

Cytomorphology: Gitte Birk Kerndrup, M.D.


Pathology: Gitte Birk Kerndrup, M.D.


Oncogenetics: Gitte Birk Kerndrup, M.D. Institute of Pathology Odense University Hospital 5000 Odense C, Denmark


Switzerland Regional Coordinator: Eva Bergsträßer, M.D. Department of Oncology University Children’s Hospital Steinwiesstr.75 CH - 8032 Zürich Switzerland Cytomorphology: Eva Bergsträßer, M.D.

Department of Oncology University Children’s Hospital Steinwiesstr.75 CH - 8032 Zürich Switzerland

Pathology: Irith Baumann, M.D.


Oncogenetics: David Betts

Department of Oncology University Children’s Hospital Onkologielabor- Steinwiesstr.75 CH - 8032 Zürich Switzerland


2. Introduction 2.1 Significance and Background Myelodysplastic and myelodysplastic/myeloproliferative disorders in childhood are separated into three groups: juvenile myelomonocytic leukemia (JMML), myelodysplastic syndrome (MDS), and Down syndrome leukemia (2). Myeloid leukemia in Down syndrome has unique features and patients should be included in the appropriate protocols for treatment of de novo AML with special consideration of drug doses. The protocol EWOG-MDS 2005 includes only patients with JMML and MDS. 2.2 Classification The first classification of MDS was introduced in 1982 by the French-American-British (FAB) group (3). It divided MDS according to morphology in 5 subgroups (table 1). The FAB classification is based on the number of blasts in the PB and BM, the presence of ring sideroblasts in the BM and the absolute PB monocyte count. Subsequently, it was recognized that the presence of Auer rods is not always an indicator of fast progression of the disease; many investigators like EWOG-MDS omitted Auer rods as a criterion for classification.

Type

Blood

Bone Marrow

Refractory anemia RA <1% blasts < 5% blasts

Refractory anemia with ring sideroblasts RARS <1% blasts < 5% blasts and ring sideroblasts > 15%

Refractory anemia with excess of blasts RAEB < 5% blast 5 - 19% blasts

Refractory anemia with excess of blasts in transformation

RAEB-T

> 5% blasts, or Auer rods

20 - 29% blasts or Auer rods

Chronic myelomonocytic leukemia CMML Monocytes > 1x109/L. < 20% blasts

Table 1 FAB classification as initially described in 1982 In 2000, the World Health Organization (WHO) classification of neoplastic diseases of the hematopoietic and lymphoid tissues incorporating both morphology and genetic changes was introduced (4). It recognizes JMML as distinct entity and places the disorder in a group of myelodysplastic/myeloproliferative disorders. For the definition of MDS, the WHO classification eliminated RAEB-T by reducing the threshold of blasts required to make the diagnosis of AML to 20% (5). At the same the subtype of RAEB was redefined, now accommodating all cases with up to 20% blasts in PB. Six MDS subtypes are described by WHO (table 2).

• Refractory anemia • Refractory anemia with ringed sideroblasts

• Refractory cytopenia with multi-lineage dysplasia • Refractory anemia with excess blasts

• Myelodysplastic syndrome, unclassifiable • Myelodysplastic syndrome associated with isolated del(5q) chromosome abnormality

Table 2 WHO classification of MDS


In children, there are no data to indicate whether a blast threshold of 20% is better than the traditional 30% to distinguish MDS from de novo AML. In addition, the subdivision of MDS does not reflect the hematological and clinical picture of MDS in childhood: Ringed sideroblasts are very infrequently seen in children, the importance of multi-lineage dysplasia is unknown and the unique 5q- syndrome has not been described. The last category, “MDS not otherwise categorized” may not be very useful. Therefore, a pediatric modification of the WHO classification MDS and myelo-dysplastic/myeloproliferative diseases (table 3) was developed (2).

Myelodysplastic / Myeloproliferative Disease • Juvenile myelomonocytic leukemia (JMML) • Chronic myelomonocytic leukemia (CMML) (secondary only) • BCR-ABL negative chronic myeloid leukemia (Ph- CML)

Myelodysplastic Syndrome (MDS)

• Refractory cytopenia (RC) (PB blasts <2% and BM blasts <5%) • Refractory anemia with excess blasts (RAEB) (PB blasts 2-19% or BM blasts 5-19%) • RAEB in transformation (RAEB-T) (PB or BM blasts 20-29%)

Table 3 Classification of MDS and myelodysplastic/myeloproliferative disorders of childhood JMML incorporates those disorders previously referred to as jCML (6) or chronic myelomonocytic leukemia (CMML) (7) of infancy, as well as some cases of the infantile monosomy 7 syndrome (8). Because there is no evidence that monosomy 7 represents a discrete entity, it should no longer be referred to as the monosomy 7 syndrome (9;10). In MDS secondary to chemo- or radiation therapy a hematological picture best described as CMML is occasionally noted. BCR-ABL negative chronic myeloid leukemia (Ph- CML) is exceedingly rare in children. In adult patients with refractory anemia (RA) anemia is generally the main presenting symptom. Children differ in their hematological presentation because neutropenia and thrombocytopenia are more frequently observed. Therefore, the term “refractory cytopenia” (RC) was felt to be more appropriate (2;11). The category of RAEB-T was kept. To have consistency with the definition of RAEB in the WHO classification, cases with up to 20% blasts in PB were incorporated in this subgroup. Disease evolving from MDS with a blast count of > 30% will be referred to as myelodysplasia-related AML (MDR-AML). Table 4 gives the composition of study patients in EWOG-MDS 98 according to the pediatric classification (interim analysis 2005).

JMML and MDS MDS only

N % %

JMML 119 23

RC 207 40 52

RAEB 137 27 35

RAEB-T/MDR-AML 51 10 13

Table 4 Study patients of EWOG-MDS 98 according to the pediatric MDS classification The genetic changes predisposing children to MDS at a young age are largely unknown. The presumed underlying mechanism may also give rise to subtle phenotypic abnormalities noted in many children with MDS. Therefore, there was a need to clearly define “secondary” MDS which will only refer to MDS following neoplasia, congenital or acquired bone marrow failure disorders or MDS in familial disease (table 5) (2).


Secondary MDS - prior malignancy and/or prior chemo- or radiation therapy - known congenital bone marrow failure disorder

- prior acquired aplastic anemia - MDS in a first degree relative (familial MDS) Primary MDS all others Table 5 Definition of primary and secondary MDS 2.3 Epidemiology Combined population-based data from Denmark and British Columbia in Canada identified 38 cases of MDS, representing 4% of all hematological malignancies in children (Table 6), which corresponds to an annual incidence of MDS of 1.8 per million children aged 0-14 years (12;13). MDS and JMML combined constituted 7.7% of the Japanese cases of childhood leukemia8 with a high proportion of therapy-related cases (23%). Recent data from the United Kingdom suggest a considerably lower annual incidence of MDS, 0.8 per million (Table 6) (14). The UK study excluded secondary MDS, partly explaining the lower incidence.

N

%

Annual incidence per million

UK incidence per million

ALL 815 79 38.5 nd

AML1 115 11 5.4 5.8

MDS1 38 4 1.8 0.8

Myeloid leukemia of DS 19 2 0.9 0.6

JMML 25 2 1.2 0.6

CML 13 1 0.6 0.5

PV/ET2 3 0 0.1 nd

Unclassified 3 0 0.1

Total 1030 100 48.7 1Excluding Down syndrome (DS); 2 PV: polycythemia vera; ET: essential thrombocythemia Table 6 Annual incidences of hematological malignancies in children 0 - 14 years. Combined data from Denmark 1980 - 1991 and British Columbia 1982 - 1996 (12;13) and, for comparison, UK data from 1990-1999 (14). 2.4 Juvenile Myelomonocytic Leukemia (JMML) Clinical presentation Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder afflicting young children (2;7;15). JMML predominates in infants (median age at diagnosis, 2 years) (7;16). There is a male predominance with a male: female ratio of 2:1. Pallor, fever, infection, skin bleeding and cough are the most commonly presenting symptoms. There is generally marked splenomegaly and hepatomegaly. Unlike acute monoblastic leukemia, JMML rarely involves the central nervous system (CNS). Hematological picture and karyotype


Leukocytosis, anemia and thrombocytopenia are common findings in JMML patients. The median white count (WBC) is 33 x 109/L (7). An absolute monocyte count exceeding 1 x 109/L is required for the diagnosis of JMML (17). The median blast cell percentage in PB smears is less than 2% (16) (7) and rarely exceeds 20%. The morphological evaluation of the peripheral blood smear is often the most important step in establishing the diagnosis. JMML lacks the Philadelphia chromosome and the BCR/ABL fusion gene. Chromosomal studies of leukemic cells show monosomy 7 in about 25% of patients, other abnormalities in 10%, and a normal karyotype in 65% (7;8;18). Clinical characteristics of patients with monosomy 7 do not differ from those of patients with a normal karyotype (7). However, patients with monosomy 7 present with a lower median WBC but similar absolute monocyte count, as they have a higher percentage of monocytes on the differential count. Red blood cells are often macrocytic, and erythropoiesis in bone marrow is more pronounced than in cases with a normal karyotype. In addition, patients with monosomy 7 present with a normal or only moderately elevated HbF, which is often elevated in patients with normal karyotype (7) (figure 1).

Figure 1 Hemoglobin F concentration for patients with JMML and either normal karyotype or monosomy 7 (7). Molecular pathophysiology JMML myeloid progenitors show a characteristic hypersensitivity for granulocyte-macrophage colony stimulating factor (GM-CSF) (19). This hypersensitivity is mediated by the RAS-RAF-MAP (mitogen-activated protein) kinase signaling pathway, which is pathologically activated by mutations in RAS, NF1 (the gene for Neurofibromatosis 1 [NF1]), and PTPN11 (20-24) (Figure 2). Mutations in PTPN11, RAS and NF1 are mutually exclusive in JMML, suggesting the importance of the pathological activation of RAS dependent pathways in the pathophysiology of the disease.


SHC

GRB2

SOSNF1

RAS

GDP RAS

GTP

GM-CSFreceptor

GAB2

RAF

MAPK

SHP-2

GM-CSF / RAS Pathway

Figure 2. Model outlining the roles of SHP-2, RAS, and NF1 in the GM-CSF signal transduction pathway. Oncogenic point mutations in codons 12, 13 and 61 of NRAS and KRAS are observed in leukemic cells of about 15% to 20% of children with JMML (21;24-27). Neurofibromin, the protein encoded by the gene for NF1, functions as GTPase activating protein and negatively regulates RAS. A clinical diagnosis of NF1 can be made in up to 11% of patients with JMML (8;16). Moreover, about 15 % of JMML patients without the clinical diagnosis of NF1 have mutations in the gene for NF1 (22;28) suggesting that approximately 25% to 30% of JMML cases are associated with NF1. Analysis of JMML cells from patients with NF1 revealed homozygous NF1 inactivation due to somatic loss of the normal allele, which results in hyperactivation of RAS (29-32). The somatic mutation in PTPN11, a gene that encodes the non-receptor protein tyrosine phosphatase SHP-2, is found in about 35% of the JMML patients (33). SHP-2 contains 2 src homology 2 (SH2) domains and a catalytic PTPase domain. The SHP-2 PTPase is activated by binding to phosphotyrosyl peptides through its N-SH2 domain. In JMML the mutations are located in exon 3 and 13 which encode segments of the N-SH2 and PTPase domains, respectively. All mutations cause a gain of function in SHP-2 through preferential occupation of the activated state. JMML-like disorder in Noonan Syndrome Heterozygous germline missense mutations in PTPN11 are known to cause Noonan syndrome, a developmental disorder characterized by dysmorphic facial features, growth retardation and heart disease (33). The mutations are found in exon 3 (most common), 7, 8, and 13, which encode segments of the N-SH2 and PTPase domains. Although many of the germ line PTPN11 mutations identified in Noonan syndrome and somatic mutations in JMML alter the same codons, the spectrum is distinct with respect to the pattern of amino acid substitutions (34). A small number of patients with Noonan syndrome develop a JMML-like disorder (21;35-39). In these patients with Noonan syndrome and JMML-like disorder, the spontaneous remission of the disorder is well documented. The distribution of mutations in PTPN11 in Noonan syndrome with JMML-like disorder also showed specificity, as most of the cases harbored the C218T mutation, which was observed in only 2% of isolated Noonan syndrome and none of isolated JMML patients. It has been demonstrated that the gain-of-function mutations in PTPN11 identified in JMML and Noonan syndrome have distinct effects. The specific mutations in isolated JMML which occur as somatic changes but are not


observed as germ line defects are stronger and most likely associated with embryonic lethality. Conversely, most of the mutations in isolated Noonan syndrome are milder and sufficient to perturb development processes are not fully leukemogenic. In this scheme, mutants in Noonan syndrome with JMML-like disorder are predicted to have intermediate effects, potentially explaining the milder course of the disorder. Although the JMML-like disorder in Noonan syndrome may not be a clonal, and the majority of cases are not fatal, children with this disorder will be included in this study. Neurofibromatosis 1 (NF1) A clinical diagnosis of NF1 can be established in about 11% of patients with JMML (7;8). Up to 15 % of JMML patients without the clinical diagnosis of NF1 have mutations in the NF1 gene (22;28), suggesting that approximately 25% of JMML cases are associated with NF1. NF1 is an autosomal dominant disorder affecting between 1/2000 and 1/4500 individuals (40). About half of all NF1 cases are familial cases, while the other half is caused by de novo mutations. Similarly, about 50% of JMML patients with the clinical diagnosis of NF1 are known to have an affected parent (own unpublished data). Because the NF1 gene is large and mutations do not cluster in certain “hot spots”, mutational studies are still challenging. Therefore, the diagnosis of NF is generally based on the clinical diagnostic criteria proposed by the National Institute of Health (NIH) (Table 7).

Cardinal clinical features (any two or more are required for diagnosis) • 6 or more café-au-lait macules over 5 mm in greatest diameter in prepubertal individuals and

over 15 mm in greatest diameter in post pubertal individuals • 2 or more neurofibromas of any type or 1 plexiform neurofibroma • Freckling in the axillary or inguinal regions • Optic glioma • 2 or more Lisch nodules (irishamartomas) • A distinctive osseous lesion such as sphenoid dysplasia or thinning of the long bone cortex

with or without pseudarthrosis • A first degree relative (parent, sibling, or offspring) with NF1 by the above criteria

Table 7 NIH Diagnostic Criteria for NF1 Applying these criteria, some individuals, who are later shown to have NF1, cannot be diagnosed in early childhood (41). Thirty percent of NF1 patients <1 year of age have only one of the cardinal clinical features (41) and must have an affected first degree relative to be diagnosed with NF1 by these criteria. Café-au-lait macules are usually the first appearing clinical feature; 99% of patients show > 6 café-au-lait macules > 5 mm in diameter by 1 year of age (Table 8).

• Café-au-lait macules: 99% of patients > 6 café-au-lait macules (> 5 mm) by 1 year of age

• Inguinal or axillary freckling: 90% of patients by 7 years old. • Lisch nodules: >70% of patients by 10 years old. • Neurofibromas: 48% of patients by 10 years old

84% of patients by 20 years old • Symptomatic optic glioma: 1% of NF1 patients by 1 year old

It reaches maximum frequency (~4%) by 3 years old. • Characteristic osseous lesions: Usually apparent within the first year of life.

They occur in ~14% of patients. Table 8 The age of appearance of each cardinal clinical feature of NF1


In addition to the features listed among the NIH criteria we suggest to consider JMML as one of the cardinal clinical features of NF1. With this adjustment a diagnosis of NF1 can be made in infants with the following criteria shown in table 9.

In patients with a confirmed diagnosis of JMML a clinical diagnosis of NF1 can be made in the presence of • ≥ 6 café-au-lait macules greater than 5 mm in diameter

or • a first degree relative (parent or sibling) with NF1.

Table 9 Diagnostic criteria for NF1 modified for JMML patients Diagnostic Criteria of JMML The diagnostic criteria proposed by the International JMML Working Group in 1998 have been widely applied (17). They are based on clinical and laboratory findings. Recently, molecular studies have greatly facilitated the diagnostic approach. As described above, somatic mutations in PTPN11 and oncogenic point mutations in RAS are found in 34% and 25% of JMML patients (21;25). In addition, approximately 25% of JMML cases are associated with NF1. In summary, one of these abnormalities can be detected in about 80% of JMML patients. Therefore, molecular studies to detect mutations in PTPN11, RAS, and if available NF1, have to be introduced in the diagnostic process of JMML. Splenomegaly, monocytosis (> 1x109/L) in PB, and less than 20% of blasts count in the BM are the essential clinical and hematological criteria for JMML. In patients fulfilling these criteria, JMML can be diagnosed in the presence of one of the following criteria: PTPN11 / RAS / NF1 mutation, clinical diagnosis of NF1, or monosomy 7. For patients without PTPN11 / RAS / NF1 mutation, clinical diagnosis of NF1, or monosomy 7, the diagnosis of JMML should be made by the classical criteria (17). In these patients, the colony assay for spontaneous growth and hypersensitivity to GM-CSF and the exclusion of BCR/ABL rearrangement (Philadelphia chromosome) are mandatory.

I. Clinical and hematological features (all three features mandatory) • Peripheral blood monocyte count > 1x109/L

• Blast percentage in PB and BM < 20% • Splenomegaly

II. Oncogenetic studies (1 parameter sufficient) • Somatic mutation in PTPN11* or RAS • NF1 mutation or clinical diagnosis of NF1 • Monosomy 7

III. In the absence of one parameter listed under II, the following criteria have to be fulfilled: • Absence of Philadelphia chromosome (BCR/ABL rearrangement) (mandatory) And at least two of the following criteria

• Spontaneous growth or GM-CSF hypersensitivity in colony assay • Hemoglobin F increased for age

• Myeloid precursors on peripheral blood smear • White blood count > 10x109/L

• Clonal abnormality besides monosomy 7 Table 10 Revised diagnostic criteria of JMML * In cases with somatic PTPN11 mutation a germline mutation has to be excluded (Noonan syndrome). JMML-like disorder seen in Noonan syndrome is considered separately.


Differential diagnosis in JMML The clinical and morphologic pictures of JMML can be mimicked by a variety of infectious agents such as cytomegalovirus (42), Epstein-Barr virus (43), human herpes virus 6 (44) and parvovirus B19 (45). However, positive results of these viruses do not exclude the diagnosis of JMML. Clinical course of JMML JMML is a rapidly fatal disorder for most children if left untreated. Some young children with JMML (i.e., those diagnosed before 1 year of age) may experience a longer course characterized by temporary clinical improvement in the absence of therapy. The median survival time without hematopoietic stem cell transplantation (HSCT) is about 1 year (Figure 3).

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 9 10 11 12

HSCT (n=38)

No-HSCT (n=72)

p=0.0001

Years after transplantation

Figure 3 Survival with and without HSCT in patients with JMML (7) Low platelet count, age above 2 years at diagnosis and high HbF at diagnosis are the main predictors of short survival (7;8;16). In a retrospective series of 110 cases, all children presenting with a platelet count of 33 x 109/L or less had died within a year from diagnosis, while those with higher counts and age less than 2 years of age at diagnosis had a median survival of 3 years (7) (Figure 4). Blastic transformation is infrequent in JMML, and most untreated patients die from respiratory failure due to pulmonary infiltration with mature leukemic cells.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 9 10 11 12

p=0.0001

Years after transplantation

Plt ≥ 33x109/L, age < 2 yrs (n=27) Plt ≥ 33x109/L, age ≥ 2 yrs (n=19) Plt < 33x109/L (n=24)

Figure 4 Survival without HSCT in patients with JMML according to platelet count and age at diagnosis (7).


Therapy prior to HSCT for JMML Long-term survival has only been achieved with HSCT, and there are no confirmed drugs which are curative for JMML in the absence of HSCT. The role of anti-leukemic therapy prior to transplantation is currently uncertain. 6-mercaptopurine (6-MP) is probably the drug most commonly applied in JMML prior to HSCT to control the tumor burden, administered either as single-agent (50 mg/m2) (16;46) in combination with low-dose cytarabine (46;47). However, the response is transient and there are no data indicating that it influences the duration of survival. Clinical remissions and long-term survival after AML-type combination therapy have been reported in small series of children with JMML (48);(49);(10). However, other investigators pointed out that intensive chemotherapy is notably unsuccessful, especially in patients with aggressive disease (16;46;50), and durable remission may not be achievable. In a recent prospective analysis of EWOG-MDS/EBMT trial, neither EFS was improved, nor relapse incidence was reduced in patients who had received intensive chemotherapy before the allograft (51). Thus, in view of these results, intensive chemotherapy prior to HSCT cannot be recommended outside clinical trials. Castleberry et al. reported that treatment with isoretinoin (100 mg/m2/day) resulted in complete or partial response in 6 of 10 children with JMML (52). However, other investigators did not observe significant clinical responses with retinoic acid (46;53;54). The treatment with interferon-α (IFN-α) showed some clinical improvements in a limited number of patients (46;55-59). A prospective study with IFN-α, 30.000 units/m2 subcutaneously daily for 14 days followed by the same dose 3 times weekly was stopped for excessive toxicity (60). By contrast, in a JMML patient relapsing after HSCT, IFN-α induced a sustained and complete remission (61). E21R is a modified GM-CSF protein which results in antagonism of GM-CSF function via selective binding to the GM-CSF receptor complex, exerting an anti-leukemic action (62;63). There is a report on a patient with JMML who was treated with 3 courses of E21R (64). A clear efficacy was observed after 2 courses of E21R but the disease appeared completely refractory during the third course. Unpublished experience indicates transient responses to E21R as well. Farnesyl transferase inhibitors (FTIs) are a novel class of compounds that inhibit an enzymatic step (farnesylation) critical to the activation of several cellular proteins, including the RAS proteins. The Phase II window evaluation of R115777 (Zarnestra®) of the Children’s Oncology Group (COG) showed some responses. Splenectomy in JMML The benefit of splenectomy for prevention of post-transplant relapse is unknown (46;54). In the current HSCT study of the EWOG-MDS, splenectomy did not improve the survival of the patient after HSCT (51) (Figure 5).

0 1 2 3 4 5Years after transplantation

0.0

0.2

0.4

0.6

0.8

1.0

< 5 cm = 61% (n=34)

Splenectomized= 48% (n=24)

> 5 cm = 44% (n=36)

P = N.S.

Figure 5. Spleen size and the event free survival after HSCT.


Allogeneic HSCT in JMLL Allogeneic HSCT is currently the only curative treatment for JMML. The results of the most recent trials on HSCT in JMML are summarized in table 11. The analysis of the EWOG-MDS/EBMT trial of 100 patients with JMML transplanted with preparative regiment of busulfan (BU), cyclophosphamide (CY) and melphalan shows a 5-year probability of EFS was 52% (51). The EFS of patients transplanted from a matched family donor (MFD) and unrelated donor (UD) are not significantly different (Figure 6).

Study group Study

design

Nr.

pts

Study

period

EFS

(%)

Donor

(MFD/

MMFD/

UD)

Regimen

Non-TBI/

TBI

Relapse

(%)

TRM

(%)

Acute

GVHD

>II/ >III

(%)

Chronic

GVHD

(%)

EWOG prospect. 100 1993-02 52 (5 yrs) 48/ 0/52 100/ 0 35* 13* 40/17* 17*

Japan retrospect. 27 1990-97 54 (4 yrs) 12/ 4/11 9/ 18 26 11 58/ 31 45

NDMP retrospect. 46 1990-97 24 (2 yrs) 0 / 0/46 11/ 35 58* 18* 54/ 33* 34*

Table 11 Recent large studies of HSCT in JMML. EFS: event-free-survival, MFD: matched familial donor, MMFD: mismatched familial donor, UD: unrelated donor. TBI: total body irradiation, TRM: transplant related mortality, GVHD: graft versus host disease, * Cumulative incidence

Years after transplatation

P = N.S.

MFD = 55% (n=48)

UD = 49% (n=52)

0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Figure 6 Event free survival in transplant with matched family donor (MFD) and unrelated donor (UD).

Relapse is a major treatment failure which is observed in up to 50% patients (65-67). In the current EWOG-MDS/EBMT trial, the 5-year cumulative incidence of relapse was 35% (51). Relapse occurs early, at a median of 2 to 6 months from transplantation (67;68) and generally within the first year. Generally, HSCT shortly after diagnosis is recommended, and younger age at HSCT predicts improved survival (68;69). In the prospective HSCT study from EWOG-MDS, multivariate analysis


shows age greater than 4 years and female sex predicted poorer outcome (51). Cytogenetic abnormalities do not confer a worse prognosis (51). A recent study from the United Kingdom reported that monosomy 7 was associated with an outcome comparable to or even better than that of JMML patients with normal karyotype (70). The impact of acute and chronic graft-versus-host disease (GVHD) on relapse and survival rate has been reported controversially among studies (51;67;69). The result from the National Marrow Donor Program (NMDP) in the US on unrelated HSCT in JMML showed that chronic GVHD was associated with lower risk of relapse and better survival, while acute GVHD (≥ grade III) was associated with poor survival (67). This result suggests a role for a graft versus leukemia (GVL) effect in JMML. Similarly, the Japanese retrospective study showed a trend for a more favorable outcome for patients with grade 0-I acute GVHD or chronic GVHD (69), and the interim analysis of the prospective Japanese study indicated a beneficial impact of chronic GVHD (71). The prospective EWOG-MDS study, however, did not show an impact of neither acute nor chronic GVHD on outcome after HSCT. This result may be explained by the low incidence of chronic GVHD in the EWOG-MDS study cohort (17%) (51). Approach for the patients relapsing after HSCT Treatment options for patients relapsing with leukemia after HSCT are limited. Withdrawal of immunosuppressive drugs is usually the first measure, which by itself can control leukemia in a limited number of patients (72;73). In case of non-response and for all patients suffering disease recurrence after cessation of immunosuppressant agents, donor leukocyte infusion (DLI) or second HSCT may be considered. There are some case reports on the successful treatment with relapsed patients with DLI (65;67;74-76). The recent analysis from EWOG-MDS showed that 6 of the 21 JMML patients, who received DLI for mixed chimerism or hematological relapse, responded to DLI and achieved complete chimerism (77). Response rate was significantly higher in patients receiving a higher number of T cells (> 1x107/kg) and in patients with abnormal karyotype. Notably, none of the 6 patients given DLI from a matched sibling responded. The outcome of even the responders was unfavorable. Only one of the responders is alive in remission, 2 relapsed and 3 died of complications of DLI or HSCT. Infusion of a high enough number of T cells, strategies to reduce toxicity, and cytoreduction prior to DLI may possibly improve the results. Despite the generally aggressive re-emergence of the malignant clone and the short interval between first and second HSCT, a substantial number of children have been cured by a second HSCT (46;78;79). In the most recent analysis of 23 patients with JMML who relapsed after first HSCT and underwent second HSCT (BMT/PBSCT n=15/8), 10 patients are alive in remission, 8 relapsed and 5 died of transplant related mortality (unpublished data of the EWOG-MDS study). The same donor was used for both first and second HSCT in 19 of 23 patients. In the majority of patient, a Bu based regimen was used for first HSCT and a TBI based regimen for second HSCT. It is reasonable to hypothesize that less intensive GVHD prophylaxis was applied during the second allograft in these patients, thereby preserving a GVL effect. In contrast to this encouraging result, the report from the Seattle group shows only one of 6 patients after the second HSCT alive in remission. At present, second HSCT seems to offer the best chance of cure following relapse after HSCT, although the role of DLI under optimal conditions has not yet been established.


Current guidelines for therapy in JMML All children should receive an allogeneic HSCT as soon as the diagnosis is established. A busulfan-based myeloablative preparative regimen is recommended. The current EWOG-MDS HSCT trial in JMML applies busulfan, cyclophosphamide and melphalan, for details see separate protocol. There is currently no evidence that therapy prior to HSCT improves survival following HSCT, and therapy other than 6-MP or isoretinoin is generally not recommended. Conventionally, oral 6-MP (50 mg/m2) has been administered in patients with very high WBC count, pulmonary problems and/or prominent organomegaly. Some investigators administer oral isoretinoin (100 mg/m2, single dose) alone or with 6-MP. More intensive chemotherapy should only be considered in severely ill children. Low-dose intravenous cytarabine (Ara-C) (i.e. 40mg/m2 x 5 days, schedule as needed) can be administered first line. In case this fails, high dose Ara-C can be considered [e.g. fludarabine 30 mg/m2/day (day 1-5) and Ara-C 2g/m2/day (day 1-5), COG protocol]. After intensive therapy, patients should be carefully observed, because a rapid rebound of disease may cause fatal pulmonary infiltration. Patients with Noonan Syndrome and a JMML-like picture are followed closely generally without therapy. 2.5 Myelodysplastic Syndrome (MDS) The following section first describes morphology and cytogenetics for all myelodysplastic syndromes. Subsequently a division into refractory cytopenia, high grade MDS and secondary MDS is made. 2.5.1 Morphology The evaluation of the morphology on BM aspirate, BM biopsy and PB smear is the sine qua non for the diagnosis of MDS. Dysplasia Cytologically, MDS is characterized by dysplasia of at least 2 cell lines. Figure 7 indicates dysplastic features noted in MDS in the 3 cell lineages.


Figure 7 Dysplastic features in MDS, modified from (1) Differential diagnosis of refractory cyotopenia and aplastic anemia The morphological differential diagnosis, in particular the histological pattern of hypoplastic RC and aplastic anemia (AA), may be challenging. Since hypoplastic RC shows patchy distribution of hematopoiesis in an otherwise fatty marrow (Figure 8), the biopsy including only fat cells and missing hematopoiesis, may mimic AA. The bone marrow biopsy is mandatory to obtain the essential information for the diagnosis of RC, such as disruption of the regular hematopoietic topography, typical clustering of left-shifted erythropoiesis, detection of dysplastic megakaryocytes and marrow fibrosis (Figure 8).

Dysgranulopoiesis

Dyserythropoiesis

Dysmegakaryopoiesis

Normal segmented neutrophil

Pseudo-Pelger- Hüet anomaly

Macrocytosis Chromatin clumping

Hypo-, agranulation of cytoplasm

Asynchr. maturation nucleus - cytoplasm

Normal erythroblast

Nuclear bridging Nuclear lobulation

Multiple nuclei

Cytoplasmic granules

Macrocytic / megalo- blastic changes

Normal megakaryocyte

Separated single nuclei

Mikromega- karyocyte

Small binucleated megakaryocyte

Round, non-lobulated megakaryocyte

Figure 8 Histological pattern of hypoplastic refractory cytopenia (1)

Erythropoieses: Uni- or multifocal clustering of predominantly immature forms with increased mitoses Occasionally with atypia

Granulopoieses: Sparsely dispersed or lacking

Megakaryopoiesis: Usually decreased Micromegakaryocytes or other dysplastic changes


Cellularity BM cellularity can only be evaluated on BM biopsies. Therefore, a BM biopsy is suggested for all cases of MDS. It is mandatory if a hypocellular BM is suspected. In RC, many cases are hypocellular (table 12), therefore a BM biopsy is to be recommended. Similarly, in high grade MDS, progression of disease is often associated with myelofibrosis which can only be diagnosed on biopsy.

Retrospective study * Prospective study **

Cellularity n=48 % n=139 %

increased 6 13 11 8

normal 15 31 16 12

low 27 56 112 81

Table 12 Cellularity in refractory cytopenia * Kardos et al. Blood 2003;102:1977 **Interim Analysis of EWOG-MDS 98 in August 2004 Ring sideroblasts Ring sideroblasts are qualified by erythroid precursors with more than 5 iron granules which comprise more than 30% of the nuclear rim. Sideroblastic anemia refers to cases with more than 15% ring sideroblasts in BM. Refractory anemia with ring sideroblasts (RARS) is extremely rare in children and there are no data to document whether patients with RARS share distinctive clinical features. Therefore, we suggest that RARS should be included in the category of “refractory cytopenia”. The finding of sideroblasts in the bone marrow in children should prompt investigations for rare disorders like mitochondrial cytopathies (Pearson syndrome) or inherited sideroblastic anemia (80;81). In Pearson syndrome, vacuolated erythroid and myeloid precursors are observed besides ring sideroblasts. Ring sideroblasts are also occasionally seen in MDS with increase in blasts; these cases, like RC, are classified according to blast count. Description of blast cells and separation from promyelocytes To clearly define blast cells and to distinguish them from promyelocytes, the FAB group in 1982 provided the definition of blasts type I and II (table13) (3;82). In 1991 a type III blast was defined on the basis of a higher number of cytoplasmatic granules (83). Promyelocytes, promonocytes, proerythroblasts and megakaryoblasts are not included in the blast count but scored separately.

Blast type I Large central nucleus with finely dispersed uncondensed (reticular) chromatin, at least one and usually 2 - 3 prominent nuclei, slight to moderate basophilic cytoplasm, no paranuclear hof (Golgi zone), no granules.

Blast type II

As type I but a few (< 20) granules in the cytoplasm

Blast type III

As type II but > 20 granules in the cytoplasm

Promyelocyte

Large eccentric nucleus, slightly condensed chromatin, one prominent nucleus, paranuclear golgi zone, less basophilic cytoplasm with evenly dispersed granules. In MDS promyelocytes are often hypogranular. A cell can therefore be recognized as a promyelocyte when the following features are present: a paranuclear golgi zone and an eccentrically placed nucleus in combination with a low nuclear to cytoplasmic ratio compared to a blast cell (EWOG-MDS Morphology Board).

Table 13 Description of blasts type I - III and promyelocytes


Myelofibrosis BM fibrosis (myelofibrosis) refers to the abnormal deposition of the reticulin network by BM fibroblasts. The prominent myelofibrosis leads extramedually hematopoiesis (EHM), which contributes to hepatosplenomegaly. Typically tear drop shaped red cells and nucleated erythroid and myeloid precursors are observed on PB smears. Myelofibrosis can be secondary to various hematological disorders; MDS, AML (most frequently M7), myeloproliferative disorders like CML, polycythemia vera. It also occurs associated with other underlying diseases such as autoimmune diseases, Down syndrome, and metabolic disorders. In adult MDS, bone marrow biopsy frequently (17-55%) reveals focal or patchy reticulin fibrosis. It is often associated with BM hyperplasia and disturbed differentiation of megakaryopoiesis. In adults, the distribution of myelofibrosis is similar in RA, RARS, RAEB and RAEB-T, but considerably higher in CMML (84). Some reports suggests that myelofibrosis in adult MDS is associated with poor prognosis (84). Most patients show no or only mild hepatosplenomegaly. However, the minorities of cases develop striking myelofibrosis with prominent EMH and may be best described as MDS with myelofibrosis. In childhood MDS, myelofibrosis is, however, less frequent than in adult MDS. It is generally observed in advanced cases and extremely rare in low grade MDS (I Baumann, unpublished observation). Myelofibrosis in the absence of an increased blast percentage and without disturbed megakaryopoiesis is very rarely seen (85-87). Some of these patients show a stable clinical course for years, and spontaneous regression is also reported. Therefore, the initial management should be conservative (85;86). Vitamin D deficiency should be excluded (87). Some cases may respond to steroid therapy (88). The nature of these disorders remains unknown, but differs significantly from adult chronic idiopathic myelofibrosis (CIMF) which usually runs an aggressive course (85;86;89). 2.5.2 Cytogenetics and Molecular Genetics Conventional cytogenetics reveals an abnormal karyotype in about half of MDS patients (table 14) at diagnosis. However, the frequency varies dependent on whether the disease is primary refractory cytopenia, primary advanced MDS or secondary MDS. In contrast to AML, numerical abnormalities dominate; structural abnormalities are frequently part of a complex karyotype with numeric abnormalities. Monosomy 7 is the most common cytogenetic abnormality being identified in approximately 25% of cases. Constitutional trisomy 8 mosaicism may remain unrecognized (90) and should be tested for when trisomy 8 is found in the bone marrow.

Karyotype Primary MDS (%) Secondary MDS (%) All

n = 195 Refractory cytopenia n = 101

Advanced MDS n = 94

Prior chemotherapy/ radiation therapy

n = 36 Normal 113 76 37 8 Monosomy 7 (+/- 1 additional aberration)

44 14 30 7

Trisomy 8 (+/- 1 additional aberration)

7 2 5 0

Complex (>2 aberrations) 11 2 9 12 Other aberrations 20 7 13 9

Table 14 Karyotype of patients with primary and secondary MDS (EWOG-MDS unpublished)


Monosomy 7 is associated with a shorter time to progression in RC of childhood (91). In advanced MDS, monosomy 7 as the sole cytogenetic aberration has not been an unfavorable feature in most studies (7;9;10). Cytogenetic aberrations, which generally implicate a favourable prognosis like -Y, del (5q), have been often reported in adults, but these aberrations are so infrequent in children that they are of no practical importance (92). AML-specific translocations, including t(8;21)(q22;q22), t(15;17)(q22;q12), or inv(16)(p13q22), may occur in cases of de novo AML with a low blast cell count. Their response to therapy is generally favorable and they should not be considered MDS. In situ hybridization (FISH) for identification in monosomy 7 or trisomy 8 can be helpful in the absence of conventional banding cytogenetics. However, the importance of small clones (< 30%) of monosomy 7 cells remains unknown. Multicolor FISH (mFISH) can be helpful to identify unknown genetic material in cases with an abnormal karyotype. In this analysis, whole chromosome painting DNA probes are labelled with different fluorochromes or fluorochrome combinations. Special software analyses the colour information and identifies the chromosomal origin of each individual pixel within the image. Using this method, even complex rearrangements are readily detectable. Figure 9 gives an example how mFISH can provide additional information even if conventional banding cytogenetics are satisfactory. Thus, mFISH can provide new information that may help to identify special target genes which play a role in leukaemogenesis.

Figure 9 Conventional banding cytogenetics (left panel) and mFISH (right panel) of a child with primary MDS and complex karyotype The karytoype was determined by conventional cytogenetics as: 42~49,XY,t(1;11) (q43;q21),del(4)(q25),-5,del(5)(q23q34),der(6)t(3;6)(q13;q13),-7,del(13)(q13q21),-14,add(14)(p13), del(14)(q32),add(17)(p12),-20,-22,+1~3mar[cp16]. The 5q-marker recognized by conventional cytogenetics had initially been interpreted as chromosome 5 with an interstitial deletion at 5q23~q34. With mFISH it was found to contain chromosomal material translocated from chromosome 7. Moreover, the derivative chromosome 17 was recognized to present a whole-arm-translocation with a chromosome 20. Summarized we could further indentify a der(5)t(5;7) and a der(17;20). Matrix-/array-based comparative genomic hybridization (CGH) is a method that utilizes DNA chips consisting of arrayed genomic DNA fragments to screen for microdeletions and duplications. Differentially labelled patient and reference DNA is co-hybridized on DNA microarrays to identify


fluorescent signal differences due to loss or excess of genomic sequences. In a pilot study, we analyzed DNA of 10 children with primary MDS and a cytogenetically detected monosomy 7 by array-CGH to clarify if additional subtle chromosomal gains or losses were present (D. Steinemann, B. Schlegelberger, unpublished). Three patient groups could be identified: one group with sole monosomy 7, a second group with additional aberrations also detectable by conventional cytogenetics, and a third group with high chromosomal imbalances that had not been detected by karyotyping. In one example additional aberrations of chromosomes 3, 12 and 20 were detected in addition to the events involving chromosome 5, 7 and 17 that had been identified by conventional cytogenetics (see fig 10). Comparing the genomic profile in DNA from granulocytes to that obtained in mononuclear cells, subclones could be detected in the mononuclear cell fraction.

Figure 10. The genomic profile of a patient showing multiple aberrations by means of array-CGH. Gains in 3q, 12p and 20q are indicated by green arrows, losses in 3q, 5q, 7 and 17p by red arrows. 2.5.3 Refractory Cytopenia Clinical presentation and hematological picture in RC Refractory cytopenia (RC) is the most common subtype of childhood MDS accounting for about half of all MDS cases (93). Patients usually present with symptoms related to pancytopenia, such as anemia, infection, and bleeding tendency. Organomegaly is generally absent. The term “refractory cytopenia” indicates, that in children, in contrast to adults, thrombocytopenia and neutropenia are more frequently observed than anemia (93). The MCV is usually elevated in MDS, but it is also high in majority of patients with congenital bone marrow failure syndromes, in some patients with severe aplastic anemia (SAA) at diagnosis, and in most patients with SAA during their clinical course. In contrast to adult MDS, bone marrow cellularity is often reduced in RC. In an interim analysis of study EWOG-MDS 98, approximately 80% of patients with RC have a hypocellular biopsy specimen (unpublished data, see Table 12). Cytogenetics in RC Karyotype is the most important factor for progression to high grade MDS and survival (Figure 11). The median time to progression for children with RC and monosomy 7 is less than 2 years. Spontaneous disappearance of monosomy 7 and cytopenia has been noted in some infants, but remains a rare event. In contrast to monosomy 7, patients with trisomy 8 and other karyotypes may experience a long stable course of their disease.


121086420

1.0

.8

.6

.4

.2

0.0years

P

Mononsomy 7 (N=32)

Normal karyotype (N=22)

log rank: p<0.01

Trisomy 8 or other abn. (N=12)

Figure 11 Cumulative incidence of progression to high grade MDS for patients with refractory anemia and either normal karyotype, monosomy 7, or trisomy 8 or other abnormalities at the time of diagnosis. Patients who had received a stem cell transplantation were censored at time of transplantation (93). Differential diagnosis of RC MDS with less than 5% blasts in BM is particularly difficult to diagnose, because dysplasia of hematopoietic cells is frequently observed in association with infections, metabolic disorders, nutritional deficiencies, and a variety of other diseases. The congenital bone marrow failure disorders such as Fanconi anemia, dyskeratosis congenita, Shwachman Diamond syndrome, amegakaryocytic thrombocytopenia, and pancytopenia with radioulnar synostosis should be excluded by careful physical examination for skeletal and other organ abnormalities and taking past and family history. Currently many of these diseases can be diagnosed molecularly. The examination for Fanconi anemia by chromosomal breakage, G2 cell cycle arrest, Western blot or mutational analysis, is mandatory for all the patients with primary MDS. Differentiating hypoplastic RC from SAA remains an intriguing challenge. Moreover, MDS develops in 10-15% of those children with SAA not treated with HSCT (94;95). In the absence of a cytogenetic marker, the clinical course will have to be carefully evaluated; 2 BM examinations with biopsies at least 2 weeks (but not more than 3 months) apart are recommended before a diagnosis of RC can be established. Paroxysmal nocturnal hemoglobinuria (PNH) is a unique acquired clonal stem cell disorder caused by somatic mutation in the PIGA gene on the X-chromosome (Xp22.1) encoding a protein in the synthesis of the glycosylphosphatidylinositol (GPI) anchor by which many proteins are attached to the cell membrane. About 15 proteins have been found to be deficient on the abnormal blood cells in PNH. These defects can result in clinical symptoms including intravascular hemolysis, thrombotic events and bone marrow failure. The hemolysis is caused by increased susceptibility of red cells to complement-mediated lysis, as demonstrated in vitro by the acidified serum lysis test or Ham´s test, which is the classical diagnostic test for PNH. GPI-anchor deficient clones (PNH clones) can be detected more sensitively by flowcytometric screening. PNH develops as a late complication in patients with SAA and MDS. In the absence of clinical signs of PNH, PNH clones can be noted by flowcytometry in 10% - 28% of adults with MDS at diagnosis and during follow-up (96;97). These PNH clones are more frequently observed in refractory anemia than in high grade MDS. While it is unknown, how many children with RC have a PNH clone, the presence of a PNH clone does not speak against the diagnosis of MDS. PNH, the clinical disorder with hemolysis and thrombosis is very rare disease in childhood, most often seen in adolescents.


Clinical course of RC and treatment approach The therapeutic aim in children with MDS is cure and not palliation. Therefore, therapeutic efforts concentrate on HSCT rather than on novel therapeutics like anti-angiogenic therapy, farnesyl transferase inhibitors or DNA methylation inhibitors. HSCT from an HLA compatible related or unrelated donor early in the course of the disease is the treatment of choice for patients with RC and monosomy 7, 7q- or complex karyotype. For children with a normal karyotype or chromosomal abnormalities other than monosomy 7, 7q- or a complex karyotype, and absence of transfusion dependency or neutropenia a watch and wait strategy can be appropriate. If cytopenia necessitates treatment, current therapy options include HSCT with either myeloablative or reduced intensity preparative therapies. Some patients will respond to immunosuppressive therapy (IST) with cyclosporine A (CSA) and anti-lymphocyte globulin (ATG).

Figure 12 Algorithm for management of refractory cytopenia in the absence of monosomy 7, 7q- abnormality or complex karyotypes. Allogeneic HSCT in RC Myeloablative Preparative Regimen: Currently allogeneic HSCT remains the only confirmed curative treatment. In study EWOG-MDS 97 a preparative regimen consisting of busulfan, cyclophosphamide and melphalan was applied. In the interim analysis of October 2005 the probability of survival at 5 years for children was 76% for MFD and 83% for UD transplants (figure 13) (p=n.s.). Chromosomal analysis had revealed a normal karyotype, monosomy 7 or other abnormalities in 20, 12 or 5 patients, respectively; in 5 patients the karyotype was unknown. There were no relapses, all failures were treatment related.

SCT

MFD

IST

All karyotypes with the exception of monosomy 7, 7q- abnormality or ≥ 3 chromosomal abnormalities

Transfusion dependent or

ANC < 1000/µL

No transfusion and

ANC >1000/µL

SCT watch and wait

No MFD Only for hypocellular BM and normal karyotype


UD, n=24, 4 eventsp=0.83, SE=0.08

0 1 2 3 4 5

YEARS FROM SCT

0.0

0.2

0.4

0.6

0.8

.

PRO

BA

BIL

ITY

1.0

FD, n=18, 4 eventsp=0.76, SE=0.11

FD, 3 eventsp=0.18, SE=0.10

UD, 4 eventsp=0.17, SE=0.08

Log Rank: p=n.s.

Figure 13: Event free survival and transplant related mortality in patients with refractory cytopenia transplanted with the preparative regimen of busulfan, cyclophosphamide, and melphalan. Reduced intensitiy conditioning: The favorable result with absence of relapse draws attention to late clinical complications of HSCT, especially infertility caused by BU. In a pilot study for patients with normal karyotype and UD, a reduced intensive regimen consisting of thiotepa (5 mg/kg/day x 3days), fludarabine (40 mg/m² x4 days) and ATG resulted in an EFS similarly to what has been observed with the ablative regimen (EWOG-MDS unpublished). In 19 children with a hypocellular bone marrow and normal karyotype the probablility of survival 3 years after HSCT from an UD was 83% (Figure 14).

OS, N=19, 3 eventsp=0.83, SE=0.09

0 1 2 3 4 5

YEARS FROM SCT

0.0

0.2

0.4

0.6

0.8

.

PRO

BAB

ILIT

Y

1.0

TRM, 3 events,p=0.17, SE=0.09

ES, 5 eventsp=0.72, SE=0.11

Figure 14: Overall Survival, Event free survival and transplant related mortality in patients with a hypocellular bone marrow and normal karyotype.


Immunosuppressive therapy (IST) in RC Initiating events of MDS are thought to occur at a genetic level of the hematopoietic stem cell. However, early bone marrow failure can result at least in part from T-cell mediated suppression of hematopoiesis. Studies in adults demonstrated that 34% - 50% of MDS patients have clinically relevant responses to IST (98;99). In a pilot study of EWOG-MDS, IST according to the German protocol for SAA was applied [ATG (0.75ml/kg/day for 8 days), prednisolone (1mg/kg/day, day 1-14, then taper till day 28), CSA (5 mg/kg/day for > 6 months), and G-CSF (5µg/kg/day depending on neutrophil count)]. In an interim analysis (October 2005), 18 of 29 patients (69%) responded to IST (currently complete response: n=9, partial response: n=10, toxic death n=1). Response was generally observed within 3-6 months. There was no obvious correlation between the degree of neutropenia (ANC < 200/µl [n=6], 200 - 500/µl [n=9] or > 500/µl [n=14]) and response to IST. Data on long term follow-up are not yet available, and it is currently unknown whether IST can result in sustained responses in a substantial number of children with RC. It is however, reasonable to assume that survival after 5 years will not be superior to what has been reported for AA and IST. In a recent report from the German Pediatric SAA study patients with SAA and an ANC > 200µl had an 5 year survival rate of 81% (100).

Current guidelines for therapy of RC Therapy options in RC are dependend on karyotype, peripheral blood counts and bone marrow cellularity. 1. Patients with monosomy 7, 7q-, or complex karyotypes should be transplanted soon after

the diagnosis is established (generally within 3 months). The recommended preparative regimen is myeloablative consisting of busulfan, cyclophosphamid and melphalan (details see EWOG-MDS RC-SCT 2006)

2. Patients with all other karyotypes can be followed according to a watch and wait strategy if their ANC is > 1000/µl and there is no need for transfusions.

3. If these patients have an ANC < 1000 /µl or are transfusion dependent they will require therapy.

3.a. In case of hypocellularity of the BM • HSCT with reduced intensity from a sibling or unrelated HLA matched donor (8/8 or 7/8

antigens) is recommended (details see EWOG-MDS RC-SCT 2006). • Therapy with IST can be a treatment option (details see EWOG-MDS RC IST 2006) for

patients with normal karyotype or trisomy 8. For patients on IST who are non responders on day 120, an unrelated donor search is to be initiated. In the presence of non-response it is advised to transplant the patient as soon as a suitable donor is identified.

3b. Patients with normocellular or hypercellular BM are not candidates for IST, therapy consists of HSCT following a myeloablative preparative regimen.

Therapy options are outlined in the algorism depicted in Figure 12. 2.5.4 High Grade MDS MDS with increased blast count comprises the MDS-subtypes RAEB and RAEB-T (table 1). In contrast to the original FAB classification (3), RAEB includes cases with up to 19% blasts in PB, and Auer rods are no longer used for classification. The international prognostic scoring system (IPSS) for MDS in adults has recommended subdivision of RAEB according to the BM blasts into BM blasts 5-10% (RAEB I) and 11-20% (RAEB II) (101). This scheme warrants further investigation in pediatrics.


High grade MDS and de novo AML The separation of MDS with increased blast count from de novo AML remains challenging and thresholds of blast counts, whether set at 20% or 30%, are arbitrary. Assuming that the underlying genetic changes between MDS and de novo AML are different and therapy approaches will differ, the distinction between these entities becomes important. De novo AML is a chemo-sensitive disease characterized by balanced translocations, while the typical genetic changes in MDS, typically resistant to chemotherapy, are numerical aberrations. Patients with recurrent cytogenetic abnormalities typically associated with AML, e.g. t(15;17) (PML/RARα), t(8;21) (AML1/ETO), inv(16)(CBFβ/MYH11), t(9;11) (MLL/AF9), should be diagnosed and treated as de novo AML regardless of the blast count (102). The only chromosomal abnormality which may be regarded as marker of MDS-like biology is monosomy 7 (table 14). MDS progressing to disease with BM blast counts > 30% is referred to as myelodysplasia-related AML (MDR-AML). For monosomy 7, it is unknown, whether cases evolving from MDS to MDR-AML have the same biology than cases diagnosed as AML with monosomy 7. In AML studies, patients diagnosed as AML with monosomy 7 have a lower response rate to chemotherapy (9;10) and a higher relapse rate (103) compared with AML without -7. It should be emphasized, however, that most MDS patients have a blast percentage < 20% at diagnosis, while the vast majority of children with de novo AML present with a frank leukemic BM. Therefore, the blast count can be considered a surrogate marker for the underlying biology of the disease in most cases (figure 15).

Blast(%)

Time (months)

Threshold(20 or 30 %)

AML

MDS

Figure 15 Disease progression in de novo AML and MDS Modified from (104) For patients with an ambiguous blast count, organomegaly, CNS infiltration or chloroma are indicative of de novo AML (figure 16). In patients presenting with a BM blast percentage > 20% and no clinical or cytogenetic changes characteristic of MDS or de novo AML, it is recommended to repeat the BM examination after 2 weeks. If the blast count has increased to ≥ 30% the patient most likely has de novo-AML. If the blast count is stable over an arbitrary period of 4 weeks the diagnosis of RAEB-T can be made.


Figure 16 Algorithm for discrimination of de novo AML and MDS by blast count, monosomy 7 and clinical characteristics In contrast to de novo AML, leukemic cells in MDS infiltrate the central nervous system (CNS) in rare cases only. A mere 1% of the patients in EWOG-MDS 98 exhibit blasts in the CNS. Cranial nerve palsy was not observed. However, at the time of diagnosis, a diagnostic lumbar puncture is recommended. In the absence of blasts, CNS therapy is not warranted. Therapy in high grade MDS Allogeneic HSCT is the treatment of choice in high grade MDS and can rescue a large proportion of children. Whether intensive chemotherapy prior to stem cell transplantation should routinely be employed is highly controversial. Data from EWOG-MDS (EWOG-MDS trial 97, 88 patients, preparative regimen with busulfan 16 mg/kg, cyclophosphamid 120 mg/kg and melphalan 140 mg/m2) indicate that AML-type therapy prior to the grafting procedure does not prolong survival. With a median observation time after SCT of 35 mo. the EFS at 5-yrs. was 68% and 46% for SCT from MFD and UD, respectively (EWOG- MDS, Interim analysis, 2005). Overall, in the patients transplanted for RAEB/ RAEB-T/ AML the EFS at 5 years were 0.50 ± 0.08. Toxicity of the procedure and relapse rate contributed equally to the number of events. There were no significant differences in relapse incidence among the patient groups with RAEB and RAEB-T (highest FAB-type prior to SCT), but patients with MDR-AML experienced a higher relapse rate. Therefore, intensive AML-type therapy prior to HSCT is not recommended for patients with high grade MDS. Current guidelines for therapy in MDS with increased blast count Allogeneic HSCT should be performed as soon as possible. The current HSCT trial of EWOG-MDS utilizes a preparative regimen consisting of busulfan, cyclophosphamide and melphalan. Intensive chemotherapy prior to HSCT is not recommended. 2.5.5 MDS after Chemo- or Radiation Therapy Hematological, therapy-related secondary MDS (tMDS) after chemo- or radiotherapy can manifest as RC, high grade MDS, or CMML (105). Between 7% to 18% of MDS cases in children are tMDS (38;70). The pathophysiology of the development of tMDS is supposed to be similar in children, adolescents and adults. It likely depends on complex interactions between three kinds of factors: (1) The effect of the primary leukemogens, i.e., topoisomerase II inhibitors and alkylators; enhanced by (2) additional facilitating treatment-related risk factors like irradiation, asparaginase, and thiopurines; modified by (3) host factors, for example CYP3A4 polymorphisms (106). In children and adolescents, the classical distinction between alkylator-type (107) and topoisomerase II

Chromosomal changes

typical for AML

Mono - somy 7

Leukocytosis Organo-megaly

Second BM examination after 2 weeks

AML

MDS

Blasts > 30% Blasts

< 20%


inhibitor-type (108) tMDS does neither exist clinically (EWOG database, unpublished data) nor cytogenetically (109). There is little information on clinical and cytogenetic characteristics of tMDS in children and adolescents. The largest series published, a retrospective study from Japan reporting on 24 patients, found a higher proportion of abnormal karyotypes (often complex aberrations) and a worse prognosis compared to primary MDS (110). HSCT improved survival (111). The following data result from an interim analysis of study EWOG-MDS 98 (April 2005, unpublished): tMDS occurred most frequently after ALL (34%), followed by tMDS after solid tumors outside the CNS (27%) and after CNS tumors (20%). In some ALL patients signs of tMDS can already be noted during maintenance therapy. They include low platelet counts, prominent monocytosis, and unusually high hematological toxicity of maintenance therapy. These clinical features can persist for months before blasts emerge, and the CMML-like picture converts to frank high grade MDS. This observation corresponds to the short interval between diagnosis of the primary malignancy and tMDS (median 3.3 years; range, 0.5 to 11.1). Autoimmune phenomena like skin rashes or effusions are sometimes noted at presentation or during clinical course of tMDS, and may require steroid treatment. Suspected tMDS after primary AML is a diagnostic dilemma. Even if there is strong myelodysplasia and the course of the hematological disease is relatively stable over prolonged time, the disorders should be considered relapse of de novo AML with a “smouldering” course. Like in AML relapse, patients should receive intensive therapy for remission induction prior to HSCT. In the absence of an increased blast percentage, the morphological diagnosis of tMDS may be difficult. Conventional cytogenetics may be helpful in demonstrating an abnormal clone (see table 14). Early diagnosis of tMDS is vital for cure. Because many patients suddenly deteriorate after a relatively stable course, HSCT should be performed as soon as possible, preferably within 3 months from diagnosis. One of the unresolved problems is relapse of the primary malignancy after HSCT for tMDS. Current guidelines for therapy in tMDS Diagnostic procedures should be performed as soon as a suspicion of tMDS arises. If a curative therapy approach is chosen the patient should receive an allogeneic HSCT as soon as possible. Intensive therapy prior to HSCT is not recommended. The preparative regimen prior to HSCT is the same than for primary high grade MDS, currently consisting of busulfan, cyclophosphamide and melphalan. 2.5.6 MDS after Bone Marrow Failure Disorders 2.5.6.1 MDS in congenital bone marrow failure disorders A number of inherited disorders are characterized by BM failure associated with or without somatic abnormalities. They may present in infancy or thereafter, some cases as late as adulthood. The BM failure may involve all 3 lineages [e.g. Fanconi anemia (FA), dyskeratosis congenita (DC)] or a single lineage [e.g. severe congenital neutropenia (SCN), Diamond Blackfan anemia (DBA)]. Most congenital BM failure disorders (CBMF) are associated with a high preposition for secondary MDS and secondary acute myeloid leukemia. The definition of secondary MDS in CBMF without increase in blast count is difficult, because myelodysplastic features are often observed in the absence of clonal evolution. We recommend diagnosing secondary MDS after CBMF only when the criteria outlined in table 15 are fulfilled.


Secondary MDS in CBMF is defined by a consistent acquired BM abnormality such as

• increase in blasts • acquired chromosomal abnormality • increasing bone marrow cellularity in the presence of blood pancytopenia

Table 15 Definition of secondary MDS in congenital CBMF The incidence of secondary MDS in the different CBMF is summarized in table 16. FA is the disorder most frequently followed by hematological malignancy. Among congenial neutropenias, MDS/AML develops in SCN and Shwachman-Diamond syndrome (SDS), but neither in cyclic neutropenia nor glycogen storage disease Type Ib. In patients with congenital amegakaryocytic thrombocytopenia, cancer preposition is not documented. CBMF differ in their natural history and display different disease-specific problems. Consequently, the primary disorder has an impact on treatment strategies of secondary MDS (e.g. the indication of HSCT and the preparative regimen), and a uniform therapy approach to all CBMF patients is not advisable. In general, patients with CBMF should be registered in national or international registries/studies, and MDS/AML should be treated according to guidelines of the respective registries/studies.

Incidence (% of total cohort)

hematological malignancy

non-hematol. malignancy

Age at the time of MDS/AML

Ref.

FA 7-15% 5-9% median 11-14 yrs (112-115) DC 2.7% 8.8% 10, 22, 27 and 29 yrs (116) SCN 6% - median 8 -13 yrs (117;118) Cyclic neutropenia

0% - - (117;118)

SDS 5-33% - median 12-27 yrs (118-120) DBA 0%-5.2% 1.5-1.6 % median 22 yrs (121-123)

Table 16 Incidence of hematological and non-hematological malignancy in patients with congenital bone marrow failure disorder Fanconi anemia Fanconi anemia (FA) is an autosomal recessive disorder characterized by BM failure, short stature, hyperpigmentation, developmental abnormalities such as radial and thumb defects, microcephaly, and renal anomaly (124;125). FA often manifests during early childhood. Progressive BM failure affects platelets first, followed by pancytopenia (115;126). Increased HbF and high MCV are commonly observed (115). Because up to 30 % of patients show no apparent congenital defects and hematological and clinical features are highly variable, clinical diagnosis can be difficult. Therefore, FA should be considered and excluded in all children with hypoplastic cytopenias. The standard screening test for FA is based on the characteristic hypersensitivity of FA cells to cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). At least 12 complementation groups are known to date with FANCA being the most common group; genes for 8 groups have been cloned (FANCA, C, D2, E, F, G, L, and BRCA (127;128). The FA proteins cooperate in a common FA/BRCA pathway responding to DNA damage (124;129).


The median survival of patients with FA is approximately 30 years (112). Treatment with androgens can improve pancytopenia in many patients. HSCT is the only treatment for establishing normal hematopoiesis. Because FA patients are extraordinarily sensitive to the chemo-therapeutic agents and radiation, the dosage of conditioning agents have be reduced to avoid fatal toxicities. HSCT with MFD can result in a cure rate for BM failure of 60% - 90% (130-135), results with UD are less favorable with 30% - 60% survival (132;136-138) Currently, regimens with reduced intensity using fludarabine are being investigated (139). Post HSCT, there is a high incidence of malignancy, particularly cancers of the head and neck. FA patients are at increased risk for hematological and non-hematological malignancies (112). The cumulative incidence for myeloid neoplasia at the age of 40 years is 33% (113). In about one third of patients, the initial diagnosis of FA is established at the time of myeloid neoplasia, without an apparent preceding AA (115). Consequently, FA has to be excluded in all patients with MDS (excluding some cases of therapy-related MDS). As outlined above, the morphological diagnosis of MDS in FA is difficult in the absence of an increased blast percentage and should be restricted to cases with increasing cellularity and ineffective hematopoiesis. Despite these morphological limitations it is believed that most cases of myeloid leukemia in FA are preceded by an MDS phase (115). With the underlying chromosomal instability cytogenetic abnormalities are frequently observed. The relationship between fluctuating cytogenetic clones and progression to myeloid neoplasia is not always clear. The most common aberrations are monosomy 7 and trisomy 1 (140). Gains of the chromosomal segment 3q26q29 have been shown to be an adverse risk factor (141). Although only HSCT offers the potential for cure in patients with FA and myeloid neoplasia, the survival is generally poor. Dyskeratosis congenita DC is an inherited disease characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia (116). The median age for the onset of mucocutaneous abnormalities is 6-8 years and nail change occurs first. Dental, gastrointestinal, genitourinary, neurological, ophthalmic, pulmonary and skeletal abnormalities have also been reported. DC is caused by inherited defects in the telomerase complex. Autosomal dominant DC (10% of patients) is associated with mutations in the RNA component of telomerase, hTERC, while X-linked DC is due to mutations in the gene encoding dyskerin, a protein implicated in both telomerase function and ribosomal RNA processing (142). Immunological abnormalities can occur in a subgroup of patients, and fatal infection can be attributable to immunodeficiency rather than AA (143). BM failure is the major cause of early mortality; the median age for the onset of pancytopenia is 10 years. By age 40 years, 90% of DC patients have at least a single cytopenia, 50% have pancytopenia (116). In a number of cases AA preceded the onset of abnormal skin, dystrophic nails, or leukoplakia. HSCT for pancytopenia is generally less successful than in FA because of fatal pulmonary and vascular complications. In a report from the DC registry, malignancies developed in 13 (8.8%) out of the 148 patients, including 4 cases of MDS, which developed at age 10, 22, 27 and 29 years (116). Severe congenital neutropenia SCN (Kostmann´s Syndrome) is a CBMF disorder characterized by severe neutropenia. It is associated with a maturation arrest at the promyelocyte/myelocyte level in most cases (117). The majority of SCN patients respond to G-CSF with increasing numbers of neutrophils and absence of infections. Most SCN patients have inherited a heterozygous mutation in the gene encoding neutrophil elastase (144).


SCN patients are at risk for developing MDS/AML regardless of presence or absence of G-CSF therapy. According to data from the SCN International Registry (SCHIR) the cumulative incidence of myeloid neoplasia is 12 % - 13% without a statistically significant relationship between age at onset of MDS/AML and dose or duration of G-CSF therapy (117). In contrast, a French study group showed that high time-averaged and cumulative G-CSF doses are associated with high risk of progression to MDS/AML (118). Acquisition of a G-CSF receptor point mutation precedes MDS/AML in > 80% of cases. These nonsense mutation result in the truncated C-terminal cytoplasmic region that is crucial for G-CSF induced maturation. SCN patients with an acquired G-CSF receptor mutation have a high risk of developing MDS/AML, time to MDS/AML progression varies from months to years (117;118). Almost all patients with transformed disease show a cytogenetic abnormality such as -7, 7p-, and +21(118), and half have activating RAS mutations. HSCT is indicated for SCN patients without response to G-CSF. In patients with MDS/AML HSCT is the only curative therapy option and should be performed as soon as possible. Like in other forms of high grade MDS, intensive therapy prior to HSCT is not recommended. The outcome of HSCT in patients with SCN and myeloid neoplasia has been significantly inferior to those without an excess blast count (145). Therefore, some investigators advocate HSCT as soon as a clonal cytogenetic abnormality or a G-CSF receptor mutation is detectable. Annual BM examination with cytogenetic and molecular analysis is recommended in all SCN patients. Schwachman-Diamond syndrome SDS is an autosomal recessive disorder characterized by BM failure, exocrine pancreatic insufficiency and short stature (119). It is the second most common cause of inherited pancreatic insufficiency after cystic fibrosis and ordinarily presents in early childhood as failure to thrive. SDS can be associated with skeletal abnormality (metaphyseal chrondro dysplasia mainly affecting the hip), psychomotor retardation, liver and renal dysfunction. SDS is caused by inactivating mutations of the SBDS gene located on chromosome 7 (146). The SBDS gene encodes a 250 amino acid protein of unknown function; indirect genetic evidence suggests that SBDS may be involved in RNA processing. The diagnosis SDS is usually made clinically by exocrine pancreatic insufficiency (low serum trypsinogen and isoamylase, abnormal 72 hour fecal fat content) and characteristic hematological abnormalities. Intermitted (2/3 of patients) or persistent (1/3) neutropenia is the common hematological abnormality in SDS (119). In addition, neutrophils show impaired chemotaxis. About 20 % of patients present with pancytopenia (119;120). Increased HbF is noted in most of patients. While pancreatic insufficiency will improve later in life in about half of the patients, BM failure persists and clonal disease may develop. MDS is documented in 5% - 33% of SDS patients, but the lifetime risk of MDS/AML is still unknown (120;147). Chromosome abnormalities can be detected in 7% - 29% of SDS patients. Isochromosome 7q is a fairly specific change in SDS that may be related to the mutant SBDS gene on 7q11. The clinical course of SDS patients with abnormal cytogenetics is highly variable, and stable disease or disappearance of the abnormal clone have been observed in some patients (147). Therefore, some investigators recommend a watch and wait strategy with careful monitoring for SDS patients with chromosome abnormality and stable disease. Signs of disease progression like increase in blasts, recurrent infections, transfusion dependency etc should prompt HSCT (147). Supportive care, pancreatic enzyme replacement and G-CSF for severe neutropenia are the standard of care for SDS. HSCT is indicated in patients with severe pancytopenia or MDS/AML. Recently, the EBMT reported on 21 patients, who underwent HSCT for BM failure (n=13), MDS/AML (n=4),


or unknown reason (n=4). Overall survival was 65% (76% in patients with BM failure, 25% in MDS/AML) (148). Diamond-Blackfan anemia DBA is a congenital hypoplastic pure red cell aplasia generally diagnosed within the first year of life (121;149). In about 40% of patients with DBA diverse physical abnormalities are noted. Patients with DBA show features characteristic of fetal hematopoiesis, including persistent macrocytosis, elevated fetal hemoglobin and erythrocyte deaminase (eADA) level. DBA patients can develop neutropenia or thrombocytopenia, although infants often display thrombocytosis at diagnosis (149). The majority of patients with DBA respond to steroid therapy, non-responders will have to undergo transfusion therapy. Recent molecular studies have identified mutations in the gene encoding the ribosomal protein RPS19 on chromosome 19 in 25% of patients with DBA. In another subset of patients, linkage analysis has identified another locus on chromosome 8p in association with DBA (150). There are, however, other cases of DBA that are linked neither to the RPS19 gene nor to the locus on 8p, implying the involvement of undefined genetic defects in the cause of DBA (150). There are no genotype-phenotype correlations. DBA carries a preposition for malignancy. A review of the literature reveals 10 cases of hematological malignancy and 19 case of other malignancy (5 of them are osteogenic sarcoma) (122). Data from the DBA registries of North America and Europe indicate a incidence of frequency of malignancy of 1.3% -1.7% (121) (149). In contrast, in a single center study from Boston with the cumulative risk for the development of MDS/AML at age 30-40 years was 23% (123). Current guidelines for therapy of MDS in congenital bone marrow failure disorders Only HSCT offers cure for patients with CBMF disorders and MDS. Indications for HSCT differ between disorders. In FA, DC and possibly SDS, the underlying genetic defect will exclude treatment with standard myeloablative preparative regimens like those applied for primary high grade MDS. Treatment should be in accordance with the recommendations of the respective National/International Studies on that particular CBMF disorder. Patients with CBMF and MDS should also be registered in EWOG-MDS 2005. 2.5.6.2 MDS in acquired bone marrow failure disorders MDS after acquired aplastic anemia The prognosis of severe AA (SAA) has dramatically been improved by HSCT and IST. Although IST with ATG and CSA allows recovery of autologous hematopoiesis in 70-80% of children (151-153), patients remain at risk for clonal disease. Previous reports show that in the absence of HSCT the cumulative incidence of myeloid neoplasia is 10% - 16% (94;151;154-161). A similar incidence has been reported for hepatitis associated SAA(162). In contrast, MDS/AML after HSCT is not observed. Because the distinction between hypoplastic RC and aplastic anemia has been controversial (see 2.1.4.2 Morphology), one may assume that incorrect diagnoses may increase the incidence of subsequent clonal disorders in SAA. The initial diagnosis of SAA has to be questioned when secondary MDS/AML is diagnosed within a few months after presentation. The median interval from the diagnosis of SAA to the onset of MDS/AML is 37-56 months. Risk factors for clonal disease in SAA are not well clarified, but some reports showed that older age, splenectomy, multiple courses of IST, duration of G-CSF therapy, and unresponsiveness to IST are related with high risk of clonal evolution (94;154;163).


In some series of AA patients, cytogenetic studies at the time of diagnosis have demonstrated clonal abnormalities in 4% -12% patients (164;165). Numeric aberrations (trisomy 8, trisomy 6 and monosomy 7) were the most frequent abnormalities observed. When conventional cytogenetics is unsuccessful, FISH analysis for monosomy 7 and trisomy 8 may be helpful. Patients with trisomy 8 often respond to IST, and the prognosis is favorable. In contrast, monosomy 7 generally implies a poor prognosis with a high likelihood of transformation to leukemia. Currently there is no consensus as to whether certain chromosome abnormalities such as trisomy 8 or monosomy 7 exclude the diagnosis SAA. In cases of clearly secondary MDS/AML, abnormalities of chromosome 7 are most commonly detected (94;151). Current guidelines for therapy of MDS after acquired bone marrow failure disorders HSCT without prior intensive chemotherapy is recommended as soon as the diagnosis is established. Current preparative regimens of EWOG-MDS utilize a preparative regimen consisting of busulfan, cyclophosphamid, and melphalan.


3. Study Objectives 3.1 Rationale of the study The aim of the study is to improve the accuracy of diagnosis for children and adolescents with MDS by a standardized review of morphology and standardized cytogenetic and molecular analyses. 3.2 Primary Objectives The primary objectives of the study are: - To evaluate the frequency of the different subtypes of MDS in childhood and adolescence by

a standardized diagnostic approach - To evaluate the frequency of cytogenetic and molecular abnormalities:

Specifically using array-CGH to evaluate the frequency of subtle chromosomal imbalances, i.e. gains and losses of defined chromosomal regions, and amplifications. Specifically using mFISH to identify unknown chromosomal aberrations, particularly subtle translocations involving new candidate genes, and to better define chromosomal breakpoints.

3.3 Secondary Objectives The secondary objectives of the study are: - To assess survival for children and adolescents with MDS and JMML - To evaluate relapse rate, morbidity and mortality in children with MDS and JMML treated by

HSCT.


4. Investigational Plan 4.1 Study Design This is a prospective, non-randomized, multicenter study. Around 260 patients will be enrolled. Patients who are identified as being eligible according to the Inclusion and Exclusion Criteria will enter the study. The study will take place at centers in 12 European countries. The final analysis of the trial will be performed at the beginning of 2011. Based on the assumption of a recruitment of 65-70 patients per year, the total study duration will be approximately 5 years. Timetable: Start of Study: 01.01.2006 Enrollment: 48 months End of Study: Fourth quarter 2010 Data available: First quarter 2011 Study report: Second quarter 2011 4.2 Participating centers The center of Freiburg, Germany, is the Coordinating Study Center (CSC). Patients for this study are recruited in European centers which are located in the following countries: Austria, Czech Republic, Denmark, Finland, Germany, Iceland, Italy, the Netherlands, Norway, Poland, Sweden and Switzerland. Centers and the locally responsible investigators are listed in Appendix 1. All study centers are tertiary care centers with sufficient experience in clinical trials in pediatric oncology. In every country a Regional Coordinator is responsible for forwarding the national data to the Coordinating Study Center (CSC). For the Nordic Countries (Denmark, Finland, Iceland, Norway, Sweden) there is only one EWOG-MDS Regional Coordinator. The EWOG-MDS Regional Coordinators are solely responsible for conducting the study in their country/study group. They assure that diagnostic BM and PB samples of all study patients are evaluated by a member of the EWOG-MDS Morphology Board. Cytogenetic and molecular studies as well as immunophenotyping are generally performed in national reference laboratories and are evaluated by members of the EWOG-MDS Committee on Molecular Genetics and the Regional Coordinators. Data are collected regionally by the Regional Coordinator and transferred on a 3-monthly schedule to the Coordinating Study Center (CSC) in Freiburg, Germany. 4.3 Number of patients Approximately 260 patients are expected to enter the study during the study period. 4.4 Central and Reference Laboratories Every region has its own national reference laboratories for pathology and oncogenetic studies. They are listed in the section 1.


5. Study Population 5.1 Study Population Patients will only be allowed to enter the trial if they or their caretakers provide written informed consent about their participation (following full explanation of the trial) and if the physician has verified that the patient meets all of the Inclusion Criteria and none of the Exclusion Criteria. 5.2 Inclusion Criteria Patients enrolled in this study are to meet the following Inclusion Criteria:

• Confirmed diagnosis of MDS or JMML (morphology, cytogenetics) • Myeloid leukemia of Down syndrome (patients aged > 6 years). • Age: age less than 18 years

The caretakers will have given their written informed consent to participate in the study. Consent will be documented by the caretaker's dated signature which will be also signed and dated by the investigator in the participating center. If the patient is able to understand the meaning and consequences of the study and its procedures his/her written informed assent is also needed. Written informed consent has to be obtained prior to enrollment into the study. 5.3 Exclusion Criteria Patients who do not fulfill the Inclusion Criteria may not be included into study. Specific Exclusion Criteria are:

• Denied informed consent and/or assent by caretakers/patient. • Fanconi anemia (diagnosed by chromosomal breakage, G2 cell cycle arrest, Western blot or

mutational analysis) or other congenital bone marrow failure disorders (diagnosed clinically or by disease specific germ line mutations) without secondary MDS. Secondary MDS in congenital bone marrow failure is defined by a consistent acquired bone marrow abnormality as a) increase in blasts b) acquired consistent chromosomal abnormality c) increasing bone marrow cellularity in the presence of blood pancytopenia

• Shwachman syndrome or Fanconi anemia with a single aberration not typical of MDS. • Translocation characteristic for de novo AML like

t(8;21)(q22;q22) [AML1/ETO fusion gene] t(15,17)(q22;q12) [PML/RARα rearrangement] inv(16)(p13q22) [CBFβ/MYH11rearrangement]

• Myeloid leukemia of Down syndrome (patients aged < 6 years). • Participation in another interventional study within the last 4 weeks (except for therapy

optimizing studies in cancer or bone marrow failure, diagnostic protocols).


6. Enrollment and Patient Registration 6.1 Time of enrollment It is planned to start enrollment 01.01.2006. Enrollment is planned to be finished 31.12.2009. 6.2 Minimal requirements for enrollment The following minimal requirements have to be fulfilled before a patient can be registered in the study:

− Patient identification by name and sex − Date of birth − Date of diagnosis − White blood cell count − Bone marrow with differential count − Peripheral blood with differential count

6.3 Mode of enrollment The caring physician has to receive the written informed consent of the patient before any study specific examination. The Regional Coordinator investigator will register the patient on a patient identification list located at the regional coordinating center. The Regional investigator has to record the following information about the patient on the patient identification list: – full name – date of birth – gender – Inclusion and Exclusion Criteria of the study fulfilled yes/no The patient receives a consecutive patient identification number which is given by the Coordinating Study Center (CSC) in Freiburg. This patient identification number consists of 2 letters, 3 digits and an extra code number:

1. The first two letters are the country code. 2. The next three digits stand for successively included patients. As the patients have been

registered to the MDS-study for many years, the patient number will continue in that region as a successive number.

3. All patients recruited from 1st May 2006 receive as extra coding the digit “_06” to mark them as belonging to the EWOG-MDS study 2006.


7. Methodology 7.1 Study Schedule A patient with suspected MDS undergoes the diagnostic procedures as outlined in section 7.3.1. Once entered the study and assigned to a defined subgroup a defined data set is recorded for each patient every 12 months for minimum of 1 and a maximum of 5 years. 7.2 Written Informed Consent A written Informed Consent will be obtained from caretakers and possibly also patients prior to participation to the study. 7.3 Patient Evaluation Every patient undergoes a specific diagnostic evaluation. In regular time intervals (at least 12 months) follow-up information is requested. The diagnostic procedures are part of the routine examinations. No additional punctures are performed for the study. For study purposes 5 ml of peripheral blood and 5 ml of bone marrow are collected at the same timepoints of routine examinations. 7.3.1 Initial Diagnostic Procedures The investigator will record the following information and will perform the following diagnostic evaluations: 7.3.1.1 Demographic Assessments: ♦ Inclusion/Exclusion Criteria: Compliance with Inclusion and Exclusion Criteria must be verified

and recorded on the source documents and in the CRF prior to enrollment of the patient into the study.

♦ Demography: date of birth, sex. ♦ Medical History: complete past and current medical conditions (including neurofibromatosis) ♦ Family history ♦ Transfusion history (yes/no) ♦ Disease Details ♦ Concomitant Medication/ Therapy: steroids, immunoglobulins, growth factors, immuno-

suppression, chemo- and radiotherapy 7.3.1.2 Physical Examination: ♦ Height (cm), weight (Kg), head circumference (cm) with percentiles respectively ♦ Birth weight ♦ Congenital abnormalities, xanthomas, café au lait spots, lymphadenopathy, hepatosplenomegaly,

cranial nerve palsy, respiratory tract symptoms; signs of neurofibromatosis type I 7.3.1.3 Screening Laboratory Tests: ♦ Hematology (hemoglobin, hematocrit, MCV, reticulocyte count, CBC with differential).

Hemoglobin electrophoresis (Hb A2, Hb F) ♦ Serum Chemistry: LDH ♦ Direct and indirect Coombs-test ♦ Serology (IgG, IgM): EBV, CMV, HHV-6, HSV, Parvovirus B19, HBV, HCV, HIV 7.3.1.4 Special Examinations: All MDS and JMML:


♦ Evaluation of morphology on PB smear, BM aspirate and BM biopsy (BM biopsy optional for JMML)

♦ Standard Cytogenetics ♦ FISH analysis for complex karyotypes ♦ Comparative hybridization Refractory Cytopenia: Diagnostic Procedures ♦ Second bone marrow biopsy mandatory (reference pathology) within 3 months prior to any

therapy; for transfusion-dependent or neutropenic patients consider second biopsy within 2 weeks

♦ Exclusion of Fanconi anemia by G2 cell cycle arrest, chromosomal breakage test, Western blot or mutational analysis

♦ Exclusion of pancreatic insufficiency: Stool elastase or serum trypsinogene and serum isoamylase 1. PNH clone (for details see EWOG-MDS RC IST 2005 protocol) Differential diagnosis in refractory cytopenia: diagnostic procedures to consider if reasonable 2. Viral screening: PCR for CMV, EBV, parvovirus, HHV 6 in bone marrow and peripheral blood 3. Screening for vitamin deficiencies: folinic acid, vitamin B 12, copper, iron 4. Exclusion of Shwachman syndrome: stool elastase, transaminases 5. Exclusion of Pearson syndrome ♦ Screening for metabolic diseases: venous blood gases, screening for urinary organic acids,

lactate, glucose ♦ Exclusion of immunological disorders: complement factors, subpopulations of lymphocytes,

direct and indirect antibodies against surface of neutrophils, organ-specific and non-organ-specific auto-antibodies

JMML: Diagnostic Procedures ♦ Buccal swab or skin biopsy for germline DNA ♦ PTPN11 / RAS mutation (somatic and germline)

For patients without any of the above mutations or monosomy 7 but with the typical clinical and morphological picture of JMML the following examinations have to be performed:

♦ BCR/ABL rearrangement (Philadelphia chromosome) ♦ Colony assay: spontaneous growth or GM-CSF hypersensitivity High Grade MDS: Diagnostic Procedures ♦ Second bone marrow biopsy. ♦ Exclusion of Fanconi anemia by G2 cell cycle arrest, chromosomal breakage test, Western blot or

mutational analysis ♦ Diagnostic lumbar puncture According to the obtained results patients will be assigned to one of the following groups: 1.1) JMML 1.2) JMML-like picture in Noonan-Syndrome 2) MDS 2.1) Refractory cytopenia 2.2) MDS with increased blast count 2.3) Secondary MDS


7.3.3 Laboratory Tests/Special Examinations During Study Period For each patient the follow-up data is recorded at 12 month intervals for the whole duration of the study (minimum one year, maximum 5 years). 7.3.3.1 Patients Receiving Chemotherapy or No Therapy The following data is recorded for this subgroup of patients:

− Treatment other than AML-therapy: weight, length; begin/end/ongoing therapy, drug, dosage − Splenectomy (date), splenic irradiation − AML-therapy, remission − Hematological data:

Every bone marrow examination along with peripheral blood counts. In the absence of a bone marrow examination the first peripheral blood count indicating progress. In the absence of progress the last peripheral blood count.

o complete blood count with differential (including reticulocytes), transfusion history o bone marrow with differential

− Cytogenetics: Every analysis before HSCT (metaphase and FISH)

− Date of last examination − Karnofsky score − Survival: stable disease, complete remission (CR), relapse (date, site, kind of relapse) − Secondary malignancy (date of diagnosis, diagnosis) − Death (date, cause)

7.3.3.2 Patients Undergoing HSCT For patients with HSCT the recorded data corresponds to the EBMT-follow-up but adding MDS-specific topics.

− Initial data at transplantation until day 100: o Pre-Transplant Treatment: subclassification at primary treatment, therapy, complete

remission o At Transplantation: clinical features, subclassification at transplantation, status of

disease, bone marrow investigation, cytogenetics, hematological values before start of conditioning regimen

o Allograft data: patient, donor, graft manipulation, conditioning, transplantation, engraftment, acute GvHD

o Status at day 100: best response of disease; complications within the first 100 days (infection/ non infection related); additional treatment post-transplant; donor leukocyte infusion (DLI)

− Follow-up year one: o Complications after day 100: infection/ non infection related, chimerism studies,

immunosuppressive therapy, chronic GvHD (onset, treatment, resolution), relapse, treatment of relapse, follow-up (disease status, death)

− Follow-up yearly from year two: o complications (infection/ non infection related); events sine last follow-up (chronic

GvHD, chimerism, first relapse or progression after transplant, secondary malignancy, death); disease status


7.4 Duration of Study Participation All patients who have entered the study are followed for a maximum of 5 years according to this protocol. They will most probably be followed for a longer period according to a study following in 2010. There are no criteria for planned drop-outs. For patients lost to follow-up the last known data should be retrieved and recorded. 7.5 Use of Patients’ Material Peripheral blood, bone marrow aspirate, bone marrow biopsies and fibroblasts are retrieved from the patient for diagnostic procedures. This material may be partially stored for study purpose. A separate consent has to be obtained from the patient/ caretakers if this material is stored for later research work. Every patient/ caretaker may state if he wants to be informed about the research results. No patient is undergoing an additional invasive procedure just to gain material for research. It has to be stated that the material will only be used for doing research on the disease and that genetic analysis will only concern the biology of the disease. The research is not commercial. The material is stored nationally. Material will be sent to other laboratories anonymously. The right to perform analysis lies within the national coordinator’s research group. 7.6 Asservation of Patients’ Material At diagnosis, prior to HSCT and at relapse material form peripheral blood and bone marrow will be retrieved and stored for research purposes. If there are other diagnostic bone marrow examinations at other time points (prior to HSCT), the material will be handled the same way. The following material will be retrieved:

• 8 smears from PB • 8 smears from BM • at least 5 ml of heparinized PB • at least 5 ml of heparinized BM

The material will be stored as follows:

• Smears from PB and BM will be frozen at -80 °C. • Cells in PB and BM will be separated by a Ficoll procedure, mononuclear cells (MNC) will

be frozen according to standard procedure • DNA and RNA from MNC will be extracted according to standard procedure and stored

adequately • DNA from granulocytes in the Ficoll pellet will be extracted after red cell lysis and stored

adequately To obtain germ-line DNA a buccal swab is to be obtained from all patients with JMML at diagnosis. Prior to HSCT a skin biopsy is recommended to allow for culturing of fibroblasts. Optimally, the biopsy is taken when general anesthesia is required for therapeutic purposes (e.g. with central line placement). Cytogenetic material is stored at the reference laboratory. Material should be stored at the attention of the regional coordinator. Details concerning laboratory procedures can be found in the study manual.


8. Data Handling and Reporting 8.1 Reporting and Recording of Data Follow-up information is asked for on a 12 month basis. Data is requested every four months. The follow-up list with the due patients will be sent automatically from the Coordinating Study Center (CSC) in Freiburg to the Regional Coordinator. The Regional Coordinator will stay in contact with the local center and will return the completed form within 3 months. HSCT patients will be documented the same way as non-HSCT patients. Once a year, the Coordinating Study Center (CSC) in Freiburg will provide the Regional Coordinators with the data base of their respective national data. All protocol-required information collected during the study must be entered and signed by the investigator, or designated representative in the CRF. The data has to be complete, clear, accurate, legible, and plausible. Missing examinations or data have to be marked along with a justification/explanation. Documentation of data on any study paper forms should be made with a black pen and well readable note. Potential corrections are to be made by the investigator or an authorized person (according to the centers' signature form). Corrections are to be made according to the GCP-guidelines, i.e.: • the version that has to be corrected will be crossed in a way that it is still readable • the correct version will be written above or beside the first version • the correction (or any remark) will be marked with date, initials, and a justification by the

investigator or an authorized person. 8.2 Data Management and Handling Once the diagnosis of MDS and JMML is confirmed, the registration form is completed by the local center and sent to the regional Coordinator. The Regional Coordinator is obliged to register the patient within 3 months of diagnosis to the Coordinating Study Center (CSC) in Freiburg. The data will be transferred into the target data base at the CSC. Data will only be entered in the data base by advice of the regional coordinator. Advices are:

1. completed MDS forms 2. information about events, status of the patient sent by mail or email

The CSC will not enter any information received by local centers. For yearly follow ups the Coordinating Study Center (CSC) will contact the regional coordinator in January. For state of the art the data manager will send a list of all patients diagnosed since 01.07.98 (start of EWOG-MDS) by email. On this list the patients who need an actual FUP form (clinical course, event) will be marked. HSCT patients will be documented the same way as non-HSCT patients. For security reasons the email will be sent with a password-based data protection. Although the CSC would appreciate to receive the completed forms until the end of March, forms can be sent at any time. To inform the national coordinators about changes in the national data the CSC will send an email every 4 months (January, May, Sepember). This email will include:

1. Patient’s name, birth date, presumptive diagnosis for material that has been sent to Freiburg for in vitro or molecular studies

2. Events the CSC heard of by email from local centers 3. Forms the CSC received within the last 4 months


9. Quality assurance In the framework of the clinical trial quality-control and quality-assurance will be guaranteed by a data supervision board a steering committee and an authorized supervision. 9.1 Data Supervision Board The Data Supervision Board consists of all Regional Coordinators and two independent members. Their duty is to supervise the Coordinating Study Center (CSC) at least every two years. Correct data handling from the CRF to the database is their main area of monitoring. They will have access to all study documents including the trial master file and the standard operating procedures. 9.2 Steering Committee All Regional Coordinators are part of the Steering Committee in their role as principal investigators for their countries/ regions. The Committee meets once a year in one of the member countries. Study problems and interim data and analysis are discussed. The Committee guarantees the scientific value and actuality of the study. 9.3 Authorized Supervision During the course of the study, the study assistant located at the Coordinating Study Center (CSC) will have the duty of an authorized supervisor together with the study coordinator and the principal investigator. The authorized supervisor will stay in regular contact with the study centers to get information about the compliance with the study protocol requirements, consensus of the data in the CRF and the originals, the updated patient identification lists, and the archiving system. The contacts will be mostly done by e-mail and telephone and are supposed to control the progress of the trial, realize problems early and potentially solve them. The authorized supervisor will review the case report forms of the patients in the study to make certain that the items have been completed and that the data provided are plausible and obtained in the manner specified in the protocol. The authorized supervisor signs to handle all data that are under professional secrecy or show the patient’s identity confidentially and will use the data only for the purpose the patient gave informed consent for. No data disclosing the identity of patients should leave the study center as a result of the monitoring procedure. 9.4 Data Verification There will not be a source data verification in the sense of controlling the recorded data of the CRFs in regard to correctness and completeness compared to the original data. There will be an inherent plausibility check contained in the principal data set. If primary data is suspected to be incorrect a query form is going to be generated. The local investigator has to respond to the query and provide written information as soon as possible. The authorized supervisor will also generate written queries if data entered into the data base does not seem plausible. A query trail will document all changes to the data set. 9.5 Auditing Procedures In addition to the quality assurance procedures outlined above, audits can be done in the framework of the auditing system according to the ICH-GCP-guidelines. It can be an inspection initialized by authorities (even after the study has been completed). In the context of an audit it will be checked if planning, conduction and analysis of a clinical trial are in agreement with the law and the requirements of the ICH-GCP-guidelines. This includes controlling of the data keeping and organization of the study center as well as controlling of laboratories and the original documents. The aim of auditing is to assure that all results and conclusions written in the final report can be drawn from the raw data.


All persons who are auditing are obliged to sign to handle data that are professional secrecy or show the patient’s identity confidentially and use the data only for the purpose the patient gave informed consent for. The investigator will be informed in time about planned audits. The investigator is required to inform the Coordinating Study Center (CSC) immediately of an inspection requested by a regulatory authority.


10. Statistics 10.1 Trial design This is a prospective, non-randomized, multicenter study. The aim of the study is to improve the accuracy of diagnosis for children and adolescents with MDS by a standardized review of morphology and standardized cytogenetic and molecular analyses. The primary and secondary objectives are defined in chapters 3.2/ 3.3. 10.2 Patients included in the analyses Based on the assumption of a recruitment of 65-70 patients per year and a recruitment time of 5 years around 260 patients will be enrolled. Patients who are identified as being eligible according to the Inclusion and Exclusion Criteria will enter the study (see 5.2). The study will take place at centers in 12 European countries. The final analysis of the trial will be performed in autumn 2010. 10.3 Statistical analysis The final analysis of the EWOG-MDS 2005 study will be performed within six months after the end of the study. One Interim analysis two years after study start is planned. Overall survival (OS) is defined as the probability of survival from date of diagnosis or the beginning of treatment with only death as event; children alive were censored at their last follow-up. Event-free survival (EFS) is defined as the probability of survival following treatment without any events including disease progression, relapse or death of any cause. Graft rejection or failure were considered events. Survival times will be calculated according to the Kaplan-Meier method and comparisons between probabilities in different patient groups will be performed using the log-rank test. Relapse incidence (RI) is defined as the probability of having a relapse before time t; death without experiencing a relapse is considered a competing event. On the contrary, transplant-related mortality (TRM) is defined as the probability of dying without previous occurrence of a relapse, which was the competing event. Both these probabilities will be estimated as cumulative incidence curves. All results will be expressed as 5-year probability or 5-year cumulative incidence (%) and 95% confidence interval (95% CI). Univariate analyses of EFS, RI and TRM will be performed. For multivariate analyses, the Cox proportional hazard regression model will be used, including in the models all the variables with P < 0.1 in univariate analysis. Statistical analyses will be done using the statistical software SPSS (Statistical Package for Social Sciences) and SAS (Statistical Analysis System). All analyses will be documented and saved. The transfer of the data from the database hold in the Coordinating Study Center (CSC) will be performed after checking the data for plausibility. The procedures of later transformation of data during the statistical analysis will be validated by check of random samples. The statistical analysis will be done under the supervision of the responsible statistician.


11. Conditions for protocol amendments 11.1 Changes in Protocol Any change or addition to this protocol requires a written protocol amendment. No change to the protocol may be made without the joint agreement of the Principal Investigator and the Regional Coordinators. Any amendment has to be signed by all parties before the change of or addition to the final protocol is effective. If an amendment significantly affects the safety of the patients, the scope of the investigation or the scientific quality of the study, it should be formally approved by the Ethics Committee, and communicated to the regulatory authority, as required by local law. After approval, an amendment becomes an integral part of the protocol. All Regional Coordinators will be informed immediately after approval by the Coordinating Study Center (CSC) in Freiburg. The Principal Investigator is authorized to decide the discontinuation of the study due to relevant medical or administrative reasons. The above-mentioned requirements do not preclude any immediate action taken by the investigator in the interests of the patient’s safety. In the case where such an immediate change to the protocol is implemented and the principal investigator should be notified immediately.


12. Ethical and Legal Considerations The study will be conducted in accordance with the Declaration of Helsinki (Appendix 2), the current revision of ICH Topic E6 (Appendix 3), Guideline for GCP: "Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95), and the legal requirements of each participating country in its valid version. It is mandatory that all considerations regarding the protection of the patients be carried out in accordance with the Declaration of Helsinki. The data protection will be granted according to the local law. To ensure compliance the investigator agrees, by written consent to this protocol, to fully cooperate with compliance checks by allowing access to all documentation by authorized individuals. 12.1 Patient Information and Informed Consent All patients must sign and personally date an approved Informed Consent Form after receiving detailed written and verbal information about the reason, the nature and the methods of the study. The information comprises also information about the patient insurance and the conditions subsequent to this policy. The Informed Consent complies with regulatory requirements. The written informed consent must be obtained before the entry of the patient into the study! Furthermore, the patient must be notified that participation is voluntary and that he/she may withdraw from the study at any time and that withdrawal of consent will not affect his/her right to the most appropriate medical treatment or affect the doctor/patient relationship. A written patient information leaflet will be handed to the patient, whose contents have to be discussed with the patient by the investigator. The investigator will provide the patient ample time and opportunity to inquire about details of the study and to decide whether or not to participate in the study. All questions about the trial will be answered to the satisfaction of the patient. The patient should be given sufficient time to read and understand the statement him/herself before signing his/her consent and dating the document. Neither the investigator nor the trial staff will coerce or unduly influence a patient to participate or to continue to participate in the trial. Personal information will be treated as strictly confidential and will not be publicly available. The patient will receive a copy of the written informed consent once signed, and the original version of the informed consent has to be kept in the investigator file. 12.1.1 Patient Withdrawal A patient may withdraw from the study at any time, at his or her own request, for any reason, specified or unspecified, and without penalty or loss of benefits to which the patient is otherwise entitled. Patients who are withdrawn from the study will not be allowed to re-enter later. Date of discontinuance, all recorded results at this time and, if known the reasons for discontinuance are to be documented in the CRF. If possible a final examination has to be done. 12.1.2 Premature discontinuation of the study by the investigator The responsible investigator has the right to exclude patients from the study if the diagnosis of MDS/JMML turns out to be wrong. Centers may be excluded from the study if there is no appropriate data documentation or data transmission. 12.2 Disclosure and Confidentiality Throughout this study all data will be treated confidentially. Throughout the whole data-recording and -analysis patients will be identified only by a patient identification number and medication number - never by their full name, initials and date of birth. The legal provisions by the respective Laws will be heeded. The investigator is responsible for keeping sufficient information for every patient (name, date of birth, internal clinic number, patient identification number, gender, informed consent), in order to


identify the patient. According to the ICH-GCP-guidelines these documents (Patient Identification List) have to be archived for at least 15 years. By conducting this study, the investigator agrees that he and his staff will maintain all information in strict confidence. The investigator is requested to insist on similar confidentiality for this information from other bodies such as the Hospital Scientific Committees and Ethic Committees/Institutional Review Boards that have been consulted by the investigator. Study documents provided by (protocols, CRFs and other material) will be stored appropriately to further ensure their confidentiality. It is understood that the confidential information provided to the investigator will not be disclosed to others without direct written authorization from the patient. Such information will not be communicated by telephone to potential or enrolled patients or to any other individual. The evaluation of the study will be done by the Coordinating Study Center (CSC) in Freiburg. 12.3 Independent Ethics Committee (IEC) / Institutional Review Board Prior to implementation of this study, the protocol, Patient Information Sheet and the proposed Informed Consent must be reviewed and approved by the Ethics Committee. Signed and dated approval by the Ethics Committee must be obtained by prior to study initiation and patient enrollment. The investigator is committed in accordance with local requirements to inform the IEC of any emergent problem and/or protocol amendments. 12.4 General Disclosure Duty Before starting the clinical trial authorization from the national authorities has to be obtained. All regional authorities will be informed. According to the law of participating centers in other countries the corresponding authorities will be informed correctly as well. 12.5 Insurance The aim of this study is the collection of epidemiological data based on a standardized diagnostic approach and not the investigation of clinical or pharmacological properties of drugs. The study is therefore exempt from clinical trials insurance coverage according to law. Patients are covered by the public liability insurance of their hospitals.


13. Study Documents and Archiving of Records 13.1 Investigator’s File The investigator’s file contains all essential and relevant documents (e.g. regulatory and study documents, correspondence with ethics committee and general information). The investigator’s file has to be accessible during audits and authorized inspections. After finishing the trial the investigator’s file has to be kept within the study center according to the ICH-GCP-guidelines and legal regulations at least for 15 years. 13.2 Documentation of Patient Data 13.2.1 Case Report Form (CRF) The investigator or his representatives (according to the signature form) document the data currently and continuously on the trial relevant CRF. If possible, documentation should be made immediately. No section of the CRF is to be left blank without an appropriate explanation by the Investigator. All data have to be entered in an accurate, plausible and complete way by the investigator or a person authorized by him. 13.2.2 Documentation of data in the patient’s file The investigator documents in the patient’s file the participation to the study, the frequency of the trial visits, all relevant data of disease, all examinations and diagnostic evaluations and concomitant treatment. 13.2.3 Patient Identification List According to the ICH-GCP-guidelines the investigator has to keep a patient identification list which allows an accurate relation of the patient’s identity to his/her enrollment into the trial. The following information will be recorded on the patient identification list: – full name – date of birth – gender – Inclusion and Exclusion Criteria of the study fulfilled yes/no 13.3 Archiving of Records According to the German law, all investigational records must be retained at the investigational site for a minimum of 15 years. Patient files and other source documents must be kept for the maximum period of time permitted by the hospital/institution, but for not less than 15 years. Originals of all documentation and copies of outgoing correspondence concerning the study will be stored and retained in a safe area in the Master File at the Coordinating Study Center (CSC) in Freiburg.


14. Administrative Considerations 14.1 Financing The study is supported by public grants. There is no support from the industry. The main supporter in Germany is the Deutsche Krebshilfe. In every country the study is supported by national public grants. 14.2 Final Report The Coordinating Study Center (CSC) in Freiburg will perform the statistical analysis and present a written statistical and medical report. The final report of the trial will be written by the Coordinating Study Center (CSC) in Freiburg in cooperation with the Regional Coordinator. Except for cogent reasons nobody will pass on data to third persons unless all parties agreed upon the analysis and interpretation of the results. 14.3 Publication of Study Results Any formal presentation or publication of data collected as a direct or indirect result of this trial will be considered as a joint publication by the investigators. It requires the agreement of the Principal Investigator and all Regional Coordinators. Authorship will be determined by mutual agreement. The results of the study may be presented during scientific symposia or published in a scientific journal only after review and written approval by the Principal Investigator and all regional coordinators. Investigators participating in multicenter studies must agree not to engage in presentations based on data gathered individually or by a subgroup of centers before publication of the first main publication, unless this has been agreed otherwise by all other investigators. Every clinical study should be published to avoid the problem of 'Publication Bias'. At least within 1 year of termination of the study, a manuscript for publication has to be jointly finalized.


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16 Protocol Approval

Coordinating Investigator

(LKP)

15.03.2006

Signature Date

Study-Coordinator

(Germany)

15.03.2006

Signature Date

Statistician

15.03.2006

Signature Date

The original of this page is to be filed in the Central Trial Master File


17 Investigator Statement

Regional Coordinator:

Trial center and country:

COMMITTMENTS By signing this document, I agree to conduct the trial as outlined in the protocol and in accordance with the Declaration of Helsinki (Appendix 2) as well as all applicable government regulations and GCP. I declare: 1. I am well qualified by scientific training and experience to conduct investigational studies in the

clinical area of the proposed trial and I am affiliated with a recognized medical school or with an independent institution recognized for its excellence.

2. I shall provide information to all staff members involved in the trial about their obligations as described in this document.

3. I shall submit the protocol, Informed Consent form/Information Sheet and other required documentation to the EC for review and approval.

4. I shall make no changes to the protocol without formal amendment (signed by the principal investigator and submitted to the EC for notification/approval), except when necessary to protect the safety, the rights or welfare of patients. In this last case I will inform the principal investigator of the change.

5. I shall require Informed Consent from each patient prior to enrollment into the study. The Informed Consent shall be documented by use of a written consent form approved by the national authority and the EC.

6. I shall complete the Study's Case Report Form (CRF) in a timely and legible manner. 7. I shall maintain accurate source records (hospital or other institutional records), which will

support the data entered into Case Report Forms and I shall maintain these as specified by the protocol.

8. I shall allow monitoring visits by representatives of the supervising boards as needed. 9. I shall allow any Regulatory Authorities to inspect the facilities and pertinent records at

reasonable times and in a manner which ensure patients confidentiality. Following completion of the study, the data may be considered for reporting at a scientific meeting and/or for publication in a scientific journal. A copy of the manuscript or abstract will be provided to all main regional co-investigators for review before submission to a scientific journal for publication and/or a scientific meeting selection committee for oral or poster presentation. Investigator Signature Date The original of this page is to be filed in the Central Trial Master File. A copy is kept by the Investigator.


Appendix 1 List of participating study centers Zie lijst deelnemende studie centra vermeld op de website van SKION: www.skion.nl

http://www.skion.nl


Appendix 2 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 and the 52nd WMA General Assembly, Edinburgh, Scotland, October 2000 Note of Clarification on Paragraph 29 added by the WMA General Assembly, Washington 2002 Note of Calrification on Paragraph 30 added by the WMA General Assembly, Tokyo 2004 A. INTRODUCTION

1. 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.

2. 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.

3. 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."

4. Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects.

5. 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.

6. 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.

7. In current medical practice and in medical research, most prophylactic, diagnostic and therapeutic procedures involve risks and burdens.


8. 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.

9. 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.

B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH

10. It is the duty of the physician in medical research to protect the life, health, privacy, and dignity of the human subject.

11. 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.

12. 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.

13. 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.

14. 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.

15.

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.


16. 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.

17. 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.

18. 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.

19. 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.

20. The subjects must be volunteers and informed participants in the research project.

21. 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.

22. 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.

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH MEDICAL CARE

28. 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.

29. 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.1

30. 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.2

31. 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.

32. 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.

___________________________ 1 Note of clarification on paragraph 29 of the WMA Declaration of Helsinki The WMA hereby reaffirms its position that extreme care must be taken in making use of a placebo-controlled trial and that in general this methodology should only be used in the asbsence of existing proven therapy. However, a placebo-controlled trial may be ethically acceptable, even if proven therapy is available, under the following circumstances: - Where for compelling and scientifically sound methodological reasons its use is necessary to determine the efficacy or safety of a prophylactic, diagnostic or therapeutic method; or - Where a prophylactic, diagnostic or therapeutic method is being investigated for a minor condition and the patients who receive placebo will not be subject to any additional risk of serious or irreversible harm. All other provisions of the Declaration of Helsinki must be adhered to, especially the need for appropriate ethical and scientific review. 2 Note of clarification on paragraph 30 of the WMA Declaration of Helsinki The WMA hereby reaffirms its position that it is necessary during the study planning process to identify post-trial access by study participants to prophylactic, diagnostic and therapeutic procedures identified as beneficial in the study or access to other appropriate care. Post-trial access arrangements or other care must be described in the study protocol so the ethical review committee may consider such arrangements during its review. 9.10.2004


Appendix 3 CH-GCP-Guidelines http://www.ich.org E6 Good Clinical Practice: Consolidated Guideline E11 Clinical Investigations of Medicinal Products in the Pediatric Population

http://www.ich.org


Appendix 4 CRFs De formulieren zijn verkrijgbaar bij het Centraal Bureau van de SKION, afdeling Datamanagement, tel.: 070 – 367 45 45. www.ewog-mds.org

http://www.ewog-mds.org


Appendix 5 Invoice Forms

EWOG MDS - 2006 Invoice Form for Research Material Prof. Dr. C. Niemeyer University of Freiburg Department of Pediatrics and Adolescent Medicine Division of Pediatric Hematology and Oncology Mathildenstr.1 Tel: + 49- 761- 270 - 4515 (Laboratory) 79106 Freiburg Fax: + 49- 761- 270 - 4616 email: [email protected] Physician: __________________________________________________________________________ Institution: __________________________________________________________________________ Address: __________________________________________________________________________ Email: __________________________________________________________________________ (arrival of sample will be confirmed by email) Phone: ________________________________________________________________________________ Fax: _________________________________________ Patient Name: ____________________________________ First Name:__________________________ Date of Birth: |____|____||____|____||____|____| Presumptive Diagnosis: ___________________________________________________________________ Clinical Signs Splenomegaly o Yes o No Hepatomegaly o Yes o No Lymphadenopathy o Yes o No Additional Findings ___________________________________________________________________ Hematological Findings Hb (g/dl) ________ MCV (fl) ________ WBC (109/L) ________ Platelets (109/L) ________ Reticulocytes (‰) ________ Transfusions within the last 4 weeks o no o yes o Ery. Tx o Plt. Tx Material Sent

o heparinized bone marrow (min. 3-5 ml) Date |____|____||____|____||____|____| (please enclose 1 unstained bone marrow smear)

o heparinized blood (min. 3-5 ml) Date |____|____||____|____||____|____| (please enclose 1 unstained blood smear)

o buccal swab (special indications only) Date |____|____||____|____||____|____| Intended analyses

o JMML: Mutational analysis for RAS and PTPN11 In the absence of a RAS or PTPN11 mutation: GM-CSF hypersensitivity will be tested

o Other: ______________________________________________________________________ Date |____|____||____|____||____|____| Stamp Signature _____________________________ In general, a report will be available within 3 weeks. It will be faxed (provided fax number given) with initials only. The original report will be sent by mail.



Appendix 6 Patient /Parent Information/ Informed Consent

Include the name and address of Local Center Patients’ Information Leaflet Dear Patients, Your treating physician has informed you that you are suspected to suffer from a disease called refractory cytopenia. This disease is a subtype of myelodysplastic syndrome involving blood and bone marrow. You have already been asked to participate in a study called EWOG-MDS 2006. Patients with refractory cytopenia who participate in this study are also asked to participate in a corresponding study called EWOG-MDS RC 06. The following information concerns the study EWOG-MDS RC 06. This information leaflet summarizes the main points of the discussion with your treating physician. When you have understood the principles of the study and when you agree to participate in the study, you will be asked to declare your written consent by signing the “informed consent form”. You alone decide in favour or against participating in the study. Please note that participation in the study is voluntary. Should you decide now or later against participation in the study, this will not lead to disadvantages for you. Please take your time to carefully evaluate the decision and do not hesitate to ask additional questions if necessary. What does this study investigate? This study EWOG-MDS RC 06 is a type of research study called clinical trial. It does not concern your treatment. This study analyses scientific problems independent of treatment. For exact diagnosis, determining the best therapy, blood and bone marrow samples must be taken for different analyses independent of this study RC 06. For this study, small volumes of extra blood and extra bone marrow (5 ml each) will be taken at the same time. For all patients with refractory cytopenia these extra samples will be taken at the time of diagnosis. In patients with refractory cytopenia receiving immunosuppressive therapy, additional material (5 ml blood and 5 ml bone marrow) will be obtained at day 120 and at day 240 after start of therapy. In summary, this means that no additional punctures or biopsies are performed for the study EWOG-MDS RC-06. The extra study samples of blood and bone marrow for this study will be stored at the laboratory of Dr. Marry van Heuvel-Eibrink and her co-workers at the Erasmus Medical School, Rotterdam, the Netherlands. You can also decide whether this material may be stored for future non-commercial research on your disease.. You can always ask for information about the research results. Maar een deel ervan is toch blinded?

Study EWOG-MDS RC 06 Prospective non-randomized multi-center study for TCR Vbeta repertoire and PNH analysis

(Version: November 06, 2006)


In addition, your tissue type (HLA-type) will be determined. This analysis will be performed in a local blood bank. For most patients with refractory cytopenia the analysis of the HLA-type is necessary independent of this study to determine the best form of therapy. For HLA-typing 5-20 ml of blood is necessary depending on blood counts. Blood drawing for HLA-typing can be done at any time with a diagnostic procedure. Why is this study being done? The purposes of this study are to answer the following questions in order to know more on the myelodysplastic diseases and to develop better ways of therapy for them. It is known that a proportion of patients with refractory cytopenia benefits from treatment with immunosuppressive therapy. This study investigates whether the response to immunosuppressive therapy is correlated with the presence of auto-immune abnormalities and which white blood cells subtypes are associated with the occurrence of this auto-immune phenomenon. For the future this would implicate that a prediction can be made which patients would benefit from this treatment. Moreover, we know that in a proportion of patients with refractory cytopenia PNH clones can be found. PNH clones are blood forming cells that lack certain proteins on their cell surface that makes them thereby more vulnerable for attacks of the immune system. It is not known how often these clones are present in children and adolescents with refractory cytopenia . Specific questions are: How often do we find auto-immune abnormalities in children with refractory cytopenia? Which types of blood cells are involved in these auto-immune abnormalities? In children who receive immunosuppressive therapy, how does the response to that therapy correlate with the autoimmune abnormalities tested in the laboratory? How often do we find PNH clones in children with refractory cytopenia? How does the size of these PNH clones change during immunosuppressive therapy? Is there a correlation between the HLA-type and auto-immune abnormalities? How many people will take part in this study? At diagnosis, all patients with refractory cytopenia can be included in the study. Response to immunosuppressive therapy can only be investigated in patients treated with this form of therapy. How will the different centers involved in this European study work together? Like the study EWOG-MDS 2006, in which you are already enrolled, this integrated study is being conducted in 12 European countries. Your locally treating physician is responsible for your care. To participate in this study he will send samples from blood and bone marrow to the laboratory at the Erasmus Medical School in Rotterdam, the Netherlands. Samples will be labelled with your child’s full name. The results of the presence of PNH clones in your blood samples will be transmitted to the Regional Coordinator of this study and to your treating physician, so that follow-up examinations can be planned individually. For the Netherlands, Dr. M. van den Heuvel or Dr. E. Korthof should be notified for registration of the patient in the IPIG database! The results on autoimmunity will be kept blinded and not transmitted to your physician or the regional coordinator. Instead they will be correlated with the course of the disease at the time of completion of this study. For this purpose the results of the laboratory research of this study will be analyzed together with the data on diagnosis and follow-up collected in study EWOG-MDS 2006 and transmitted to the Coordinating Study Center in Freiburg. There data on your diagnosis and clinical course are being collected and saved in a protected data base and analysed anonymously.


Guidelines concerning professional discretion and data protection are followed. Names and addresses of the responsible persons in the laboratory of the Erasmus Medical Center, Rotterdam, the Netherlands, of the Regional Coordinators of this study and the Coordinating Study Center in Freiburg, Germany, are listed at the end of this information. How long will you be in the study? Information on the course of your health status will be collected yearly until the end of 2012, but you can stop your participation at any time before. If you have decided that the extra samples may be stored for future research, these samples will be stored anonymously for 15 years. If you change your mind and do not want any more that the samples are being stored, the remaining samples will be destroyed. It is important to know that any research that has already been done on the samples cannot be undone. What are the risks of this study? Since blood and bone marrow samples need to be taken anyway for routine analyses in order to determine the best treatment for you, there are no additional risks for you because of the participation in the study. Are there benefits of taking part in the study? Children in whom a PNH clone is identified will be followed closely with serial measurements of these clones in blood (not bone marrow) samples, because some of these patients can develop a disorder with increased blood clotting (thrombosis) and increased destruction of red blood cells. Should this happen, early diagnosis and close follow-up within this study might be an advantage for you. For all other patients participating in this research study, there will be no direct benefit when participating in this study. The results of this study may benefit other patients with your disease in the future. There is no financial remuneration for participation in this study. What are the costs? There will be no costs for the additional research analyses done for this study. Your parents or your parents health insurance company will be billed for the routine analyses, as would be the case when you would not participate in the study. What are my rights as a participant? Taking part in the study is voluntary. You may choose not to take part in the study, or may remove you from the study at any time. Leaving the study will not result in any disadvantage. We will tell you about any new information that might affect your health, welfare or willingness to stay in this study. What about confidentiality? Confidentiality is maintained throughout the whole study. For research purposes in laboratories outside the regional reference laboratories, samples will be identified by a number and not by name. vorige blz: labelled met de naam, voor lab in R’dam! Identifying information will be on file at the regional coordinating center and at the coordinating study center in Freiburg, Germany. These data are only accessible for specialists for scientific research. The results of this study will be published, but your name will not be used in any publication. All persons involved in the study are under professional discretion and have to follow the data protection law. Your decision concerning the transmission of data is voluntary and will have no impact on your treatment. You can cancel your assent at any time. On demand we can show you which data is collected.


We ask for your consent to process, e.g. storage, transmission, modification, deletion, your personal data (name, date of birth, residence, diagnosis, therapy and other medical data). The processing of data serves the purpose of medical documentation in the framework of cooperation between different hospitals to guarantee a better diagnostic approach and a monitoring of therapy in the single hospitals. This documentation is therefore an important support of modern treatment. At the end of this leaflet you will find the centers to which data is transmitted. What about ethical questions? The ethical guidelines of the Declaration of Helsinki and of the Good Clinical Practice are followed. What other options are there? You may choose not to participate in this study. You can still be treated for his disease at this center, even if you do not take part in this study. Whom do I call if I have questions or problems? For questions about the study or a research-related injury, contact ___________________________ (add name of regional coordinator) at _______________________________________. For questions about your child’s rights as a research participant, contact the Institutional Review Board, contact _________________________ at ___________________________________ (add name and address of ethics committee at the institution of the regional coordinator) Names and addresses of the scientist responsible for the research performed in this study, the Regional Coordinator and the Study Coordinating Center Coordinating investigator Dr. M.M. van den Heuvel-Eibrink, Sophia Children’s Hospital, Erasmus Medical Center, Rotterdam, The Netherlands. Regional Coordinator add name and address of regional coordinator Study Coordination Prof. Dr. Charlotte Niemeyer; Department of Pediatrics and Adolescent Medicine; Division of Pediatric Hematology and Oncology; University of Freiburg; Mathildenstr 1; D-79106 Freiburg; Germany.


Parents’ Information Leaflet Dear Parents, The treating physician of your child has informed you that your child is suspected to suffer from a disease called refractory cytopenia. This disease is a subtype of myelodysplastic syndrome involving blood and bone marrow. You have already been asked to participate in a study called EWOG-MDS 2006. Patients with refractory cytopenia who participate in this study are also asked to participate also in a corresponding study called EWOG-MDS RC 06. The following information concerns the study EWOG-MDS RC 06. This information leaflet summarizes the main points of the discussion with your child’s treating physician. When you have understood the principles of the study and when you agree that your child participates in the study, you will be asked to declare your consent in writing, by signing the “informed consent form”. You as parents decide alone for or against participation in the study when your child is unable to judge because he/she is too young. If your child is able to understand the nature and importance of the study his/her decision on giving assent or denial to the study should be considered together with your opinion. Please note that participation in the study is voluntary. Should you and/or your child decide now or later against participation in the study, this will not lead to disadvantages for you and your child. Specifically, your child will be treated carefully independent from this decision. Please take your time to carefully evaluate the decision and do not hesitate to ask additional questions as necessary. What does this study investigate? The study EWOG-MDS RC 06 is a type of research study called clinical trial. It does not concern the treatment of your child. This study analyses scientific problems independent of treatment. For exact diagnosis, determining the best therapy, blood and bone marrow samples must be taken for different analyses independent of this study. For this study, small volumes of extra blood and extra bone marrow (5 ml each) will be taken at the same time. For all patients with refractory cytopenia these extra samples will be taken at the time of diagnosis. In patients with refractory cytopenia receiving immunosuppressive therapy, additional material (5 ml blood and 5 ml bone marrow) will be obtained at day 120 and at day 240 after start of therapy. In summary, this means that no additional punctures or biopsies are performed for the study EWOG-MDS RC 06. The extra study samples of blood and bone marrow for this study will be stored at the laboratory of Dr. Marry van Heuvel-Eibrink and her co-workers at the Erasmus Medical School, Rotterdam, the Netherlands.

Include the name and address of Local Center




You can also decide whether this material may be stored for future non-commercial research on your child’s disease. You can always ask for information about the research results. In addition, the tissue type (HLA-type) of your child will be determined. This analysis will be performed in a local blood bank. For most patients with refractory cytopenia the analysis of the HLA-type is necessary independent of this study to determine the best form of therapy. For HLA-typing 5-20 ml of blood is necessary depending on blood counts. Blood drawing for HLA-typing can be done at any time with a diagnostic procedure. Why is this study being done? The purposes of this study are to answer the following questions in order to know more on these diseases and to develop better ways of therapy for them. It is known that a proportion of patients with refractory cytopenia benefits from treatment with immunosuppressive therapy. This study investigates whether the response to immunosuppressive therapy is correlated with the presence of auto-immune abnormalities and which white blood cells subtypes are associated with the occurrence of this auto-immune phenomenon. For the future this would implicate that a prediction can be made which patients would benefit from this treatment. Moreover, we know that a in a proportion of patients with refractory cytopenia PNH clones can be found. PNH clones are blood forming cells that lack certain proteins on their cell surface that makes them thereby more vulnerable for attacks of the immune system. It is not known how often these clones are present in children and adolescents with refractory cytopenia . Specific questions are: How often do we find auto-immune abnormalities in children with refractory cytopenia? Which types of blood cells are involved in these auto-immune abnormalities? In children who receive immunosuppressive therapy, how does the response to that therapy correlate with the autoimmune abnormalities tested in the laboratory? How often do we find PNH clones in children with refractory cytopenia? How does the size of these PNH clones change during immunosuppressive therapy? Is there a correlation between the HLA-type and auto-immune abnormalities? How many people will take part in this study? At diagnosis, all patients with refractory cytopenia can be included in the study. Response to immunosuppressive therapy can only be investigated in patients treated with this form of therapy. How will the different centers involved in this European study work together? Like the study EWOG-MDS 2006, in which your child is already enrolled, this integrated study is being conducted in 12 European countries. The physician treating your child locally is responsible for your child’s care. To participate in this study he/she will send samples from blood and bone marrow to the laboratory at the Erasmus Medical School in Rotterdam, the Netherlands. Samples will be labelled with your child’s full name. The results of the presence of PNH clones in your child’s blood samples will be transmitted to the Regional Coordinator of this study and to your child’s treating physician, so that follow-up examinations can be planned individually. The results on autoimmunity will be kept blinded and not transmitted to you, your child’s physician or the regional coordinator. Instead they will be correlated with the course of the disease at the time of completion of this study. For this purpose the results of the laboratory research of this study will be analyzed together with the data on diagnosis and follow-up collected in study EWOG-MDS 2006 and transmitted to the Coordinating Study Center in


Freiburg. There data on your child’s diagnosis and clinical course are being collected and saved in a protected data base and analysed anonymously. Guidelines concerning professional discretion and data protection are followed. Names and addresses of the responsible persons in the laboratory of the Erasmus Medical Center, Rotterdam, the Netherlands, of the Regional Coordinators of this study and the Coordinating Study Center in Freiburg, Germany, are listed at the end of this information. How long will your child be in the study? Information on the course of your child’s health status will be collected yearly until the end of 2012. You can stop your child from participating at any time. Or the child can stop having reached the age of 12? Years. If you have decided that the extra samples may be stored for future research, these samples will be stored anonymously for 15 years. If you/your adult? child change(s) your mind and do not want any more that the samples are being stored, the remaining samples will be destroyed. It is important to know that any research that has already been done on the samples cannot be undone. What are the risks of this study? Since blood and bone marrow samples need to be taken anyway for routine analyses in order to determine the best treatment for your child, there are no additional risks for your child because of the participation in the study. Are there benefits of taking part in the study? Children in whom a PNH clone is identified will be followed closely with serial measurements of these clones in blood (not bone marrow) samples, because some of these children can develop a disorder with increased blood clotting (thrombosis) and increased destruction of red blood cells. Should this happen, early diagnosis and close follow-up within this study might be an advantage for your child. For all other children participating in this research study, there will be no direct benefit when participating in this study. The results of this study may benefit other patients with your child’s disease in the future. There is no financial remuneration for participation in this study. What are the costs? There will be no costs for the additional research analyses done for this study. You or your health insurance company will be billed for the routine analyses, as would be the case when your child would not participate in the study. What are my child’s rights as a participant? Taking part in the study is voluntary. You may choose not to allow your child to take part in the study, or may remove your child from the study at any time. Or the grown up child may remove him/herself Leaving the study will not result in any disadvantage. We will tell you about any new information that might affect your child’s health, welfare or willingness to stay in this study. What about confidentiality? Confidentiality is maintained throughout the whole study. For research purposes in laboratories outside the regional reference laboratories, samples will be identified by a number and not by name. vorige blz naam op samples r’dam! Identifying information will be on file at the regional coordinating center and at the coordinating study center in Freiburg, Germany. These data are only accessible for specialists for scientific research. The results of this study will be published, but your child’s name will not be used in any publication


All persons involved in the study are under professional discretion and have to follow the data protection law. Your decision concerning the transmission of data is voluntary and will have no impact on your child’s treatment. You your grownup child etc. can cancel your assent at any time. On demand we can show you which data is collected. We ask for your consent to process, e.g. storage, transmission, modification, deletion, your child’s personal data (name, date of birth, residence, diagnosis, therapy and other medical data). The processing of data serves the purpose of medical documentation in the framework of cooperation between different hospitals to guarantee a better diagnostic approach and a monitoring of therapy in the single hospitals. This documentation is therefore an important support of modern treatment. At the end of this leaflet you will find the centers to which data is transmitted. What about ethical questions? The ethical guidelines of the Declaration of Helsinki and of the Good Clinical Practice are followed. What other options are there? You may choose not to allow your child to participate in this study. Your child can still be treated for his disease at this center, even if your child does not take part in this study. Whom do I call if I have questions or problems? For questions about the study or a research-related injury, contact ___________________________ (add name of regional coordinator) at _________________________________. For questions about your child’s rights as a research participant, contact the Institutional Review Board, contact _________________________ at ___________________________________ (add name and address of ethics committee at the institution of the regional coordinator) Names and addresses of the scientist responsible for the research performed in this study, the Regional Coordinator and the Study Coordinating Center Coordinating Investigator Dr. M.M. van den Heuvel-Eibrink, Sophia Children’s Hospital, Erasmus Medical Center, Rotterdam, The Netherlands. Regional Coordinator add name and address of regional coordinator Study Coordination Prof. Dr. Charlotte Niemeyer; Department of Pediatrics and Adolescent Medicine; Division of Pediatric Hematology and Oncology; University of Freiburg; Mathildenstr 1; D-79106 Freiburg; Germany.


Include the name and address of Local Center

Name: _________________________________________________ Date of Birth: ____________________________________________

I / We,

• declare that we have been informed by the treating physician orally and in written form (Information Leaflet) on the EWOG-MDS RC 06 Study. Specifically, we have been informed on the nature and the importance of the study, the risks and benefits, the rights of the participants, confidentiality issues, data protection and where to get answers on questions concerning the study.

• declare that we had enough time to evaluate the decision on participating in the study. • declare that we have understood that participating in the study is voluntary, that we can ask,

without giving reasons, that participation ends in the future, and that there are no disadvantages, for me or for our child, when not participating in the study.

• declare that we have read all of the above, asked questions and received answers concerning areas I did not understand.

• declare that we give consent/assent for this study, as follows (initial/date for each item] Item 1 Sample Collection: Blood Samples and Bone Marrow Samples, Communication of Results, and Analysis Check YES if you agree that extra blood and extra bone marrow (about 5 ml each) are taken for research on autoimmunity and PNH clones. In case you/ your child will receive immunosuppressive therapy additional samples will be taken on day 120 and day 240 of therapy to correlate the results of autoimmunity and PNH clones with therapy response. You also agree that the samples are being sent to the scientists performing that research at the Erasmus Medical Center in Rotterdam, the Netherlands. You are informed that the results of the research on PNH clones will be communicated to your/your child’s physician, while the result of autoimmunity will only be analyzed after the study is completed. # 1: Yes _____ No _____ Initials ____________ Date ____________ Item 2 Data: Collection, Storage, Modification, Transmission


(Version: November 06, 2006) Informed Consent




Check YES if you agree that information on the course of disease, on the health status of your child and the results of PNH clones are transmitted in identifiable for whom? form to the coordinating study center located in Freiburg, Germany, where this information is stored in a protected database and analyzed in the future. All researchers having access to the database are under professional discretion. Your / your child’s name will not be used in any publication resulting from this study. Blood samples will not be labelled with your/your child’s full name, only with initials. You also agree that information stored in the databases of the regional coordinating center and the coordinating study center in Freiburg, Germany, can be viewed by members of the regional and national public health authorities responsible for quality assurance, who are as well under professional discretion. For this monitoring of data personal data are separated? Niet helemaal duidelijk. #2: Yes _____ No _____ Initials ____________ Date ____________ Item 3 Research Material for Future Research Check YES if you agree that blood and bone marrow that has been already obtained can be used to answer additional research questions. This material will be used for future research concerning the cause of your/ your child’s disease or for optimizing treatment. This research (as molecular genetic studies?) is not commercial. All samples will be stored in the treating center or regional reference laboratories to the conditions described above. All research will be supervised by the steering committee.is deze naam al elders genoemd? Information on this research can be obtained at the Coordinating Study Center in Freiburg, Germany. # 3: Yes _____ No _____ Initials ____________ Date ____________ SIGNATURES ____________________________________________________________________________ (Patient Signature) (Date) ____________________________________________________________________________ (Parent / Guardian Signature) (Date) ____________________________________________________________________________ (Parent / Guardian Signature) (Date) ____________________________________________________________________________ (Physician Signature) (Date) By initializing the following, the parents / guardian and physician indicate their opinion that the patient is too young to give consent / assent at that time. __________ Parents / Guardian __________ Physician __________ Age of patient The original version of this informed consent sheet is in the patient chart. The patient /his her parents get(s) a copy.


Appendix 7 Approval by Ethics Committee


18 Nederlandse bijlagen Deze bijlage dient in samenhang te worden bekeken met:

• het SKION protocol: Diagnostiek Beenmergfalen voor het uitsluiten van andere oorzaken van beenmergfalen (Appendix 9: stap 2 en 3)

• het “masterprotocol” EWOG-MDS 2006 (blz. 8-10, en blz. 57-59), voor diagnostiek en follow-up specifiek gericht op JMML, RC of high grade MDS.

18.1 Bijlage I: Diagnostiek fase van JMML/ MDS

18.1.1 Anamnese Medische geschiedenis voorafgaand aan MDS Transfusie anamnese Huidige en voorafgaande medicatie

18.I.2 Onderzoek bij diagnose 1) Lichamelijk onderzoek (waaronder lengte, gewicht, lichaams-oppervlak), en onderzoek naar

congenitale afwijkingen juveniele xanto granuloma’s, tekenen van NF1, zie pag. 27 en 28 van het master protocol).

2) Hb, leukocyten, MCV, differentiatie, trombocyten, reticulocyten, bloedgroep, Rhesus, Directe en indirecte Coombs, HbF.

3) Beenmerg- en bloedonderzoek (uitstrijken) en behalve bij JMML ook biopt. 4) Cytomorfologie, cytochemie 5) Immunologisch onderzoek (+ hemoblok naar SKION). 6) Cytogenetisch onderzoek, inclusief FISH en Comparative Hybridisation. 7) Liquor-onderzoek: celaantal, cytologie (+ liquor-blok naar SKION)

(bij trombocyten < 50 x 109/L eerst thrombocytentransfusie). 8) Bloedchemie: ureum, kreatinine, urinezuur, Na, K, Ca, P, Cl, Mg, bilirubine, totaal eiwit,

glucose, SGOT, SGPT, LDH, alkalische fosfatase. 9) Stollingsonderzoek. 10) Infectie-inventarisatie. 11) Antistoffen (IgG, IgM): EBV, CMV, HHV-6, Parvovirus B19, HBV, HCV, HIV, VZV. 12) Bacteriologische inventarisatie zoals gebruikelijk in het centrum. 13) PTPN11/RAS mutatie analysis in Freiburg bij verdenking JMML. Tevens buccal swab voor

germline mutatie analysis. Tevens GM hypersitiviteits analysis zeker bij germline PTPN11/RAS en geen cytogenetische clonale afwijking. (zie invoiceformulier in appendix 5).

14) ECG en echocardiogram. 15) Röntgenonderzoek: X-thorax. 16) HLA-typering van patiënt, broers, zusters en ouders. 17) Bij RC elastase in de faeces of serum trypsinogeen/isoamylase en vitamine B12 en foliumzuur

spiegels. Tevens excluderen van metabole ziektes, en Pearson disease. (zie pag. 58 en 59 van het “masterprotocol” EWOG-MDS 2006).

18) Bij RC, PB en BM voor PNH screening en Tcel repertoire analyse naar het imm.lab in Rotterdam zie RC-IST protocol voor advies en invoice formulieren.

18.2 Laboratoriumonderzoek (SKION) Beenmergonderzoek: Cytomorfologisch, en immunologisch Tijdstippen afname:

• Diagnose en indien MDS (RAEB/RAEB-t): bij 2e Beenmergonderzoek • vόόr BMT • bij recidief (of verdenking recidief) • verder op momenten aangegeven in de behandelingsprotocollen


18.2.1 Cytologische diagnostiek (uitstrijkpreparaten) Benodigd materiaal:

• Voorafgaande aan de behandeling worden 6 ongekleurde beenmerguitstrijkjes en 3 ongekleurde bloeduitstrijkjes gestuurd naar het laboratorium van de SKION. De uitstrijkjes moeten zijn afgenomen vóór eventuele transfusie van bloed of bloedprodukten.

• Tijdens en na behandeling volstaan 3 ongekleurde beenmergpreparaten en 1 ongekleurd bloedpreparaat. Bij (verdenking) recidief dient dezelfde procedure te worden gevolgd als bij diagnose.

Richtlijnen voor het vervaardigen van bloeden beenmerguitstrijken Ter realisatie van de gewenste uniformiteit van bloeden beenmergpreparaten gaarne aandacht voor de volgende richtlijnen voor bloeden beenmerguitstrijken:

• het opbrengen van slechts een kleine druppel op het objectglas. • het uitstrijken met een glaasje dat smaller is dan het objectglas onder een hoek van 45º, langzaam

uitstrijken. • zodanig uitstrijken dat het einde van de film ongeveer halverwege het objectglas komt te liggen.

Pathologische cellen zijn vaak erg kwetsbaar en vallen spoedig uiteen bij snelle verplaatsing. De hoeveelheid plasma dient gering te zijn. Indien het plasma meer dan enkele seconden nodig heeft om op te drogen, gaan de cellen door osmotische invloed schrompelen. Men gebruike een geslepen dekglaasje van een telkamer of een geslepen objectglas. Doel Op de uitstrijkpreparaten wordt standaard een May-Grünwald-Giemsa kleuring gedaan voor het tellen van het percentage blasten. Voor het klassificeren van de leukemie worden tevens een Sudan-Black B, een Peroxidase en gecombineerde naftol AS-D chloroacetaat esterase en α-naftyl-acetaat-esterase verricht. Beoordeling en typering geschiedt volgens de Pediatrische modificatie van de WHO-classificatie (zie: Hoofdstuk 2, table 3, van dit protocol). Uitslag De uitslag wordt telefonisch en schriftelijk doorgegeven aan de behandelend kinderarts.

18.2.2 Biopsie ten behoeve van ( SKION review) Benodigd materiaal: Bij advanced MDS altijd 2e botbiopt. Naast de locale patholoog die het biopt van het beenmerg zal bekijken zal tevens een nationale en internationale review plaastvinden van het gebiopteerde materiaal. Hiervoor dient u uw patholoog te verzoeken 1 HE en 10 blanco coupes te sturen naar de secretaris van het pathologen panel:

Prof. Dr. J.G. van den Tweel Academisch Ziekenhuis Utrecht, afd. Pathologie, H 04123 Postbus 85500 3508 GA Utrecht

Onder vermelding van: Protocol EWOG-MDS 2006. Uitslag De uitslag wordt schriftelijk doorgegeven aan de behandelende kinderarts, met copie aan de SKION. Het BMF CRF (op te vragen bij de SKION) dient ingevuld binnen 2 weken na insturen BM/PB bij de SKION te zijn.

18.2.3 Immunophenotypering and Celbank (Hemoblok – SKION) Benodigd materiaal:

• heparine beenmerg: 5 ml • heparine bloed: 10 ml

Werkwijze:


Hiervoor zijn in het zgn. hemoblok heparinebuizen aanwezig; na afname goed mengen om stolling te voorkomen. Zonodig een tweede beenmergpunctie op een andere plaats uitvoeren, om teveel bloedbijmenging te voorkomen. Het materiaal dient te worden bewaard en getransporteerd op kamertemperatuur. Doel

• Immunofenotypering geschiedt op het laboratorium van de SKION in meervoudige labeling en volgens de richtlijnen van de SIHON (www.sihon.nl) in een gefaseerde aanpak. De volgende markers worden in elk geval gebruikt:

o Niet specifiek CD45, CD34, CD117, TdT en HLA-DR o B-cel markers CD19, CD10, CD20, o T-cel markers CD2, CD3, CD7 o Myelo-monocytaire marker CD13, CD33, CD14, CD15, MPO, (CD61, CD235a)

Uitslag De uitslag van de PNH screening wordt schriftelijk aan de behandelend arts meegedeeld. Deze samples voor PNH screening (5-10 ml. zoals in het RC-protocol staat aangegeven) kunnen worden ingestuurd naar Vincent v.d. Velden en Ton Langerak in Rotterdam volgens de procedure zoals beschreven in het RC-IST protocol. Aanmelden via P. Schneider 010-4088084 of R. Stigter 010-4636233. Maak voor het versturen gebruik van de invoice form in appendix 12.

• Materiaal voor toekomstig onderzoek: o DNA en RNA van MNC o DNA van granulocyten (Ficoll pellet) zal na erythrocytenlysis worden geëxtraheerd

voor CGH, zie p 58 par 7.3.1.4. van het masterprotocol. o Heparine plasma o Bij verwijderen van de milt altijd materiaal “vers” opslaan en “vers” versturen naar

Rotterdam: lab. kinderoncologie P. Schneider tel. 010-4088084 of via R. Stigter 010-4636233.

Overweeg deelname aan de add-on studie. Zie hiervoor appendix 11.

http://www.sihon.nl)


18.3 Insturen materiaal naar SKION bij Beenmergfalen (appendix 9 vanaf stap2), diagnose en

follow-up JMML/MDS Patient telefonisch aanmelden bij de SKION (070-3674545). Hierbij worden naam, geboortedatum en indien van toepassing de (voorlopige) diagnose gemeld, alsmede gegevens over het afgenomen materiaal. Afnames bij diagnose en follow-up: Tijdstip Preparaten

(ongekleurd) Hemoblok Botbiopt

BM Bloed Beenmerg (heparine buis)

Bloed (heparine buis)

Beenmerg

Diagnose en (verdenking) recidief

6 3 5 ml

10ml 1 HE en 10 blanco coupes

Overige tijdstippen

3 3 2 ml 10 ml

PA review Ten behoeve van de PA review dient biopt materiaal verstuurd te worden naar de secretaris van het pathologen panel:

Prof. Dr. J.G. van den Tweel Academisch Ziekenhuis Utrecht, afd. Pathologie, H 04123 Postbus 85500 3508 GA Utrecht

Onder vermelding van: Protocol EWOG-MDS 2006. PNH screening en TCR Vβ repertoire analyses Hiervoor wordt 3-5 ml. PB en BM ingestuurd naar lab immunologie in Rotterdam voor procedure zie het RC IST protocol (invoice form zie appendix 12). Review cytogenetische uitslagen Ten behoeve van de cytogenetische review dienen de karyotypering en de FISH uitslagen in copie naar de SKION te worden verstuurd. Add-on studies: Voor de add-on studie in het LUMC wordt geadviseerd contact op te nemen met dr L.Ball voorafgaande aan afname van heparine bloed en BM (Zie add-on studie in bijlage).


18.4 Verzenden per koerier naar het SKION laboratorium Instructies voor verzenden van patiëntenmateriaal naar het laboratorium van de SKION Materiaal naar de SKION: Hemoblok: Bloed en/of beenmerg kunnen in een, door de SKION verstrekt hemoblok worden verstuurd. Liquorblok: Voor het verzenden van liquor is een liquorblok beschikbaar. Deze zijn in de regel in het laboratorium van het ziekenhuis van de kinderarts aanwezig (evt. aanvragen bij het laboratorium van de SKION). In de blokken zit een formulier met instructies waarop tevens enkele gegevens van de patiënt moeten worden ingevuld. Controle preparaten: deze kunnen in de daartoe ontworpen goedgekeurde verpakking maar per gewone post kunnen worden verstuurd. Melding van verzenden van patientenmateriaal: Telefonisch of per fax wordt de verzending vóóraf gemeld aan de SKION: Op werkdagen van 08.30-17.00 uur: telefoon 070 - 367 45 45 (buiten deze uren kan een boodschap worden ingesproken op het antwoordapparaat) of faxnr. 070 - 367 08 68 In het weekend op zaterdag van 08.30-17.30 uur en op zondag tot 13.00 uur via telefoonnummer 06- 5120 12 97 de dienstdoende analist(e) waarschuwen. Indien de analiste niet reageert via dit telefoonnummer (b.v omdat ze bezig is), dan graag uw boodschap op de voice-mail inspreken, opdat men u kan terugbellen. Insturen van patientenmateriaal:

• Op werkdagen via Fiege (BLS koerier) o aanmelding voor vervoer doet u per fax: 075 - 650 16 98 of per email

([email protected]), bij voorkeur vóór 16.00 uur en met een speciaal daarvoor ontworpen opdrachtformulier. Eventueel tot 21.00 uur bellen naar nummer: 075 - 650 16 19; bgg 06-432 71 741, of via nummer 06-432 71 751.

o Verpakking: in SKION verpakking, lekdicht en met absorberend materiaal verpakt. o Plaats van ophalen: U zorgt dat het pakket klaar ligt op de met de koeriersdienst

afgesproken plaats. o Opdrachtformulieren zijn indien nodig aan te vragen bij het SKION laboratorium.

• In het weekend op zaterdag en op feestdagen o Vervoer: Na telefonisch overleg met de dienstdoende analist moet het materiaal per

taxi (voor eigen rekening) naar het SKION laboratorium worden vervoerd. o Alléén materiaal dat vóór 14.00 uur binnen is op het SKION laboratorium kan

nog diezelfde dag worden verwerkt. Materiaal dat niet vóór 14.00 uur bij ons binnen kan zijn, kunt u dus beter de volgende dag afnemen en versturen. Wij hebben er dan weer alle tijd voor.

mailto:([email protected])


18.5 Cytogenetisch onderzoek Het chromosomenonderzoek (karyotypering en FISH) geschiedt in 9 cytogenetische laboratoria in Nederland. Deze zijn hiervoor een gezamenlijk te volgen procedure overeengekomen voor het verkrijgen van materiaal. Voor het doen verrichten van cytogenetisch onderzoek neme men telefonisch contact op met één der onderstaande personen en instituten: Dr C Mellink Dr. M. Stevens-Kroef Academisch Medisch Centrum UMC St.Radboud Afd. Klinische Genetica 848 sectie Cytogenetica Meibergdreef 15 Postbus 9101 1105 AZ Amsterdam ZO 6500 HB Nijmegen Tel: 020 - 566 51 69 Tel: 024-3614107/024-3610867/024-3668934 Fax 020-6918626 Fax:: 024-33668751 e-mail: [email protected] e-mail: [email protected] Dr. E. van den Berg-de Ruiter Mevr. Dr. H.B. Beverloo Universitair Medisch Centrum Groningen Erasmus Universiteit Rotterdam Hoofd Tumor Cytogenetisch Laboratorium Afd. Klinische Genetica Dept. of Genetica Postbus 1738 Postbus 30.001 3000 DR Rotterdam 9700 RB Groningen Tel: 010 - 408 83 15/ 010-4087196 Tel : 050 - 361 71 34 Fax: 010- 408 94 92 Fax : 050- 361 72 30 e-mail: [email protected] e-mail: [email protected] Mw dr. J Janssen Dr. A. Buijs Afdeling Klinische Genetica AZM Afdeling Medische Genetica Postbus 5800 UMC, locatie Wilhelmina Kinderziekenhuis 6202 AZ Maastricht Huispostnr. KC 04.084.2 Tel: 043 - 387 58 44 Postbus 85090 Fax: 043- 387 78 77 3508 AB Utrecht e-mail: [email protected] Tel: 030- 250 38 00/ 3866 Fax: 030- 250 38 01 e-mail: [email protected] Mevr. Drs. W Kroes Klinische Genetica (LDGA) Mw. dr. J.Jansen Afdeling Cytogenetica Afdeling Klinische Genetica AZM Postzone S-06-P Postbus 108 Leids Universitair Medisch Centrum 5500 AC Veldhoven Postbus 9600 Tel. 040- 8888300 2300 RC Leiden Fax: 040- 8888303 Fax: 071-5268276 Tel: 071- 526 98 26/ 071-5269800 e-mail: [email protected] e-mail: [email protected] Mevr. Drs A.W.M. Nieuwint VU medisch centrum Laboratorium voor Chromosoomdiagnostiek Afdeling Klinische Genetica en Antropogenetica Ruimtenummer PK OX )11 Postbus 7057 1007 MB Amsterdam Tel: 020-4440745/ 020-4440157 Fax: 020-4440744 e-mail: [email protected] De uitslagen worden rechtstreeks aan de behandelend kinderarts toegestuurd. De SKION ontvangt de uitslag van het desbetreffende cytogenetisch laboratorium.











18.6 Patiënten informatie formulieren Informatie over registratie en diagnostiek bij kinderen met Myelodysplastisch Syndroom (MDS) en Juveniele Myelomonocytaire Leukemie (JMML) volgens het EWOG-MDS 2006 protocol Officiële titel: Prospectieve niet-gerandomiseerd multicenter onderzoek naar epidemiologie en karakteristieken van MDS en JMML op de kinderleeftijd (protocol EWOG-MDS 2006). Informatieformulier voor kinderen vanaf 12 jaar Beste , Je behandelend arts heeft je verteld dat je vermoedelijk lijdt aan één van de volgende aandoeningen: Myelodysplastisch Syndroom (MDS), Juveniele MyeloMonocytaire Leukemie (JMML), of het Noonan Syndroom met een JMML-achtige afwijking. Dit zijn ziektes van het bloed en het beenmerg. Om de behandelingsmogelijkheden te blijven verbeteren en verder te ontwikkelen, wordt wetenschappelijk onderzoek gedaan. Samen met je ouder(s)/verzorger(s) heb je een gesprek gehad met je behandelend arts over je ziekte en de behandeling ervan. Je behandelend arts heeft jou en je ouders over het EWOG-MDS 2006 protocol verteld. In aansluiting op dit gesprek krijg je hierbij schriftelijke informatie, waarin verdere uitleg wordt gegeven. In deze informatiebrief kan je nalezen wat jouw dokter verteld heeft over de behandeling en vragen we je om mee te doen aan wetenschappelijk onderzoek. Wij vragen je niet meteen een beslissing te nemen, neem maar rustig wat bedenktijd en praat erover met mensen om je heen. We willen je toestemming vragen voor registratie van je gegevens volgens het EWOG-MDS 2006 protocol. Als je na het lezen van deze informatiebrief toestemming geeft om mee te doen aan dit registratieprotocol, word je gevraagd het toestemmingsformulier te ondertekenen. Wat zijn Myelodysplastisch Syndroom en Juveniele Myelomonocytaire Leukemie (JMML)? Alle cellen in ons bloed hebben verschillende taken. Bloedcellen moeten eerst uitrijpen (ontwikkelen) om hun specifieke taak goed uit te kunnen voeren. Bij MDS lukt het de bloedcellen niet (meer) om normaal uit te rijpen. Onder een microscoop zien we dat de rode en witte bloedcellen niet normaal uitgerijpt zijn en er anders uit zien dan normaal; ze hebben afwijkende vormen. Het kan ook zijn dat we een toename zien van onrijpe cellen (blasten). Naarmate de ziekte vordert, neemt het aantal blasten in het beenmerg toe en kan het beenmerg geen normale rode bloedcellen, witte bloedcellen en bloedplaatjes meer aanmaken. De rijpe cellen die aanwezig zijn kunnen hun werk niet goed meer uitvoeren. Juveniele Myelo Monocytische Leukemie (JMML) is een bijzondere vorm van MDS waarbij één van de witte bloedcellijnen abnormaal uitrijpt. Bij patiënten met JMML wordt meestal een grote milt, een laag aantal bloedplaatjes en een hoog aantal witte bloedcellen in het bloed gevonden. Diagnostiek & registratie Kinderartsen die zich bezig houden met ziektes van het bloed en beenmerg heten kinderhaemato-oncologen. Kinderhaemato-oncologen in Nederland zijn overeengekomen kinderen met MDS of JMML te onderzoeken en registreren volgens internationaal gemaakte afspraken. Deze afspraken staan in protocollen. Een protocol is een beschrijving van de diagnostiek- en registratiemethode voor een patiënt met een bepaalde aandoening. Het EWOG-MDS 2006 protocol is het protocol wat op dit moment gebruikt wordt voor de registratie van kinderen en adolescenten met MDS of JMML. Protocol EWOG-MDS 2006 is opgesteld door de European WOrking Group voor MDS op de kinderleeftijd (EWOG-MDS). De EWOG werd in 1993 opgericht door leden uit verschillende Europese landen. Het doel van de EWOG is om artsen en onderzoekers met MDS als aandachtsgebied zo goed mogelijk te laten samenwerken en kennis met elkaar te delen. Meer informatie over de EWOG-MDS kun je lezen op de website van de EWOG: www.ewog-mds.org (Engelstalig).


In Nederland nemen de behandelend kinderartsen deel via de SKION (Stichting Kinderoncologie Nederland). De SKION is een landelijk samenwerkingsverband van alle Nederlandse kinderartsen en andere hulpverleners, die gespecialiseerd zijn in onderzoek en behandeling van kinderen met kanker of aandoeningen die kunnen leiden tot een vorm van kanker. De SKION registreert gegevens over de ziekte en behandeling van kinderen en jongeren met kanker om zo de kwaliteit van de behandeling te kunnen controleren. Daarnaast verzamelt de SKION informatie ten behoeve van wetenschappelijk onderzoek. Meer informatie over de SKION kun je lezen in de informatiebrief over de ‘SKION basisregistratie’ en op de website www.skion.nl Waar richt dit protocol zich op? Dit protocol wordt ook wel klinische studie genoemd. Het gaat bij EWOG-MDS 2006 studie niet om de behandeling van je ziekte. Het EWOG-MDS 2006 protocol richt zich wel op twee aspecten onafhankelijk van de behandeling:

1. Diagnose: Om voor jou de beste behandeling te kunnen bepalen, worden bloeden beenmergmonsters afgenomen (in het geval van JMML of Noonan Syndroom wordt aanvullend een klein huidbiopt afgenomen of wordt wat wangslijmvlies afgenomen met behulp van een wattenstokje). Voor de studie worden tegelijkertijd met de gewone afnames kleine hoeveelheden bloed en beenmerg (5 ml per keer) afgenomen. Kortom: dit betekent dat er geen extra prikken of ingrepen worden gedaan voor het protocol. De afgenomen monsters worden opgeslagen in ons ziekenhuis of in één van de door de EWOG-MDS aangewezen laboratoria. Deze monsters zijn bedoeld voor diagnostiek. Je kunt apart toestemming geven voor opslag van het lichaamsmateriaal voor toekomstig onderzoek naar MDS/JMML. Het gaat hier dan dus om overgebleven materiaal (‘restmateriaal’) wat voor onderzoeksdoelen wordt gebruikt.

2. Registratie van ziektebeloop: gegevens over ziektebeloop en je gezondheidstoestand worden via de SKION doorgegeven aan het coördinerend studiecentrum in Freiburg, Duitsland.

Als uit verder onderzoek blijkt dat de diagnose MDS of JMML toch niet op jou van toepassing is, worden de tot dan toe verzamelde gegevens en het verzamelde materiaal (zoals bloed en beenmerg) uiteraard niet voor onderzoek volgens het EWOG-MDS 2006 protocol bewaard. Doel van het onderzoek Het doel van dit protocol is het verzamelen van informatie die mogelijk in de toekomst kan leiden tot betere behandeling van kinderen met MDS of JMML. Hoe verloopt de registratie? De registratieprocedure wordt in tientallen ziekenhuizen uitgevoerd, verdeeld over 12 verschillende landen. De verwachting is dat er in totaal zo’n 260 patiënten zullen worden geregistreerd. De behandelend arts is verantwoordelijk voor de medische zorg. Er zullen bloeden beenmergmonsters (en bij JMML of Noonan Syndroom het al eerder genoemde huidbiopt of wangslijmvlies monster) worden opgestuurd naar de daarvoor aangewezen laboratoria. Na analyse worden de uitslagen doorgestuurd naar het coördinerend centrum in Freiburg. Daar worden alle gegevens verzameld, bewaard en geanalyseerd in een beveiligd computerbestand (‘database’). Deze gegevens worden uiteraard beschermd en vallen onder medische geheimhouding. Voor- en nadelen Omdat de afnames voor de studie altijd gecombineerd worden met de gewone afnames die nodig zijn voor het bepalen van de beste behandeling vinden geen extra afnames plaats. Er zijn daarom dan ook geen risico’s verbonden aan deelname aan dit protocol. Door dit protocol wordt door alle ziekenhuizen op dezelfde manier gewerkt. We noemen dat ook wel ‘uniform’ of ‘gestandaardiseerd’. Behalve deze gestandaardiseerde diagnostische benadering en de uniforme manier van behandelen, brengt deelname aan de studie voor jou geen voordelen met zich mee. De resultaten van deze registratie kunnen ook voor toekomstige patiënten van belang zijn. Er is geen financiële vergoeding voor deelname aan deze studie. Toestemming

http://www.skion.nl


Als je ouder bent dan 12 jaar moeten jij en je ouders/verzorgers samen beslissen of je toestemming geeft voor deelname aan dit registratieprotocol. Zowel jij als je ouders - verzorgers moeten een handtekening zetten onder het toestemmingsformulier. In dit formulier staat dat je weet wat de behandeling inhoudt en wat de mogelijke voor- en nadelen zijn. Wanneer je niet dezelfde mening hebt als je ouders - verzorgers kun je dit bespreken met je behandelend arts. Verantwoording en vertrouwelijkheid We behandelen alle verzamelde gegevens zorgvuldig en vertrouwelijk. Persoonlijke gegevens (zoals je naam of adres) zullen we niet gebruiken op studiedocumentatie, in rapporten of publicaties over dit onderzoek. Jouw medische gegevens krijgen een codenummer. De gecodeerde gegevens worden opgeslagen in een computerbestand van het bureau voor de statistiek van de EWOG. Binnen Nederland worden de gegevens opgeslagen en verwerkt bij de SKION. De gegevens worden, als jij en je ouders daar toestemming voor geven, gedurende 15 jaar bewaard. Lichaamsmaterialen die tijdens deze studie worden verzameld, worden opgeslagen onder een codenummer. Na afloop van de studie worden de opgeslagen lichaamsmaterialen vernietigd of, als jij en je ouders daarvoor toestemming geven, gedurende maximaal 15 jaar na afloop van de studie bewaard. Het opgeslagen lichaamsmateriaal kan dan eventueel in de toekomst worden gebruikt voor onderzoek naar MDS/JMML. De onderzoeksresultaten worden gerapporteerd in medisch-wetenschappelijke literatuur en/of op medische congressen. In het kader van zorgvuldigheidstoetsing is het EWOG-MDS 2006 protocol voorgelegd aan de METC van het Erasmus MC. De voor dit onderzoek geldende internationale richtlijnen zullen nauwkeurig in acht worden genomen. Vrijwillige deelname Zoals al werd gezegd, is het EWOG-MDS 2006 protocol momenteel de standaard voor registratie en diagnostiek voor kinderen en adolescenten met MDS/JMML. De vrijwilligheid waar in deze patiënten informatie brief over wordt gesproken betreft de keuze of je toestemming geeft om je gegevens gecodeerd te laten registreren, om gegevens te bewaren en om restweefsel na diagnostiek te mogen gebruiken voor wetenschappelijk onderzoek naar MDS/JMML bij kinderen. Mocht je tijdens het onderzoek beslissen dat je niet wilt doorgaan met het onderzoek, dan kun je je toestemming altijd zonder opgave van redenen weer intrekken. Wij vragen je wel om dat met de behandelend arts te bespreken. Eventuele twijfels kun je bespreken met een onafhankelijke arts die zelf niet bij het onderzoek is betrokken maar wel deskundig is op dit gebied. Ook is het belangrijk dat je eventuele twijfels met je ouders/verzorgers bespreekt. Je mag uiteindelijk zelf beslissen of je wilt doorgaan of wilt stoppen. Als je niet wilt meedoen hoef je daar geen reden voor te geven. Dit verandert niets aan de verdere behandeling die je krijgt. Er is een team beschikbaar om jou en je ouders ook tijdens het onderzoek zo goed mogelijk te begeleiden. Dit team bestaat onder andere uit gespecialiseerde verpleegkundigen en een psycholoog. Het behandelteam ziet nauwlettend toe op de belasting die deelname aan een onderzoeksprotocol voor kinderen vormt, hoewel van belasting geen sprake is in het geval van het EWOG-MDS 2006 protocol. We werken zoveel mogelijk volgens de landelijke afspraken zoals die door de Nederlandse Vereniging voor Kindergeneeskunde (NVK) zijn vastgelegd ter bescherming van kinderen en jongeren. Voor meer informatie hierover verwijzen wij je naar www.ccmo.nl (weten regelgeving/gedragscode verzet: minderjarigen). Verzekering Voor dit registratieprotocol is de verzekering voor onderzoeksdeelnemers niet van toepassing. Het gaat hier om een registratie- en diagnostiek protocol in plaats van een onderzoeksprotocol: er is geen verschil in behandeling vergeleken met de standaardbehandeling van MDS/JMML . Contactpersonen

http://www.ccmo.nl


Bij vragen of opmerkingen kun je contact opnemen met dr. M.M. van den Heuvel-Eibrink, kinderarts-oncoloog, tel. 010-4636691, of met Eline Visser of Inekee van der Vaart, researchverpleegkundigen van de afdeling kinderoncologie, tel. 010-4636402. Als je twijfelt over deelname kun je een onafhankelijk arts raadplegen die niet zelf betrokken is bij dit onderzoek maar wel deskundig op dit gebied is: Dr. J.B. van Goudoever, kinderarts, tel. 010-4636363. Ook kun je als je voor of tijdens het onderzoek vragen hebt die je liever niet aan de onderzoekers stelt contact opnemen met de onafhankelijke arts. Als je niet tevreden bent over het onderzoek of de behandeling dan kun je terecht bij de onafhankelijke klachtencommissie van het ziekenhuis. Je kunt je daartoe wenden tot: de secretaris van de klachtencommissie Postbus 2040 3000 CA Rotterdam tel. 010-4633198 Neem gerust de tijd om deze informatie door te spreken en aarzel niet je behandelend arts vragen te stellen. Wanneer je besluit deel te nemen krijg je een kopie van dit document, nadat jij en je behandelend arts beiden getekend hebben. Met vriendelijke groet, Dr. M.M. van den Heuvel -Eibrink, Kinderarts-oncoloog


Toestemmingsformulier voor kinderen van 12 t/m 17 jaar

behorende bij de patiënteninformatie over registratie en diagnostiek bij kinderen met Myelodysplastisch Syndroom (MDS) en Juveniele Myelomonocytaire Leukemie (JMML)

volgens het EWOG-MDS 2006 protocol Titel van het onderzoek:” Prospectieve niet-gerandomiseerd multicenter onderzoek naar epidemiologie en karakteristieken van MDS en JMML op de kinderleeftijd” (protocol EWOG-MDS 2006). Ik bevestig, dat ik het informatieformulier voor kinderen heb gelezen. Ik begrijp de informatie. Ik heb de gelegenheid gehad om aanvullende vragen te stellen. Deze vragen zijn naar tevredenheid beantwoord. Ik heb voldoende tijd gehad om over deelname na te denken. Ik weet dat deelname geheel vrijwillig is en dat ik mijn toestemming op ieder moment kan intrekken zonder dat ik daarvoor een reden hoef te geven. Ik geef toestemming voor deelname aan de registratie en diagnostiek volgens het EWOG-MDS 2006 protocol onder de omstandigheden zoals die mij zijn uitgelegd. Ik geef wel/geen* toestemming om mijn gegevens gedurende 15 jaar na afloop van de studie te bewaren. Ik geef wel/geen* toestemming voor het langdurig (maximaal 15 jaar) bewaren van restweefsel, waar mogelijk in de toekomst verder onderzoek naar MDS en/of JMML mee gedaan wordt. Ik geef toestemming, dat daartoe bevoegde medewerkers van het onderzoeksteam, medewerkers van de Inspectie voor de Gezondheidszorg, bevoegde inspecteurs van een buitenlandse overheid of leden van de medisch-ethische toetsingscommissie inzage kunnen krijgen in de medische gegevens en onderzoeksgegevens van mijn kind. Ik geef toestemming voor het gecodeerd verzamelen en verwerken van de gegevens over het verloop van de behandeling, zoals in deze informatiebrief beschreven is. De gegevens zullen worden opgeslagen in een database. De resultaten van het onderzoek zullen voor wetenschappelijke doeleinden worden gebruikt waarbij de vertrouwelijkheid gewaarborgd wordt. Naam : Handtekening: Datum : __ / __ / __ Naam onderzoeker/behandelend arts: Handtekening: Datum : __ / __ / __ * Doorhalen wat niet van toepassing is.


Informatie over registratie en diagnostiek bij kinderen met Myelodysplastisch Syndroom (MDS) en Juveniele Myelomonocytaire Leukemie (JMML) volgens het EWOG-MDS 2006 protocol Officiële titel: Prospectieve niet-gerandomiseerd multicenter onderzoek naar epidemiologie en karakteristieken van MDS en JMML op de kinderleeftijd (protocol EWOG-MDS 2006). Geachte ouders - verzorgers De behandelend arts van uw kind heeft u er van op de hoogte gebracht dat uw kind vermoedelijk lijdt aan één van de volgende aandoeningen: Myelodysplastisch Syndroom (MDS), Juveniele MyeloMonocytaire Leukemie (JMML), of het Noonan Syndroom met een JMML-achtige afwijking. Deze bij kinderen zeer zeldzame aandoeningen hebben betrekking op het bloed en het beenmerg. Myelodysplastisch Syndroom en JMML Bloedcellen moeten eerst uitrijpen om hun specifieke taak goed uit te kunnen voeren. Bij MDS hebben de bloedcellen hun vermogen om normaal uit te rijpen verloren. Onder een microscoop zien we dat de rode en witte bloedcellen niet normaal uitgerijpt zijn en afwijkende vormen hebben. Het kan ook zijn dat we een toename zien van onrijpe cellen (blasten). Naarmate de ziekte vordert, neemt het aantal blasten in het beenmerg toe en kan het beenmerg geen normale rode bloedcellen, witte bloedcellen en bloedplaatjes meer aanmaken. De rijpe cellen die aanwezig zijn kunnen hun werk niet goed meer uitvoeren. Juveniele Myelo Monocytische Leukemie (JMML) is een bijzondere vorm van MDS waarbij één van de witte bloedcellijnen (de monocytaire lijn) abnormaal uitrijpt. Bij patiënten met JMML wordt meestal een grote milt, een laag aantal bloedplaatjes en een hoog aantal witte bloedcellen in het bloed gevonden. Om de behandelingsmogelijkheden te blijven verbeteren en verder te ontwikkelen, worden alle kinderen in Europa geregistreerd na uniforme diagnostiek. Om meer inzicht te krijgen in MDS en JMML wordt in Europa klinisch onderzoek gedaan door de EWOG-MDS (European Working Group on Childhood MDS). De behandelend arts van uw kind heeft u en uw kind geïnformeerd over bovengenoemd protocol. In aansluiting op dit gesprek, ontvangt u hierbij schriftelijke informatie, waarin nadere toelichting wordt gegeven. U wordt uitgenodigd toestemming te verlenen voor registratie van uw kind volgens het EWOG-MDS 2006 protocol geheten. Voor toestemming of weigering is goede voorlichting van onze kant nodig, en een zorgvuldige afweging van uw kant. Vandaar dat u deze schriftelijke informatie ontvangt. U kunt die rustig (her)lezen en in eigen kring bespreken. Ook daarna kunt u nog altijd vragen stellen aan de contactpersonen die aan het einde van deze informatiebrief genoemd staan. Als u na het bestuderen van deze informatie toestemming geeft voor deelname van uw kind aan dit protocol, wordt u gevraagd het toestemmingsformulier te ondertekenen. In geval de diagnose MDS of JMML niet wordt bevestigd, worden de dan reeds verzamelde gegevens en het verzamelde materiaal (zoals bloed en beenmerg) uiteraard niet voor onderzoek bewaard. Diagnostiek & registratie Kinderhaemato-oncologen in Nederland zijn overeengekomen kinderen met MDS of JMML te onderzoeken en registreren volgens internationaal gemaakte afspraken. Deze afspraken staan in protocollen. Een protocol is een beschrijving van de diagnostiek- en registratiemethode voor een patiënt met een bepaalde aandoening. Het EWOG-MDS 2006 protocol is het huidig geldige protocol voor de registratie van kinderen en adolescenten met MDS of JMML. Protocol EWOG-MDS 2006 is opgesteld door de European WOrking Group voor MDS op de kinderleeftijd (EWOG-MDS). De EWOG werd in 1993 opgericht door leden uit verschillende Europese landen en heeft tot doel om artsen en onderzoekers met MDS als aandachtsgebied, informatie te laten uitwisselen, de samenwerking te intensiveren en te fungeren als kennisbron voor andere behandelaars. Meer informatie over de EWOG-MDS kunt u lezen de website www.ewog-mds.org (Engelstalig).


In Nederland nemen de behandelend kinderartsen deel via de SKION (Stichting Kinderoncologie Nederland). De SKION is een landelijk samenwerkingsverband van alle Nederlandse kinderartsen en andere hulpverleners, die gespecialiseerd zijn in onderzoek en behandeling van kinderen met kanker of aandoeningen die kunnen leiden tot een vorm van kanker. De activiteiten van de SKION betreffen kinderen en adolescenten in de leeftijd van 0 tot 18 jaar. De SKION stelt zich ten doel: • optimale diagnostiek en behandeling van kinderen en adolescenten met kanker en voorstadia daarvan; • bevordering van het wetenschappelijk onderzoek naar kanker op kinderen adolescenten leeftijd. De SKION heeft een centraal bureau waar gegevens over de ziekte en behandeling van deze groep kinderen en adolescenten worden geregistreerd om zo de kwaliteit van de behandeling te kunnen controleren. Daarnaast verzamelt de SKION informatie ten behoeve van wetenschappelijk onderzoek. Meer informatie over de SKION kunt u lezen in de informatiebrief over de ‘SKION basisregistratie’ en op de website www.skion.nl Waar richt dit protocol zich op? Dit protocol wordt ook wel klinische studie genoemd. Het gaat bij EWOG-MDS 2006 studie niet om de behandeling van de ziekte van uw kind. Het EWOG-MDS 2006 protocol richt zich wel op twee aspecten onafhankelijk van de behandeling:

1. Diagnose: Om voor uw kind de beste behandeling te kunnen bepalen, worden bloeden beenmergmonsters afgenomen (in het geval van JMML of Noonan Syndroom wordt aanvullend een klein huidbiopt afgenomen of wordt wat wangslijmvlies afgenomen met behulp van een wattenstokje). Voor de studie worden tegelijkertijd met de reguliere afnames kleine hoeveelheden bloed en beenmerg (5 ml per keer) afgenomen. Kortom: dit betekent dat er geen extra prikken of ingrepen worden gedaan in het kader van de studie. De afgenomen monsters worden opgeslagen in ons ziekenhuis of in één van de door de EWOG-MDS aangewezen laboratoria. Deze monsters zijn bedoeld voor diagnostiek. U kunt apart toestemming geven voor opslag van het lichaamsmateriaal van uw kind voor toekomstig onderzoek naar MDS/JMML. Het gaat hier dan dus om overgebleven materiaal (‘restmateriaal’) wat voor research doeleinden wordt gebruikt.

2. Registratie van ziektebeloop: gegevens over ziektebeloop en de gezondheidstoestand van uw kind worden via de SKION doorgegeven aan het coördinerend studiecentrum in Freiburg, Duitsland.

Doel van het onderzoek Omdat MDS een zeer zeldzame ziekte is op de kinderleeftijd, willen we door middel van deze uniforme registratie en diagnostiek meer inzicht krijgen in MDS en JMML. Er wordt in het bijzonder gekeken naar: • het vóórkomen (de frequentie) van de verschillende subtypes van MDS bij kinderen en adolescenten door een

zogenaamde ‘gestandaardiseerde diagnostische aanpak’. Praktisch betekent dit dat het protocol een richtlijn vormt voor de manier waarop de onderzoeken worden gedaan en alle testen op dezelfde manier worden uitgevoerd.

• Wat is de frequentie van chromosoomafwijkingen in de bloeden beenmergcellen? Dit wordt uitgevoerd met behulp van de nieuwste technieken, om ons meer inzicht te geven in de zogenaamde cytogenetische en moleculaire afwijkingen van de cellen.

• Hoeveel kinderen genezen van MDS en JMML? • Hoeveel kinderen met MDS of JMML genezen na behandeling met hematologische stam cel transplantatie

(meestal ‘beenmergtransplantatie’ genoemd)? • Hoe is het klinisch beloop bij patiënten met het Noonan Syndroom? Het doel van dit protocol is het verzamelen van klinische informatie die mogelijk in de toekomst kan leiden tot een richtlijn voor de behandeling van kinderen met MDS of JMML en het verbeteren van de ziektevrije overleving. Op basis van de resultaten van voorgaande protocollen voor de behandeling van MDS, is besloten tot het huidige protocol. Hoe verloopt de registratie?

http://www.skion.nl


De registratieprocedure is internationaal en wordt in tientallen ziekenhuizen uitgevoerd, verdeeld over 12 verschillende landen. De verwachting is dat er in totaal circa 260 patiënten zullen worden geregistreerd. De behandelend arts is verantwoordelijk voor de medische zorg aan uw kind. Om het diagnostische proces te verbeteren, zal hij/zij bloeden beenmergmonsters (en bij JMML of Noonan Syndroom het al eerder genoemde huidbiopt of wangslijmvlies monster) opsturen naar de daarvoor aangewezen laboratoria. Na analyse worden de uitslagen met betrekking tot de diagnose en het ziektebeloop doorgestuurd naar het coördinerend centrum in Freiburg. Daar worden alle gegevens verzameld, bewaard en geanalyseerd in een beveiligde database. De richtlijnen met betrekking tot medische geheimhouding en bescherming van gegevens worden hierbij natuurlijk in acht genomen. Voor- en nadelen Omdat de afnames voor de studie altijd gecombineerd worden met de routine afnames die noodzakelijk zijn voor het bepalen van de beste behandeling voor uw kind, vinden zoals gezegd, geen extra afnames plaats. Er zijn daarom dan ook geen risico’s verbonden aan deelname aan deze studie. Behalve de gestandaardiseerde diagnostische benadering en de uniforme manier van behandelen, brengt deelname aan de studie voor uw kind geen voordelen met zich mee. De resultaten van deze registratie kunnen ook voor toekomstige patiënten van belang zijn. Er is geen financiële vergoeding voor deelname aan deze studie. Toestemming Indien u toestemming geeft voor deelname van uw kind aan het onderzoek, vragen we u een toestemmingsformulier te ondertekenen waarin staat dat u weet wat dit registratie- en diagnostiek protocol inhoudt en wat de consequenties zijn. Er is een multidisciplinair team beschikbaar om u en uw kind gedurende de behandeling zo goed mogelijk te begeleiden. Dit team bestaat o.a. uit gespecialiseerde verpleegkundigen en een psycholoog. Het behandelteam ziet nauwlettend toe op de belasting die deelname aan een onderzoeksprotocol voor kinderen vormt, hoewel van belasting geen sprake is in het geval van het EWOG-MDS 2006 protocol. We werken zoveel mogelijk volgens de landelijke afspraken zoals die door de Nederlandse Vereniging voor Kindergeneeskunde (NVK) zijn vastgelegd ter bescherming van minderjarige onderzoeksdeelnemers. Voor meer informatie hierover verwijzen wij u naar de volgende website: www.ccmo.nl (weten regelgeving/gedragscode verzet: minderjarigen). Verantwoording en vertrouwelijkheid Tot uw kind herleidbare registratiegegevens kunnen slechts met uw toestemming door daartoe bevoegde personen worden ingezien. Deze personen zijn medewerkers van het onderzoeksteam, medewerkers van de Inspectie voor de Gezondheidszorg of bevoegde inspecteurs van een buitenlandse overheid en leden van de Medisch Ethische Toetsings Commissie (METC). Inzage kan nodig zijn om de betrouwbaarheid en kwaliteit van het onderzoek na te gaan. Onderzoeksgegevens zullen worden gehanteerd met inachtneming van de Wet bescherming persoonsgegevens en het privacyreglement van het Erasmus MC. Persoonsgegevens die tijdens deze studie worden verzameld, zullen worden vervangen door een code, bestaande uit een studienummer en de initialen van uw kind. De sleutel van deze code zal alleen toegankelijk zijn voor de onderzoeker, de behandelend arts of de researchverpleegkundige/ datamanager. Alleen deze gecodeerde gegevens zullen gebruikt worden voor studiedocumentatie, in rapporten of publicaties over dit onderzoek. De vertrouwelijkheid van de gegevens blijft hierbij gewaarborgd. De gecodeerde gegevens worden opgeslagen in een computerbestand van het bureau voor de statistiek van de EWOG. Binnen Nederland worden de gegevens opgeslagen en verwerkt bij de SKION. De gegevens worden, indien u daar toestemming voor geeft, gedurende 15 jaar bewaard.

http://www.ccmo.nl


Lichaamsmaterialen die tijdens deze studie worden verzameld, worden gecodeerd opgeslagen. Na afloop van de studie worden de opgeslagen lichaamsmaterialen vernietigd of, indien u daarvoor toestemming geeft, gedurende maximaal 15 jaar na afloop van de studie bewaard. Het opgeslagen lichaamsmateriaal kan dan eventueel in de toekomst worden gebruikt voor onderzoek met een zelfde onderzoeksdoel. De onderzoeksresultaten worden gerapporteerd in medisch-wetenschappelijke literatuur en/of op medische congressen. In het kader van zorgvuldigheidstoetsing is het EWOG-MDS 2006 protocol voorgelegd aan de METC van het Erasmus MC. De voor dit onderzoek geldende internationale richtlijnen zullen nauwkeurig in acht worden genomen. Vrijwillige deelname Zoals al werd gezegd, is het EWOG-MDS 2006 protocol momenteel de standaard voor registratie en diagnostiek voor kinderen en adolescenten met MDS/JMML. De vrijwilligheid waar in deze patiënten informatie brief over wordt gesproken betreft de keuze of u toestemming geeft om de gegevens van uw kind gecodeerd te laten registreren, om gegevens te bewaren en om restweefsel na diagnostiek te mogen gebruiken voor wetenschappelijk onderzoek naar MDS/JMML bij kinderen. Deelname aan bovenstaande aspecten (registratie, weefselopslag) is geheel vrijwillig. Als u niet wilt dat uw kind hieraan gaat deelnemen, hoeft u daarvoor geen reden te geven. Als u dit besluit, zal dat geen enkele verandering brengen in de verdere behandeling of begeleiding. Ook indien u nu toestemming geeft, kunt u die te allen tijde zonder opgave van redenen weer intrekken. Ook zult u op de hoogte worden gehouden van eventuele nieuwe informatie die beschikbaar komt tijdens het onderzoek en die ertoe zou kunnen leiden dat u uw besluit om verder te gaan met het onderzoek wilt herzien. Mocht u besluiten tot deelname van uw kind aan het protocol, dan zal de behandelend arts u vragen dit document te ondertekenen en te dateren. Het ondertekenen van dit formulier is niet van invloed op de wettelijke rechten van uw kind, maar dient ter bevestiging van het feit dat u volledig geïnformeerd bent over het onderzoek, dat u het doel ervan en uw betrokkenheid erbij begrepen hebt en tenslotte, dat u vrijwillig instemt met deelname van uw kind. Het originele toestemmingsformulier dat door u is ondertekend, wordt bewaard in het patiëntendossier; u ontvangt een kopie van het door u ondertekende informatie- en toestemmingsformulier. Verzekering Voor dit registratieprotocol is de verzekering voor onderzoeksdeelnemers niet van toepassing. Het gaat hier om een registratie- en diagnostiek protocol in plaats van een onderzoeksprotocol: er is geen verschil in behandeling vergeleken met de standaardbehandeling van MDS/JMML . Contactpersonen Bij vragen of opmerkingen kunt u contact opnemen met dr. M.M. van den Heuvel-Eibrink, kinderarts-oncoloog, tel. 010-4636691, of met de researchverpleegkundigen van de afdeling kinderoncologie, Inekee van der Vaart of Eline Visser, tel. 010-4636402. Als u twijfelt over deelname van uw kind aan deze studie dan kunt u een onafhankelijke arts raadplegen die zelf niet bij het onderzoek is betrokken maar wel deskundig is op dit gebied: Dr. J.B. van Goudoever, kinderarts, tel. 010 4636077. Ook indien u voor of tijdens het onderzoek vragen heeft die u liever niet aan de onderzoekers stelt dan kunt u contact opnemen met de onafhankelijke arts. Indien u niet tevreden bent over het onderzoek of de behandeling dan kunt u terecht bij de onafhankelijke klachtencommissie van het ziekenhuis. U kunt zich daartoe wenden tot: de secretaris van de klachtencommissie Postbus 2040 3000 CA Rotterdam tel. 010-4633198


Neemt u de tijd om deze informatie door te spreken en aarzel niet uw behandelend arts te raadplegen als u vragen heeft. Met vriendelijke groet, Dr. M.M. van den Heuvel -Eibrink, Kinderarts-oncoloog


Toestemmingsformulier ouders/voogd

behorende bij de patiënteninformatie over registratie en diagnostiek bij kinderen met Myelodysplastisch Syndroom (MDS) en Juveniele Myelomonocytaire Leukemie (JMML) volgens het EWOG-MDS 2006

protocol Titel van het onderzoek:” Prospectieve niet-gerandomiseerd multicenter onderzoek naar epidemiologie en karakteristieken van MDS en JMML op de kinderleeftijd” (protocol EWOG-MDS 2006). Ik bevestig, dat ik het informatieformulier voor mijn kind heb gelezen. Ik begrijp de informatie. Ik heb de gelegenheid gehad om aanvullende vragen te stellen. Deze vragen zijn naar tevredenheid beantwoord. Ik heb voldoende tijd gehad om over deelname van mijn kind na te denken. Ik weet dat deelname geheel vrijwillig is en dat ik mijn toestemming op ieder moment kan intrekken zonder dat ik daarvoor een reden hoef te geven. Ik geef toestemming voor deelname van mijn kind aan de registratie en diagnostiek volgens het EWOG-MDS 2006 protocol onder de omstandigheden zoals die mij zijn uitgelegd. Ik geef wel/geen* toestemming om de gegevens van mijn kind gedurende 15 jaar na afloop van de studie te bewaren. Ik geef wel/geen* toestemming voor het langdurig (maximaal 15 jaar) bewaren van restweefsel, waar mogelijk in de toekomst verder onderzoek naar MDS en/of JMML mee gedaan wordt. Ik geef toestemming om de huisarts van mijn kind op de hoogte te brengen van zijn/haar deelname aan dit onderzoeksprotocol. Ik geef toestemming voor het gecodeerd verzamelen en verwerken van de gegevens over het verloop van de behandeling, zoals in deze informatiebrief beschreven is. De gegevens zullen worden opgeslagen in een database. De resultaten zullen voor wetenschappelijke doeleinden worden gebruikt waarbij de vertrouwelijkheid gewaarborgd wordt. Ik geef toestemming, dat daartoe bevoegde medewerkers van het onderzoeksteam, medewerkers van de Inspectie voor de Gezondheidszorg, bevoegde inspecteurs van een buitenlandse overheid of leden van de medisch-ethische toetsingscommissie inzage kunnen krijgen in de medische gegevens en onderzoeksgegevens van mijn kind. Naam kind: Geboortedatum: __ / __ / __ Naam ouder/voogd **: Handtekening: Datum : __ / __ / __ Naam ouder/voogd **: Handtekening: Datum : __ / __ / __ Naam onderzoeker/behandelend arts: Handtekening: Datum : __ / __ / __ * Doorhalen wat niet van toepassing is. ** Dit formulier moet worden ondertekend door de ouders die het gezag uitoefenen of de voogd, wanneer het

kind jonger dan 18 jaar is. Voor kinderen van 12 tot en met 17 jaar, die wilsbekwaam zijn, dient tevens het formulier voor kinderen door het kind zelf worden ondertekend.


18.7 Gegevens verzameling per Case Record Form De registratie en follow-up gegevens van kinderen met MDS of JMML worden verzameld op een Case Report Form. Het CRF wordt toegestuurd door het datamanagement van de SKION. De ingevulde CRF’s worden gecontroleerd en het origineel wordt doorgestuurd naar het coördinerend datacentrum in Freiburg, Duitsland. Daarnaast worden de gegevens ingevoerd in de ProMISe database en de kopieën van de CRF’s worden bewaard in de patiëntenfile bij SKION (centraal bureau). Een aantal puntjes die aandacht behoeven: Vul bij het item “Patient Name” alleen de initialen in. Vul bij het item “Patient Identification Number” het SKIONnummer in. De follow-up dient 1 keer per 12 maanden te worden ingevuld. Zie voor nadere informatie m.b.t. het invullen van het CRF ook sectie 8.1 van het protocol. Deze registratie studie zal vervolgd worden door een aantal behandelprotocollen, voor deze vervolgbehandelingen zullen aparte CRF’s worden samengesteld.


18.8 Ondersteunende maatregelen bij protocol EWOG-MDS-2006 Inleiding De behandeling van maligniteiten vergt een aantal maatregelen in de ondersteunende behandeling. Deze worden ingegeven door de betreffende medicatie, de toedieningsweg, toedieningsperiode en de dosering. Bij radiotherapeutische behandeling zijn het bestralingveld, volume, de dosis en de fractoinering bepalend voor de noodzakelijke ondersteunende therapie. De basale preventieve ondersteunende maatregelen treft u navolgend aan. Een deel van de maatregelen is niet gerelateerd aan een specifiek onderdeel van de behandeling maarmaar geldt als ondersteunend in algemene zin. Deze zijn het laatste onderdeel van deze paragraaf. Uiteraard bestaat een breed spectrum aan bijwerkingen en complicaties van elk betreffend medicament. Deze zijn onder andere terug te vinden in het Farmacotherapeutisch Kompas en kinderoncologische handboeken. Overigens wordt verwezen naar het werkboek “Ondersteunende behandeling in de kinderoncologie”, onder redactie van W.A. Kamps, M.C. Naafs-Wilstra, A.Y.N. Schouten-van Meeteren en W.J.E. Tissing, eindredactie C.M.F. Kneepkens. Therapie van JMML 6-Mercaptopurine individuele controleer TPMT deficientie igv sterke aplasie gevoeligheid toediening avonddosis op 1 uur nuchtere maag niet innemen met melkproducten hepatotoxiciteit transaminasen stijgingen tot 500 U / l behoeven geen dosisaanpassing leucopenie mn voor onderhoudstherapie 6-mercaptopurine

voorkom te hoge / lage leucocytenwaarden: streefwaarden volgens protocol VOD Bij 6-TG is er een verhoogde kans op het ontstaan van een VOD. (Trias: pijnlijk

vergrote lever, vochtretentie en icterus) Cis retinoic acid (isotretinoin) Gevoeligheid huid Toegenomen lichtgevoeligheid oppassen met de zon Hepatotoxiciteit en elektrolyte stoornissen Monitoren van leverfuncties, Triglyceriden en calcium Calcium>3.0 stoppen van de cis retinoic acid Aplasie mogelijk bij hoge dosering PAS OP met zwanger schap Roaccutan geeft ernstige geboorte defecten, dus anticonceptie is verplicht Cytosine arabinoside lage dosis < 1000 mg / m2 / kuur geen aanvullende maatregelen Cytosine arabinoside hoge dosis > 1000 mg / m2 / kuur hydratie 2,5 l/m2 emesis anti-emeticum 5HT3-antagonist keratitis/conjunctivitis oogdruppels corticosteroïden 4 dd tijdens kuur infektie streptococ vir profylaxe pheniticilline G 50 mg / kg in 3 dd tot na herstel uit neutropenie Igv peni-resistente streptococ in keelkweek claritromycine overwegen CZS & mucosa i.t.t. voorgaande protocollen wordt geen pyridoxine meer geadviseerd aangezien

er geen enkele evidence is dat dit profylactisch werkt Alle vormen MDS streven naar BMT met conditionering Busulfan, cyclo, melphalan


Busulfan Complicaties Kans op veno-occlusive disease (VOD).

Convulsies, evt preventief antiepilepticum geven Alkylerend chemotherapeuticum, dus verhoogde kans op verminderde vruchtbaarheid.

Maatregelen rond de kuur Spiegel controle noodzakelijk emesis anti-emeticum 5HT3-antagonist Cyclofosfamide emesis anti-emeticum 5HT3-antagonist nefrotoxiciteit 1 - hyperhydratie 3 l / m2 vanaf 3 uur voor start 2 - evt geforceerde diurese mbv furosemide bij mictie < 3 ml/kg/u Melfalan emesis anti-emeticum 5HT3-antagonist + dexa complicaties Anafylactische reactie

Mucositis, gastro-enteritis waarbij oa veel electrolyt verlies (vooral Natrium en bicarbonaat)

Aplasie diep met grote kans mucositis en verhoogde slijmproductie Algemene maatregelen emesis indien 5 HT3 antagonist ontoereikend is, overweeg dexamethason

10 mg/m2 in 3 dd en toevoeging van lorazepam pneumocysitis infektie cotrimoxazol profylaxe 3 dagen / week, 3/15 mg / kg / gift op 3 aaneengesloten

dagen Hypo- Gammaglobulinaemie in geval van infekties in tijdens de behandeling, overweeg gamma-globuline

substitutie obv IgG titer Transfusies bestraalde bloedtransfusieprodukten bij lymfopenie < 500.106/l, tot 6 maanden

na totaal lichaamsbestraling en na Fludarabine Infertiliteit semenpreservatie igv beenmergtransplantatie Teratogeniciteit De meeste chemotherapeutica zijn (potentieel) teratogeen. Bij oudere kinderen

is het daarom soms zinvol hiervoor te waarschuwen en anticonceptive maatregelen te nemen

Neutropenie en koorts start breed spectrum antibiotica indien de temperatuur een aantal uur achtereen

> 38,5 °C is Overweeg een hydrocortison stress schema indien de patient veel steroiden

heeft gehad in het recente verleden. Overweeg gamma globuline substitutie op geleidde van de spiegel Beenmergtransplantatie na de beenmergtransplantatie zijn aanvullende supportive care maatregelen

nodig. Deze verschillen per soort transplantaat e.d. en zullen dus per patient verschillen

Infectie profylaxe overweeg SDD en schimmel / gist profylaxe gezien de zeer langdurige neutropenie.


Appendix 8 MEC-Verklaring


Appendix 9 SKION protocol diagnostiek beenmergfalen Doelstellingen van dit protocol:

• Differentiaal diagnostisch handvat voor beenmergfalen • Efficiënte diagnose stelling • Criteria voor registratie bij het SKION • Criteria voor aanlevering van materiaal (bloed, beenmerg, botbiopt) voor revisie en cell-banking bij

de SKION Er wordt onderscheid gemaakt tussen single-cel cytopenie versus uitval van meerdere cellijnen. Voor cytopenie is de factor tijd van belang, waarbij beenmergfalen waarschijnlijker wordt bij langer bestaande cytopenie (>3 maanden). Stappenplan: Stap 1: Diagnoses uit te sluiten vóór start diagnostiek beenmergfalen In dit stadium wordt patiënt nog niet aangemeld bij de SKION Stap 2: Diagnostiek gericht op beenmergfalen

Patiënt gegevens en materiaal (bloed, beenmerg, botbiopt) naar SKION Stap 3: Bevestiging met moleculaire diagnostiek Opmerkingen:

• Het protocol dient als overzicht en zal worden samengevoegd met de diverse ziekte-georiënteerde protocollen van de SKION. In die protocollen staat de verdere diagnostiek en behandeling per ziektebeeld omschreven.

• Disclaimer: dit protocol dekt niet de volledige differentiaal diagnose, is gericht op beenmergfalen

diagnostiek en zeer zeldzame ziekte beelden zijn bewust buiten beschouwing gebleven. I. Single cel cytopenie van de rode reeks Stap 1: Diagnoses uit te sluiten vóór start diagnostiek beenmergfalen In dit stadium wordt patiënt nog niet aangemeld bij de SKION

Afbraak Hemolytische anemie Hb-pathie Auto-immuun Membraan defecten

Microangiopathie Enzym tekorten

Verbruik Hypersplenisme

Deficiënties IJzer Vit B12 Foliumzuur

Viral infecties CMV, EBV, ParvoB19 Medicatie Chronische ziekten

Aanmaak

Lood-intoxicatie Minimale diagnostiek:

Volledig bloedbeeld, reticulocyten, bili, haptoglobine, LDH, Vit B12, foliumzuur, HbF, TIJBC, ferritine, virusserologie


Stap 2: Diagnostiek beenmergfalen (persisterende anemie > 3 maanden!) Patiënt gegevens en materiaal (bloed, beenmerg, botbiopt) naar SKION

DBA MDS/RA SA Pearson CDA Anamnese

Pre-dysmaturiteit +/- - - + - Leeftijd 1e klachten <1 jaar Variabel >1 jaar Post-nataal >1 jaar

Groeistoornis +/- - - + - Familie +/- - +/- - +/-

Steatorrhoe - - - + -

Lichamelijk onderzoek

kleine lengte Cong. Afw.

splenomegalie kan -

klein, mager, bolle buik, retardatie

icterus mogelijk

Lab

bili & LDH↑ hapto↓

HbF HbF ↑ HbF ↑ HbF ↑ HbF ↑ HbF ↑ ADA in erys ADA kan

↑

HEMPAS serum lysis test + bij type II CDA

Beenmerg Celrijkdom rood ↓↓↓ Normaal tot ↓ normaal tot ↑ normaal normaal tot ↑

Dysplasie - + + - ++

IJzerkleuring gb gb ring sideroblasten

ring sideroblasten gb

Specifieke kenmerken vacuolisatie macrocytose, bi/multinucl. cellen

Cytogenetica normaal clonaal / monosomie 7 normaal normaal normaal

Botbiopt Cellulariteit normaal ↑ of ↓ ↓ normaal tot ↑ ↑

CD34 kleuring normaal ↑ normaal normaal normaal Architectuur normaal verstoord kan verstoord normaal normaal

DBA = Blackfan-Diamond anemie SA = Heriditaire sideroblastaire anemie MDS = Myelodysplastisch syndroom CDA = Congenitale dyserythropoietische anemie RA = Refractaire anemie NB: TEC (transient erythroblastopenia of childhood) heeft een piek incidentie tussen 1-3 jaar, meestal worden geen aanvullende afwijkingen gevonden bij anamnese, lichamelijk onderzoek, bloed-, beenmerg-, en botonderzoek. Het grootste deel van de patiënten zijn na 3 maanden herstellend. Deze diagnose hoeft niet geregistreerd te worden bij SKION


Stap 3: Bevestiging met moleculaire diagnostiek Standaard DNA-diagnostiek: 1-2 buizen 5 ml EDTA-bloed (volwassenen 20 ml EDTA) Internationale diagnostiek: Altijd overleg per email/telefoon met de betreffende kontakt persoon

Ziekte Gen locatie Product % Diagnostiek DBA1 19q13.3 Ribo prot 25

DBA2 8p23.2-p22 S19 40 Diamond Blackfan

anemie (DBA) onbekend 35

VUMC (Gerard Pals of Hans Gille) [email protected] of [email protected] Spoed: 1 mnd; normaal 3 mnd (zie standaard aanvraagformulier)

ALAS2

Hospital Universitario ‘La Paz’, Madrid Head of lab : Jesus Molano Mateos, MD PhD [email protected]

Hereditaire sideroblastische anemie (SA)

ATP7 Momenteel geen gendiagnostiek beschikbaar

Pearson syndroom Deleties mitochondr DNA

100

Sheffield Children’s Hospital, UK Contact persoon: Jo Martindale MSc MRCPath [email protected]

CDAN1 15q15.1-15.3

Codanin-1

CDAN2 20q11.2 CDAN3 15q22

Medical genetics Napoli Head of lab: Prof Achille Iolascon [email protected]

Congenitale dyserythropoietische anemie (CDA)

CDNA4-7 Alleen klinisch beschreven







II. Single cel cytopenie van de megakaryocytaire reeks Stap 1: Diagnoses uit te sluiten vóór start diagnostiek beenmergfalen In dit stadium wordt patiënt nog niet aangemeld bij de SKION Afbraak Immuun-gemedieerd

Hypersplenisme Verbruik Stollingsactivatie Micro-angiopathie

Kasabach-Merritt Chronische DIC

Deficienties Vit B12 Foliumzuur

Virale infecties EBV, CMV, ParvoB19

Aanmaak

Medicatie oa anti-epileptica Wiscott Aldrich Syndroom (WAS) Thrombocytopathie Giant platelets

Minimale diagnostiek: Volledig bloedbeeld, MPV, thrombopoietine (TPO), glycocalicine (GC), Vit B12, foliumzuur, virusserologie

Stap 2: Diagnostiek beenmergfalen (persisterende anemie > 3 maanden!)

Patiënt gegevens en materiaal (bloed, beenmerg, botbiopt) naar SKION

CAMT TAR FA MDS Dysmega- karyopoiese

Anamnese Dysmaturiteit - - +/- - -

Leeftijd 1e klachten < 1 jaar < 1 jaar variabel variabel < 1 jaar

Groeistoornis - - +/- - - +/- +/- - +/- Familie

Skeletafwijking - radius duim, radius en andere - -


radius aplasie hoefijzer nier

card. afwijkingen

duim, radius aplasie en andere cong afwijkingen;

cafė-au-lait vlekken

soms splenomegalie

Lab TPO ↑↑↑ ↑↑↑ ↑ ↑ normaal of ↑

GC ↓↓ ↓↓ N/↓ N/↓↓ N/↓↓ Lab fase II

HbF Normaal/↑ normaal ↑ ↑ normaal MMC-test normaal normaal verhoogde gevoeligheid normaal normaal

Beenmerg megakaryocyten afwezig afwezig ↓ ↓ of ↑ normaal

Dysplasie - - - ++ +++

Cytogenetica normaal normaal Clonale afwijkingen Clonale afw./ monosomie 7 normaal

Botbiopt Cellulariteit verlaagd geen megakar. ↓ ↓ of ↑ normaal

CAMT : congenitale amegakaryocytaire thrombopenie TAR : thrombocytopenia with absent radius FA : Fanconi anemia MDS : myelodysplatisch syndrome


Stap 3: Bevestiging met moleculaire diagnostiek Standaard DNA-diagnostiek: 1-2 buizen 5 ml EDTA-bloed (volwassenen 20 ml EDTA) Internationale diagnostiek: Altijd overleg per email/telefoon met de betreffende kontakt persoon Ziekte Gen locatie Product % Diagnostiek Congenitale amegakaryocytaire thrombopenie (CAMT)

C-MPL 1p35 100 Masja de Haas, Sanquin (op research basis) [email protected]

Thrombocytopenia with absent radius (TAR) onbekend Momenteel geen gendiagnostiek

mogelijk FANC-A 1455 27 FANC-B 16q24.3 FANC-C 13q 558 35 FANC-D1 9q22.3 3418 5 FANC-D2 3q12.3 1451 9 FANC-E 6p21.3 536 5 FANC-F 11p15 374 9 FANC-G 9p13 622 5 FANC-I 5 FANC-J FANC-L 2p16.1

Fanconi anemie (FA)

FANC-M

De hier beschreven percentuele verdeling is de Nederlandse verdeling, deze wijkt af van de internationale verdeling. NB 1e lijns diagnostiek fanconi: Chromosoom breuk onderzoek Lab voor chromosoom diagnostiek VUMC Aanvragen indienen in overleg: Tel: 020-4440745 of 020-4440157 5-10 ml heparinebloed Standaard aanvraag duurt 4 weken Insturen op ma/di/vr voor 15.00 uur Gendiagnostiek: VUMC (Gerard Pals of Hans Gille) [email protected] of [email protected] (zie standaard aanvraagformulier)

GATA-1 FLi-1 FOG-1 NF-E2 Dysmegakaryopoiese

Gfi-1b

GATA-1 kan binnenkort worden bepaald in een Rotterdamse research setting Monique den Boer, Sophia Kinderziekenhuis [email protected] 010-46388224 of 010-46388340 (lab)






III. Single cel cytopenie van de myeloide reeks Stap 1: Diagnoses uit te sluiten vóór start diagnostiek beenmergfalen In dit stadium wordt patiënt nog niet aangemeld bij de SKION Afbraak Imuun-gemedieerd Verbruik Hypersplenisme

Virale infecties CMV, EBV, Parvo B19 Hepatitis A, B, C Deficienties Vit B12 foliumzuur Aanmaak Medicatie oa anti-epileptica

Minimale diagnostiek:

Volledig bloedbeeld, Vit B12, foliumzuur, virusserologie, antistoffen tegen neutrofielen, immunoglobulines

Stap 2: Diagnostiek beenmergfalen (persisterende anemie > 3 maanden!) Patiënt gegevens en materiaal (bloed, beenmerg, botbiopt) naar SKION

SCN, Kostmann syndroom*

Cyclische neutropenie

Reticulaire dysgenesie

Shwach-man

Dyskeratosis Congenita

Cartilage hair hypoplasia

Chediak-Higashi

Anamnese Pre-dysmaturiteit - - +/- - - - -

1e klachten < 3 maand variabel <3 maand variabel variabel variabel variabel

Groeistoornis - - +/- + - ++ -

Familie +/- +/- +/- +/- +/- - -

Overig laat afvallen navelstreng

Periodieke infecties steatorrhoe


nageldystrofie; hyperpigmentatie; leukoplakie

Dun/ongepig-menteerd haar

oculaire blindheid; albinisme

Lab

ANC variatie met periodiciteit 20-24 dgn.

lymfocyten verminderd en functie gestoord

lymfocyten verminderd en functie gestoord

granulae In de neutrofielen

fase II lab diagnostiek

exocriene pancreas; metafysaire dysostose

metafysaire dysostose

Beenmerg

Myeloide reeks

Rijpingsstop promyelocyt

nivo

kan verminderde witte reeks

hebben

afwezige myelo- en

lymfopoiese hypocellulair hypocellulair hypocellulair

dysplastisch, niet hypocellulair

Botbiopt Cellulariteit: zie beenmerg

SCN = severe congenital neutropenia NB: let op gingiva hyperplasie – dat hebben vrijwel alle patienten.


• er bestaan ook SCN (severe congenital neutropenia) beelden met eenzelfde rijpingsstop die NIET op GCSF reageren.

Stap 3: Bevestiging met moleculaire diagnostiek Standaard DNA-diagnostiek: 1-2 buizen 5 ml EDTA-bloed (volwassenen 20 ml EDTA) Internationale diagnostiek: Altijd overleg per email/telefoon met de betreffende kontakt persoon

Ziekte Gen locatie Product % Diagnostiek

ELA2 19q13.3 90

Diagnostiek wordt opgezet in het WKZ Dr Marry Bruin ([email protected]) Severe congenital

neutropenia (SCN)

GCSF-rec 10 Research Rotterdam: Prof dr Ivo Touw ([email protected] of 010-4087837)

Cyclische neutropenie ELA2 19q13.3 90

Diagnostiek wordt opgezet in het WKZ Dr Marry Bruin ([email protected])

Reticulaire dysgenesie Momenteel geen gendiagnostiek mogelijk

Shwachman-Diamond SDBS 7q11 250 90 AMC (Mariel Alders, pieper 58960) [email protected]

DKC1 Dyskeratosis congenita (DC) TERC

Imperial College London Hammersmith Hospital Tom Vulliamy ([email protected])

Cartilage Hair syndroom RMRP 267 Klinische diagnose

Chediak-Higashi CHS1 1q42.1-42.2 LYST 100

Segregatie analyse van polymorfe markers die gelinkt zijn aan Chediak-Higashi. Hospital Necker-Enfants Malades Paris Dr G. de Saint Basile ([email protected])








IV. UITVAL MEERDERE CELLIJNEN (2 OF MEER) Stap 1: Diagnoses uit te sluiten vóór start diagnostiek beenmergfalen In dit stadium wordt patiënt nog niet aangemeld bij de SKION Afbraak Imuun-gemedieerd

Hypersplenisme Verbruik Hemofagocytair syndroom (zie SKION protocol) Deficienties Vit B12 foliumzuur Medicatie oa anti-epileptica Virale infecties EBV, CMV, ParvoB19 Hepatitis A, B, C Leishmaniasis

Aanmaak

Verdringing Leukemie solide tumoren

Metabool

Osteopetrosis Methylmalaonzuur acidaemie Organoacidurie Aminoacidurie

Minimale diagnostiek:

Volledig bloedbeeld, reticulocyten, bili, haptoglobine, LDH, Vit B12, foliumzuur, ferritine, fibrinogeen, triglyceriden, virusserologie

Stap 2: Diagnostiek beenmergfalen (hier geldt geen 3 maanden criterium!)

Patiënt gegevens en materiaal (bloed, beenmerg, botbiopt) naar SKION

AA FA PNH MDS Osteopetrose Anamnese

Pre-dysmaturiteit - + - - -

1e klachten variabel variabel variabel variabel < 1 jaar Groeistoornis - + - - macrocephaal

Familie - +/- - - +/- Overig - buikpijn hersenzenuwuitval


duim radius skelet afw congenitale afw

cafe-au-lait vlekken

hersenzenuwuitval, hepato-spleno

megalie,macrocephalie Lab

MMC-test PI-

verankerde eiwitten

extramedullaire hematopoiese

(erythroblasten in perifere bloed),

hemolyse Beenmerg

cellulariteit ↓ ↓ variabel ↓ of ↑

Cytogenetica * * * clonaal + monosomie 7

Botbiopt Cellulariteit ↓ ↓ variabel ↓ of ↑ ↓ Dysplasie - - - ++ +

Reticuline - - - toename reticuline toename reticuline

AA: aplastische anemie PNH: paroxysmale nachtelijke hemoglobinurie FA: Fanconi anemie MDS: myelodysplastisch syndroom


• bij een pure aplastische anemie hoeft geen cytogenetische afwijking gevonden te worden, wat niet uitsluit dat deze later in het beloop kunnen ontstaan.

• Stap 3: Bevestiging met moleculaire diagnostiek Standaard DNA-diagnostiek: 1-2 buizen 5 ml EDTA-bloed (volwassenen 20 ml EDTA) Internationale diagnostiek: Altijd overleg per email/telefoon met de betreffende kontakt persoon Ziekte Gen locatie Product % Diagnostiek

Aplastische anemie (AA) TERT 5p15.33 Klinische diagnose, geen gendiagnostiek

FANC-A 1455 27 FANC-B 16q24.3 FANC-C 13q 558 35

FANC-D1 9q22.3 3418 5 FANC-D2 3q12.3 1451 9 FANC-E 6p21.3 536 5 FANC-F 11p15 374 9 FANC-G 9p13 622 5 FANC-I 5 FANC-J FANC-L 2p16.1

Fanconi anemie (FA)

FANC-M

De hier beschreven percentuele verdeling is de Nederlandse verdeling, deze wijkt af van de internationale verdeling. NB 1e lijns diagnostiek fanconi: Chromosoom breuk onderzoek Lab voor chromosoom diagnostiek VUMC Aanvragen indienen in overleg: Tel: 020-4440745 of 020-4440157 5-10 ml heparinebloed Standaard aanvraag duurt 4 weken Insturen op ma/di/vr voor 15.00 uur Gendiagnostiek: VUMC (Gerard Pals of Hans Gille) [email protected] of [email protected] (zie standaard aanvraagformulier)

Paroxysmale nocturneale hemoglobinurie (PNH)

PIG-A X-p22.1 GPI-AP 100 (zie SKION protocol)

V. LABORATORIA VOOR MOLECULAIRE DIAGNOSTIEK Gendiagnostiek wordt in principe aangevraagd via het eigen klinische genetisch centrum. Informatie over gendiagnostiek kan gevonden worden op de volgende websites:

Nederland: http://www.dnadiagnostiek.nl/ Europa: http://www.eddnal.com/ Verenigde Staten: http://www.genetests.org/ Aanvraagformulieren voor moleculaire diagnostiek in de Nederlandse centra: http://www.dnadiagnostiek.nl/formulieren_nl.php Voor het aanmelden van een patiënt met Beenmerg Falen wordt het CRF u door de SKION toegestuurd. Zie de volgende pagina’s voor het voorbeeld CRF.



http://www.dnadiagnostiek.nl/

http://www.eddnal.com/

http://www.genetests.org/

http://www.dnadiagnostiek.nl/formulieren_nl.php


Verdenking beenmergfalen/MDS Formulier 1, pre-registratie

Patiënt identificatie

DCOG patiënt ID Insturend ziekenhuis: ……………………………………………

Initialen patiënt Geboortedatum (dd.mm.jjjj): _ _ . _ _ . _ _ _ _

Anamnese Datum afname (dd.mm.jjjj) beenmerg: _ _ . _ _ . _ _ _ _ Botbiopt: _ _ . _ _ . _ _ _ _

Voorgeschiedenis …………………………………………………………………………………………………………

Specifieke klachten …………………………………………………………………………………………………………

Familie anamnese beenmergfalen …………………………………………………………………………………………………………

Aanvullende observaties …………………………………………………………………………………………………………

Lichamelijk onderzoek Abnormaal J/N ? Ja Nee Zo ja, specificeer:

Alg. Lich. Onderzoek ……………………………………………………………………………………………………

… Hepatomegalie ……………………………………………………………………………………………………

…

Splenomegalie ………………………………………………………………………………………………………

Lymfadenopathie ………………………………………………………………………………………………………

Dysmorfiën ………………………………………………………………………………………………………

Gewicht : kg Lengte: cm Datum - - . - - . - - - -

Aanvullende diagnostiek Volledig bloedbeeld: Datum: (dd.mm.jjjj): _ _ . _ _ . _ _ _ _

Hb . mmol/L Blasten % Neutrofielen %

Leucocyten . x 109/L Lymfocyten % Eosinofielen %

Thrombocyten . x 109/L Monocyten % Basofielen %

Reticulocyten ‰

Chemie: Haptoglobine: g/L

LDH: U/L

Hepatitis A: Hepatitis B: Hepatitis C: Virus serologie:

(IgG/IgM: pos/neg) CMV: EBV: Parvo B19:

PNH diagnostiek: CD55 pos neg CD59 pos neg PIG-A mutatie: ja nee

Cytogenetica: Karyotype: ………………………………………………………………………………………….………………

FISH monosomie 7 Verricht: ja nee Afwijkend: ja nee Toelichting: ……………………………………….

Chromosoom breuktest Verricht: ja nee Afwijkend: ja nee Toelichting: ……………………………………….

Genmutatie diagnostiek Verricht: ja nee Afwijkend: ja nee Toelichting: ……………………………………….


Classificerende diagnose: ja ……………………………………………………………………………………………………………

nee: werkdiagnose: ……………………………………………………………………………………………………………………..

Opmerkingen : …………………………………………………………………………………………………………………

Naam insturende arts ..……………………………..

Datum (dd.mm.jjjj) _ _ . _ _ . _ _ _ _ Handtekening…………………………………………...

DCOG CRF: pre-registratie verdenking beenmergfalen/MDS page 139/197 Versie 1 Datum : 18-07-07

Stuur origineel naar SKION


Verdenking beenmergfalen/MDS Formulier 2, follow up pre-registratie (na 6 maanden)

Patiënt identificatie

DCOG patiënt ID Insturend ziekenhuis: ……………………………………………

Initialen patiënt Geboortedatum (dd.mm.jjjj): _ _ . _ _ . _ _ _ _

Bestaat er nog verdenking op beenmergfalen?

Ja Nee Is de patiënt nog in leven?

Ja Nee

Toelichting:……………………..……………………………………………………………………………………………………………………

Anamnese Evt. aanvullende anamnese ……………………………………………………………………………………………………………………

…………………………………………………………………………………………………………...……….

Lichamelijk onderzoek (alleen veranderingen tov 1e CRF doorgeven) Abnormaal J/N ? Ja Nee Zo ja, specificeer:

Alg. Lich. Onderzoek ………………………………………………………………………………………………………

Hepatomegalie ………………………………………………………………………………………………………

Splenomegalie ……………………………………………………………………………………………………

…

Lymfadenopathie ………………………………………………………………………………………………………

Aanvullende diagnostiek Vervolg beenmerg (dd.mm.jjjj) _ _ . _ _ . _ _ _ _ _ _ . _ _ . _ _ _ _ _ _ . _ _ . _ _ _ _ Vervolg botbiopt (dd.mm.jjjj) _ _ . _ _ . _ _ _ _ _ _ . _ _ . _ _ _ _ _ _ . _ _ . _ _ _ _ Laatste bloedbeeld (dd.mm.jjjj) _ _ . _ _ . _ _ _ _

Hb . mmol/L Blasten % Neutrofielen %

Leucocyten . x 109/L Lymfocyten % Eosinofielen %

Thrombocyten . x 109/L Monocyten % Basofielen %

Reticulocyten ‰

PNH diagnostiek: CD55 pos neg CD59 pos neg PIG-A mutatie: ja nee

Cytogenetica: Karyotype: …………………………………………………………………………………………………………

FISH monosomie 7 Verricht: ja nee Afwijkend: ja nee Toelichting: ……………………………………………

Chromosoom breuktest Verricht: ja nee Afwijkend: ja nee Toelichting: ……………………………………………

Genmutatie diagnostiek Verricht: ja nee Afwijkend: ja nee Toelichting: ……………………………………………


Classificerende diagnose: ja ……………………………………………………………………………………………………………

nee: werkdiagnose: ……………………………………………………………………………………………………………………..

Opmerkingen : …………………………………………………………………………………………………………………

Naam insturende arts ..……………………………..

Datum (dd.mm.jjjj) _ _ . _ _ . _ _ _ _

Handtekening…………………………………………... DCOG CRF: pre-registratie verdenking beenmergfalen/MDS page 141/197 Versie 1 Datum : 18-07-07

Stuur origineel naar SKION


Appendix 10 Registratie, diagnostiek en behandelingsrichtlijn voor kinderen met PNH klonen in Nederland.

Aanbeveling ten behoeve van SKION PC-MDS, PC-BMF en sectie benigne hematologie. E.T.Korthof M.M. van den Heuvel-Eibrink Inleiding 1. Klinische presentatie en pathofysiology van PNH. Paroxysmale nachtelijke hemoglobinurie (PNH) wordt gekenmerkt door hemolyse, trombose, ernstige met name KNO infecties en verworven beenmerg falen. Bij volwassenen wordt daarnaast regelmatig abdominale pijn, dysphagie en erectie-stoornissen gezien. Het is een verworven monoklonale aandoening van een deel van de hematopoietische stamcellen met een prevalentie van 1-10 per miljoen volwassen individuen. Van patiënten met AA is bekend dat PNH klonen bij diagnose niet aanwezig zijn maar zich tijdens de ziekte ontwikkelen of juist spontaan verdwijnen (1). PNH is zelden gerapporteerd bij kinderen en de incidentie is onbekend(2-10). PNH wordt veroorzaakt door een mutatie in het Phosphatidylinositol Glycan complementation class A (PIG-A) gen gelegen op het X-chromosoom, hetgeen, in het geval dat er een overgroei van de PNH kloon optreedt, een afwijkende synthese van het glycosylphosphatidylinositol (GPI) anker eiwit op gang brengt (7-9, 11). Dit glycolipide anker in het celmembraan draagt oppervlakte markers als DAF (decay accelerating factor, CD55) en MIRL (membrane inhibitor of reactive lysis, CD59). Vooral CD59 inhibeert het MAC (complement membrane attack complex) en beschermt de cel tegen lysis(12). Bij activatie van het complement zal de afwijkende klonale populatie erytrocyten hemolyseren. Naast CD59 kunnen de erythrocyten deficiënties laten zien van CD55, CD58 en CD108. GPI-deficiënte thrombocyten en voorlopers kunnen bv. urokinase-type plasminogeen activator(u-PAR), cellulaire prion eiwitten((PrPc), CD55, CD24, CD58,CD59 en CD108-deficiënt zijn. De deficiëntie van u-PAR and verhoogde u-PAR plasma spiegels kan de celgebonden fibrinolytische activiteit remmen, terwijl een deficiëntie van CD59 ze extreem gevoelig kan maken voor de C5b-9-geinduceerde prothrombinase activiteit (13). Tenslotte veranderd door complement activatie het celmembraan van de erythrocyten dusdanig dat hij meer gevoelig wordt voor thrombose. Op het oppervlak van de (voorlopers van) granulocyten zijn normaal de GPI gebonden markers CD16, CD24, CD55, CD58, CD59, CD66b/CD67 aanwezig, deze kunnen bij PNH patiënten deficiënt zijn, waarbij tevens CD55, CD59, CD73, CD87, CD108, CD48, CD58, CD52, CD24 en CD90 deficiënte lymfocyten kunnen bijdragen aan de cytopenie en ernstige opportunistische infecties(14).

2. Klassificatie van PNH. PNH wordt volgens het recente voorstel van de Internationale PNH Interest Group (IPEG) geclassificeerd in 3 categorieën (15):

1. Klassieke PNH. Deze patiënten hebben klinisch tekenen van intravasculaire hemolyse maar een normocellulair beenmerg.

2. PNH in het kader van een gespecificeerde vorm van beenmergfalen. Deze patiënten hebben klinische en laboratorium-technische tekenen van hemolyse en daarnaast een aangetoond verworven beenmergfalen (BMF) (bijvoorbeeld aplastische anemie(AA)), of myelodysplastisch syndroom (MDS).

3. Subklinische PNH (PNH-sc). Deze groep heeft GPI-deficiënte klonen zonder aantoonbare klinisch en laboratorium-technische tekenen van hemolyse.

3. PNH bij kinderen. Pediatrische PNH series zijn schaars, tot op heden zijn ongeveer 40 kinderen goed-gedocumenteerd terug te vinden in de literatuur (2, 4, 6, 16-23). Hieruit blijkt dat PNH bij kinderen regelmatig niet wordt herkend, dat de tijd tot aan het stellen van de diagnose meestal lang is en dat er regelmatig onjuiste diagnoses worden gesteld. Er is weinig bekend over de klinische presentatie, thrombose risico, respons op therapie en overleving bij kinderen. Een retrospectieve studie bij 26 kinderen liet zien dat de presentatie vergelijkbaar is met die bij volwassenen te weten beenmergfalen, hemolyse en thrombose (2). Het lijkt echter aannemelijk dat bij kinderen de hemolytische varianten minder vaak voorkomen en dat kinderen zich vaker presenteren met beenmerg falen (17). Het is niet ondenkbaar dat bij patiënten met bijvoorbeeld een Budd-Chiari de diagnose in het verleden nooit is overwogen.


4. Gerapporteerde behandelingsstrategieën van patiënten met PNH klonen. De therapie van patiënten met PNH klonen is afhankelijk van de klinische presentatie, en therapie resultaten zijn met name bekend bij volwassen series patiënten. Behandeling van hemolyse. De anemie bij patiënten met PNH is multifactorieel. Deze kan optreden als gevolg van Coombs-negatieve hemolytische anemie, en/of deficiënte hematopoiesis ten gevolge van stamcel dysfunctie in het beenmerg. Daarnaast kan het klinisch beeld wisselend zijn door de variatie in de grootte van de GPI-deficiënte klonen. Patiënten met hemolytische anemie zijn in het verleden behandeld met corticosteroïden, androgenen, ijzer substitutie, transfusies, en splenectomy. Alhoewel individuele responsen bekend zijn is voor geen van deze therapieën ooit aangetoond dat ze zinvol zijn. Bij patiënten met de hemolytische varianten wordt wel aangeraden foliumsubstitutie te starten (15). Recente studies hebben aangetoond dat complement remmers effectief zijn als remmers van hemolyse (15, 24). Daarnaast hebben studies in volwassen patiënten met een gehumaniseerd monoclonale antistof tegen complement C5 (Eculizumab) een zeer positief effect laten zien op de hemolyse, maar ook op andere klachten zoals erectiestoornissen, gladde spier dystonieën, en slikklachten.(25, 26). Preventie en behandeling van thrombose. Het relatieve hoge risico op een thrombo-embolische complicatie bracht Hall et al. er toe warfarine prophylaxe te adviseren bij patiënten met meer dan 50% deficiënte granulocyten (13). Tot op heden is er echter geen advies te geven op basis van gerandomiseerde prospectieve studies en wordt bij elke patiënt de afweging om wel of niet prophylactisch te antistollen individeel gemaakt.. In het geval van acute, meestal veneuze, thrombose wordt aangeraden te hepariniseren, en bij Budd-Chiari kan radiologische interventie overwogen worden (15). Als thrombose is aangetoond zal de patiënt levenslang ontstold dienen te worden. Het trombocytenaantal moet nauwkeurig gevolgd worden en eventueel dmv. transfusies op peil gebracht worden Stamcel transplantatie. De internationale adviezen m.b.t. stamcel transplantatie (SCT) zijn gebaseerd op studies in volwassen patiënten. De Franse groep die het grootste aantal patiënten geregistreerd heeft tot dusver, met een mediane overleving van 12 jaar, rapporteerde de aanwezigheid van thrombose, progressie naar pancytopenie, tranformatie naar MDS en thrombocytopenie bij diagnose als de belangrijkste prognostische factoren voor overleving (27). De keuze voor transplantatie dient te worden gemaakt op basis van het type PNH, de ernst van de symptomen, zoals recidiverende thrombo-embolieën, en refractaire, transfusie- afhankelijk anemie (28, 29). Op dit moment zijn er geen algemene adviezen over optimale tijdsplanning van SCT, conditionering, donor keuze en de invloed van de ontwikkeling van nieuwe behandelings strategieën op de indicatie voor SCT te geven. SKION Registratie, Diagnostiek en Therapie Advies voor kinderen in Nederland met PNH klonen. 1. Registratie Centrale registratie van deze zeer zeldzaam voorkomende pediatrische patiënten met PNH klonen na uniforme diagnostiek is wenselijk, om een zo goed mogelijk inzicht te krijgen in de presentatie, response op behandeling en overleving van deze patiënten. Registratie wordt verricht binnen de gebruikelijke protocollen (MDS/AA) met behulp van aanvullend CRF (Appendix 1). Op het moment dat een PNH patiënt wordt geïdentificeerd (dit zal hooguit 1 patiënt per 1-3 jaar zijn, is het wenselijk deze data ook internationaal op te nemen in de database van de Internationale PNH Interest Group IPEG). De vertegenwoordiger van de SKION voor deze registratie, die PC overstijgend is, dient aangewezen te worden door het SKION bestuur. Deze vertegenwoordiger zal de individuele behandelende kinderarts benaderen en de registratie en follow-up formulieren sturen van de IPEG en toestemming voor internationale registratie vragen. De informatie op formulieren die horen bij de IPEG registratie zullen niet worden opgenomen in de SKION database maar er zullen wel kopieën worden verstuurd naar de SKION voor centrale data bewaking.


2. Diagnostiek Indicatie voor diagnostiek naar PNH klonen bij kinderen:

• Patiënten met hemoglobinurie • Patiënten met Coombs-negatieve intravasculaire hemolysis • Patiënten met onbegrepen niet lijn-gerelateerde veneuze thrombose • Patiënten met een verworven beenmergfalen (AA en MDS) • Patiënten met onbegrepen paroxysmale dysphagie • Patiënten met priapisme

Diagnostiek van PNH klonen bij kinderen.

1. Diagnostiek van PNH klonen. De huidige standaard voor het aantonen van PNH klonen is het immunophenotypisch aantonen van GPI-deficiënte klonen door middel van flowcytometrie. Deze techniek heeft de Acid Ham test en sucrose lysis test compleet vervangen. Met immunophenotypering gebruikmakend van het panel van de volgende markers kan PNH worden aangetoond: CD14,(monocyten), CD16, CD24 (granulocyten), en CD55, CD59 voor erythrocyten.

In het RC protocol van the EWOGMDS is deze diagnostiek reeds geïntegreerd. In dat kader zal voor alle Europese patiënten deze PNH screening plaats vinden in Rotterdam op het laboratorium Immunologie (V.van der Velden).

1. Minimaal te screenen hemolyse parameters: Hb, LDH, bilirubine(indirect), directe Coombs,

reticulocyten en haptoglobine, urine op hemoglobine.

2. BMF work-up. volgens SKION protocollen BMF/MDS bij kinderen, inclusief BM morphologie, histologie en cytogenetisch onderzoek.

Van patiënten met AA is bekend dat PNH klonen bij diagnose niet aanwezig zijn maar zich tijdens de ziekte ontwikkelen of juist spontaan verdwijnen (1). Daarom is het wenselijk AA en MDS patiënten die (nog) niet getransplanteerd zijn jaarlijks te screenen op PNH klonen

3. Behandelingsrichtlijn voor kinderen met PNH klonen Behandeling van hemolyse. Op de kinderleeftijd zullen de hemolytische vormen van PNH zeer zelden voorkomen. In voorkomende gevallen is er net zo min als bij volwassenen evidence dat cortocosteroïden en androgenen zinvol zijn, alhoewel incidentele reponsen zijn waargenomen, zoals in de Nederlandse serie (17). Supportive care maatregelen zoals ijzer suppletie, foliumzuur substitutie, en splenectomy dienen te worden genomen op basis van klinische gronden. In de zeldzame patiënten met hemolytisch varianten, dient eculizumab, zeker overwogen te worden. Hiervoor is het wenselijk contact te zoeken met hematologen die volwassenen behandelen in de Academische centra aangezien dit middel momenteel slechts op basis van compasionate use of in studieverband beschikbaar is (Contact persoon Dr. P Muus, Hematoloog, UMCG). Preventie en behandeling van thrombosis. Het risco op mortaliteit bij volwassenen is grotendeels bepaald door ernstige thombo-emboliën. In de kinderserie van Ware et al, had 31% van de kinderen aangetoonde thrombose, waaronder Budd-Chiari, in de Nederlande serie hadden 2/11 patiënten een niet-levensbedreigende thrombosis (2, 17). Dit suggereert dat het thrombose risico niet groter is dan bij volwassenen, het kleine aantal patiënten maakt echter uiteraard elke conclusie onmogelijk. Daarnaast is het heel wel mogelijk dat PNH nooit overwogen is bij kinderen met een onbegrepen vena porta thrombose. Aangezien grote studies in kinderen ontbreken en het risco op thrombose multifactorieel is, is er daarom geen reden om aan te nemen dan dat profylactische antistolling zoals bij


volwassenen wel geadviseerd wordt, zinvol is (13). Indien preventieve antistolling overwogen wordt, is er keus tussen subcutane toediening van laag moleculair gewicht heparine (LMWH) en orale toediening van cumarinederivaten. LMWH’s hebben als voordeel dat het effect heel makkelijk te onderbreken is voor een eventuele ingreep door een injectie over te slaan en dat er geen controle van de stolling nodig is (wel van het trombocytengetal!), als nadeel dat er dagelijks subcutaan gespoten moet worden (een insuflon kan uitkomst bieden voor zo’n 5 dagen). Orale anticoagulantia (OAC’s, mn. cumarinederivaten) daarentegen moeten wel gecontroleerd worden (INR), maar het grote voordeel is de orale toedieningsweg. Daarom zijn LMWH’s eerste keuze bij een te verwachten korte duur van de profylactische behandeling, OACs bij een langer gebruik. Voor doseringen zie appendix 2. In het geval van acute, meestal veneuze, thrombose bij kinderen wordt geadviseerd te hepariniseren, en bij Budd-Chiari kan radiologische interventie overwogen worden (15). Als trombose is opgetreden zal de patiënt tot aan transplantatie of levenslang ontstold moeten worden (appendix 2). Het trombocytenaantal moet nauwkeurig gevolgd worden en eventueel dmv. transfusies op peil gebracht worden, ‘Antistolling bij PNH patiënten’. Behandeling van BMF. Aangezien bij kinderen met PNH klonen meestal een verworven beenmergfalen zullen hebben, zullen de therapie en ondersteunende maatregelen gericht moeten zijn op het behandelen van de onderliggende AA of MDS, volgens de richtlijnen van de voorhanden zijnde SKION protocollen. In een retrospectieve studie van 26 AA kinderen met PNH klonen werd een goede response op immunosuppressieve therapie gezien bij 6 van de 9 kinderen (2). Dit kan overwogen worden bij patiënten met hypocellulair beenmerg en zonder cytogenetisch klonale afwijkingen, niet anders dan bij alle kinderen met RC nu wordt geadviseerd in het EWOG-MDS protocol. De retrospectieve serie in Nederland liet zien dat de ziekte gerelateerde mortaliteit aaanzienlijk was bij patiënten met PNH klonen, in de aanloop naar SCT, zodat men zich af kan vragen of in deze tijd van nauwkeuriger HLA typering, een MUD met goede match niet gelijkwaardig zou moeten worden overwogen als HLA identieke sibling in het geval dat een SCT wordt overwogen. Over succes van SCT bij kinderen met PNH klonen is slechts causuïstiek gerapporteerd. In Nederland zijn tot zover 5 patiënten getransplanteerd (3MUD, en 2 met een matched family donor) waarvan er momenteel nog 4 in leven zijn (17). In Duitsland zijn 2 kinderen succesvol getransplanteerd, waarvan 1 een Budd-Chiari had voorafgaande aan MUD-SCT (4).


CRF voor SKION registratie van kinderen met PNH klonen in Nederland Gegevens worden opgeslagen in de SKION database gelinked aan SKION AA en MDS database. Te registreren gegevens: PNH klasse: 1.Klassieke vorm 2.PNH/MDS of PNH/AA

3.PNH-sc Datum diagnose: MDS/AA Symptomatologie: Hemoglobinurie (macroscopisch) Thrombose Datum Thrombose Lokalisatie Thrombose Dysphagie Priapisme Uitslag PNH klonen

% of positive cells CD14 ….% of monocytes CD16 ….% of granulocytes CD24 ….% of granulocytes CD55 ….% of erythrocytes CD59 ….% of erythrocytes

Verder relevant laboratorium onderzoek (ongeveer op Datum DX PNH klonen) Urine: Hb Bloed:

Hb LDH Reticulocyten Haptoglobine Bilirubine ongeconjugeerd Dir. Coombs

Follow-up op jaarlijkse basis volgens dit CRF.


Dosis LMWH voor behandeling van veneuze trombose (CBO richtlijn) Leeftijd

Nadroparine (13) Enoxaparine (11) Dalteparine (12) Tinzaparine (10)

0 – 2 mnd 2 – 12 mnd 1 – 5 jaar 5 – 10 jaar 10– 16 jaar

90-120IE/kgsc. 2dd 85.5 IE/kg sc. 2dd 85.5 IE/kg sc. 2dd 85.5 IE/kg sc. 2dd 85.5 IE/kg sc. 2dd

1.5 mg/kg sc. 2dd 1.0 mg/kg sc. 2dd 1.0 mg/kg sc. 2dd 1.0 mg/kg sc. 2dd 1.0 mg/kg sc. 2dd

129± 43 IE/kg sc. 1dd 129± 43 IE/kg sc. 1dd 129± 43 IE/kg sc. 1dd 129± 43 IE/kg sc. 1dd 129± 43 IE/kg sc. 1dd

275 IE/kg sc. 1dd 250 IE/kg sc. 1dd 240 IE/kg sc. 1dd 200 IE/kg sc. 1dd 175 IE/kg sc. 1dd

Dosis LMWH voor preventie van veneuze trombose Nadroparine < 20 kg nadroparine 85.5 IE/kg sc 1x daags 20-30 kg nadroparine 950 IE sc 1x daags 30-50 kg nadroparine 1900 IE sc 1x daags > 50 kg nadroparine 2850 IE sc 1x daags (anti-factor Xa- spiegel: 0.1 – 0.3 kU/L) Enoxaparine < 2 maanden 2dd 0.75 mg/kg > 2 maanden 2dd 0.5 mg/kg Dosis OAC voor therapie en preventie van veneuze trombose (INR tussen 2 en 3)

Leeftijd Oplaaddosis: Acenocoumarol /phenprocoumon

< 1 jaar 1-5 jaar > 5 jaar

0.15 mg/kg po 0.1 mg/kg po 0.05 mg/kg po


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20. Endo M, Beatty PG, Vreeke TM, Wittwer CT, Singh SP, Parker CJ. Syngeneic bone marrow transplantation without conditioning in a patiënt with paroxysmal nocturnal hemoglobinuria: in vivo evidence that the mutant stem cells have a survival advantage. Blood 1996;88(2):742-50.

21. Graham ML, Rosse WF, Halperin EC, Miller CR, Ware RE. Resolution of Budd-Chiari syndrome following bone marrow transplantation for paroxysmal nocturnal haemoglobinuria. Br J Haematol 1996;92(3):707-10.

22. Lin HC, Chen RL, Wang PJ. Paroxysmal nocturnal hemoglobinuria presenting as moyamoya syndrome. Brain Dev 1996;18(2):157-9.

23. Wainwright L, Brodsky RA, Erasmus LK, Poyiadjis S, Naidu G, MacKinnon D. Paroxysmal nocturnal hemoglobinuria arising from Fanconi anemia. J Pediatr Hematol Oncol 2003;25(2):167-8.


24. Hill A, Ridley SH, Esser D, Oldroyd RG, Cullen MJ, Kareclas P, et al. Protection of erythrocytes from human complement-mediated lysis by membrane-targeted recombinant soluble CD59: a new approach to PNH therapy. Blood 2006;107(5):2131-7.

25. Hillmen P, Hall C, Marsh JC, Elebute M, Bombara MP, Petro BE, et al. Effect of eculizumab on hemolysis and transfusion requirements in patiënts with paroxysmal nocturnal hemoglobinuria. N Engl J Med 2004;350(6):552-9.

26. Hill A, Rother RP, Hillmen P. Improvement in the symptoms of smooth muscle dystonia during eculizumab therapy in paroxysmal nocturnal hemoglobinuria. Haematologica 2005;90(12 Suppl):ECR40.

27. Socie G, Mary JY, de Gramont A, Rio B, Leporrier M, Rose C, et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. French Society of Haematology. Lancet 1996;348(9027):573-7.

28. Antin JH, Ginsburg D, Smith BR, Nathan DG, Orkin SH, Rappeport JM. Bone marrow transplantation for paroxysmal nocturnal hemoglobinuria: eradication of the PNH clone and documentation of complete lymphohematopoietic engraftment. Blood 1985;66(6):1247-50.

29. Storb R, Evans RS, Thomas ED, Buckner CD, Clift RA, Fefer A, et al. Paroxysmal nocturnal haemoglobinuria and refractory marrow failure treated by marrow transplantation. Br J Haematol 1973;24(6):743-50.


Appendix 11 SKION add-on study : Determination of phenotypical and biological characteristics of mesenchymal stromal cells. NB: Deze add-on studie is nog niet goedgekeurd door de METC ten tijde van het samenstellen van dit protocol. Dit betekent dat de goedgekeurde versie kan afwijken van deze versie. De goedgekeurde versie zal zo spoedig mogelijk op de website van SKION gepubliceerd worden!

Determination of

phenotypical and biological

characteristics of mesenchymal stromal

cells in pediatric myelodysplastic

syndromes: add-on study to

EWOG-MDS: Prospective Non-Randomized Multi Center

Study

for Epidemiology and Characterization of Myelodysplastic

Syndromes (MDS) and Juvenile Myelomonocytic Leukemia

(JMML)

in Childhood.”


Protocol management

Protocol ID

Short title

File name

Study title for participants:

Date

MSC study in children with MDS

E:\RESEARCH PROTOCOLS\MSC and MDS EWOG

studie\MSC add on study.doc

Fenotype en biologische functie van mesenchymale

stamcellen in kinderen met Myelodysplastich syndroom.

Created on 28-06-2006 12:07

Principal investigator

Collaborating investigators

Data managers

Affiliations

LM Ball1

RM Egeler1, W Kollen1, MJD van Tol1, NE Annels1, MW

Schilham1, DHJ Verhoeven1, WE Fibbe2, H Roelofs3

PAM de Koning Gans,1 JDJ Bakker-Steeneveld1

Departments of 1Pediatrics, 2Hematology and 3Immunohematology and Transfusion Medicine

Albinusdreef 2,

2300 RC Leiden,

the Netherlands.

Tel: +31-71-526 4132

Fax: +31-71-524 8198

E-mail: [email protected]

[email protected]

[email protected]

[email protected]

N.E. [email protected]

[email protected]

[email protected]

[email protected]

Independent pediatrician

Center …………………..

Dr. R Sukhai

Dr. ……………………………………………










Protocol signature sheet

Name

Signature

Date

Dr. L.M. Ball, study co-ordinator

LUMC Leiden, tel 071 526 2743

Center specific clinical investigator

Hospital

Tel

Email

Administration

Submitted Date Approved Date

Commissie Medische

Ethiek (CME)

Start of the study

End of the study


TABLE OF CONTENTS

LIST OF ABBREVIATIONS 152

PROTOCOL SUMMARY 153

1.0 INTRODUCTION 156

1.1 Background 156

1.1.1 Bone marrow microenvironment 156

1.1.2 Myelodysplastic syndrome in children 157

1.1.3 Treatment of childhood MDS/JMML 157

1.1.4 Phenotypic and biological features of MDS derived MSC’s 157

1.1.5 Stem cell transplantation in MDS 160

1.2 Rationale 161

1.3 Clinical Relevance 161

2.0 OBJECTIVES 162

3.0 ELIGIBILITY CRITERIA 163

3.1 Patient inclusion criteria 163

3.2 Patient exclusion criteria 163

3.3 Donor inclusion criteria 163

3.4 Donor exclusion criteria 163

4.0 DESIGN AND CONDUCT OF THE STUDY 164

4.1 Trial design and sample size

164

4.2 Methodology 164

4.2.1 Isolation of MNC's from bone marrow 165

4.2.2 Isolation of CD34+ve cells from bone marrow 165 4.2.3 Biological characterization of MDS derived MSC’s 165

4.2.3.1 Phase I 165

4.2.3.1.a) Differentiation 165

4.2.3.1.b) T cell interaction 166

4.2.3.1.c) Natural killer cell interaction 166

4.2.3.1.d) Antigen presenting cell interaction 166

4.2.3.1.e) T regulatory cell interaction 166

4.2.3.1.f) Cytokine, chemokine and chemokine

receptor analysis 167

4.2.3.1.g) Chromosomal characterization 167

4.2.3.1.h) Growth kinetics 167

4.2.3.2 Phase II 167

4.3 Additional materials required for the study 168


4.3.1 Blood sample 168

4.3.2 Bone marrow samples 168

5.0 ENDPOINTS 169

5.1 Study endpoints 169

5.1.1 Primary endpoints 169

5.2 Patient’s discontinuation 169

5.3 End of study 169

6.0 DATA AND ADMINISTRATIVE ASPECTS 170

6.1 Data handling and record keeping 170

6.2 Data analysis 170

6.3 Statistical analysis 170

6.3.1 Planned sample size 170

6.3.2 Power calculation 170

Missing, unused and spurious data 170

6.3.4 Selection of subjects 170

6.4 Access to source data and documents 170

6.5 Budget consideration 171

6.6 Quality assurance 171

6.7 Address for biological samples 171

7.0 ETHICAL CONSIDERATIONS 172

7.1 Regulatory statement 172

7.2 Recruitment and consent 172

7.3 Compensation for injury 172

8.0 LITERATURE 173

Appendix A: CASE REPORT FORM

177

Appendix B: INFORMATION LETTERS AND INFORMED CONSENT 178


LIST OF ABBREVIATIONS

AML Acute myeloid leukemia

ANLL Acute non-lymphoblastic leukemia

BM Bone marrow

CST Center for Stem Cell Therapy

DFS Disease free survival

EBMT European Blood and Marrow Transplantation

EWOG-MDS European Working Group of MDS in Childhood

FCS Fetal calf serum

GCP Good clinical practice

HPC Hematopoietic progenitor cells

HSCT Hematopoietic stem cell transplantation

IHOBA Sub-section: Immunology, hemato-oncology, bone marrow transplantation and

autoimmune disease

i.v. / I.V. Intravenous (ly)

JMML Juvenile myelomonocytic leukemia

LTC Long-term cultures

LUMC Leiden University Medical Center

MDS Myelodysplastic syndrome

MSC’s Mesenchymal stem cells

NC Nucleated cells

NDMP National donor marrow transplant program

PBMC Peripheral blood mononuclear cells

PBSC Peripheral blood stem cells

PCR Polymerase chain reaction

QA Quality assurance

QC Quality control

RA Refractory anemia

RAEB Refractory anemia with excess blasts

RAEB (t) Refractory anemia with excess blasts in transformation

RARS Refractory anemia with ring sideroblasts

RC Refractory cytopenia

SKION Stichting Kinder Oncologie Nederlands

UPN Unique patient number

WBC White blood cell

WHO World Health Organization

WMO Wet mensgebonden medisch onderzoek

WV Werkvoorschrift


PROTOCOL SUMMARY

Title

Determination of phenotypical and biological characteristics of mesenchymal stem cells in

pediatric myelodysplastic syndromes: add-on study to EWOG-MDS: Prospective Non-

Randomized Multi-Center Study for Epidemiology and Characterization of Myelodysplastic

Syndromes (MDS) and Juvenile Myelomonocytic Leukemia (JMML) in Childhood.”

Aims

The study is designed to determine the phenotypical, biological and functional characteristics

of bone marrow derived, expanded mesenchymal stromal cells (MSC’s) from children with

myelodysplastic syndrome (MDS) compared to de-novo acute non-lymphoblastic leukaemia

(ANLL) and normal paediatric controls. The effect of haematopoietic stem cell transplantation

(HSCT) will be studied by examining MSC’s functions and their ability to support normal

haematopoiesis, both pre and post HSCT. Potential persistent MSC’s abnormalities will be

examined as predictors of relapse post HSCT.

Background

The pluripotent stromal stem cells or so called Mesenchymal Stromal Cells (MSC’s), located

in the bone marrow, give rise to cells that form the structural network in support and

maintenance of normal hematopoiesis. The growth and differentiation of hematopoietic stem

cell progenitor cells rely on instructive signals provided by a specialized micro-environment.

MSC’s provide signaling by cell-cell contact and release of soluble factors. They themselves

are influenced by the developing hematopoietic stem cell. The stromal cells isolated from

marrow can be expanded ex vivo for biological studies.

Myelodysplastic syndrome is a heterogeneous disease characterized by

hematomorphological dysplasia, cytopenia, cytogenetic abnormalities and leukemic

transformation. In children, MDS accounts for less than 10% of all cancers and has some

distinct characteristics that distinguish the disease from its adult counterpart.

The contribution of the non hematopoietic microenvironment in MDS is controversial but there

is increasing evidence to suggest that bone marrow stromal defects occur both in adult and

pediatric MDS patients and that stromal cell/hematopoietic stem cell interactions may

influence the initiation and/or progression of MDS.


Cytogenetic abnormalities have been recently reported in MSC’s isolated from adult patients

with MDS, albeit other authors have questioned this finding. No study related to functional

capacities of MSC’s as yet exists within the pediatric setting in either normal study

populations of children with MDS. Chromosomal aberrations if indeed present may differ from

those of hematopoietic stem cells in light of the different pluripotent precursor cells postulated

between the mesenchymal stromal cell and the hematopoietic stem cell.

Modification of stromal precursors could be the result of infiltrating malignant cells, which

generate conditions favorable to the development of leukemia. As such, precursor MSC’s

may be functionally different in children with MDS and within the heterogeneity of the

disease, i.e. depending upon leukemic development.

Hypothesis

It is our hypothesis, given the relationship between hematopoiesis and MSC's, that functional

dysregulation and changes in the cell to cell signaling could be implicated in the disease

process of MDS and its progression.

It is expected (based upon the published data in adults, albeit scarce) that phenotypic

characteristics of MSC’s isolated from MDS patients will not significantly differ from normal

controls. Intrinsic chromosomal aberrations might be evident although the published data in

adults is conflicting and there is no available data in children.

Dysregulation of MSC’s may lead to functional differences especially regarding the ability of

MDS derived stroma to support normal hematopoiesis, as well as differences in MDS derived

MSC cytokine production and immune interactions compared to normal controls. Chemokine

receptor analysis of MDS derived MSC’s may show changes in comparison to published

data, which to date have not been explored in pediatric MDS patients.

Immune modulation by normal MSC’s has been well documented and changes in immune

modulatory capacity may reflect functional abnormalities of MDS derived MSC’s

All of these changes may well be more pronounced as the disease progresses toward AML in

relation to further disruption of the MSC/CD34+ cell interactions suggestive of MSC/HSC

disruption as a requisite for the development and/or maintenance of the MDS condition.

If our hypothesis is true normal signaling and interaction may be restored following

successful hematopoietic stem cell transplantation. If so pre and post transplant analysis may

be of benefit in prediction of relapse.

Co-transplantation of donor derived MSC’s may well enhance success of transplant albeit

their engraftment numbers are relatively low, sufficient correction of the patients’ stromal cell

compartment may be achieved with normal hematopoietic stem cells from the donor graft.

Study design


All patients registered with MDS/JMML in the European Working Group of MDS in Childhood

(EWOG-MDS/JMML) 2006 study are eligible to participate. Following informed consent 10-15

cc of additional bone marrow aspirate will be withdrawn at or around the time of diagnosis or

immediately before hematopoietic stem cell transplantation (HSCT). At the same time 20 cc

of EDTA blood and 5cc plasma will be withdrawn with the routine diagnostic blood sampling.

All samples will be shipped to the LUMC, Leiden, the Netherlands for subsequent analysis.

For children <10kg the blood sampling will be reduced to 10cc EDTA blood and 5cc plasma.

CD 34+ve hematopoietic stem cells (HSC’s) will be isolated from bone marrow and used in

the subsequent experiments. MSC’s will be isolated and expanded ex vivo to provide

sufficient cells for analysis. Phenotypic and biological parameters will be investigated,

inclusive of cytokine production, chemokine receptor analysis, chromosome changes and

interaction with immune regulatory cells. These results will be compared to CD34+ve HSC’s

and MSC’s obtained at the time of routine bone marrow harvest from consenting normal

pediatric bone marrow donors. The study is designed to recruit a minimum of 25 patients and

10 controls. Statistical analysis of the results will be undertaken depending upon the data

sets obtained.

In children undergoing HSCT for MDS, MSC’s will be isolated pre-transplant and their

functional characteristics will be compared to MSC’s isolated at routine follow up 3-12 months

post transplant, to determine the influence if any of hematopoietic stem cell transplantation.

The ability of the marrow stroma to support normal hematopoiesis will also be tested at the

same time points.


1.0 INTRODUCTION 1.1 Background.

1.1.1 Bone marrow microenvironment

The bone marrow consists of hematopoietic cells and adherent stromal non-hematopoietic

cells that form a structural network known as the microenvironment or bone marrow stromal

compartment.

The pluripotent stromal stem cells or so call Mesenchymal Stem Cells (MSC’s), located in the

bone marrow, give rise to cells that form the structural network in support and maintenance of

normal hematopoiesis. There they give rise to chondrocytes, osteoclasts, fibroblasts, and

adipocytes, which form the cellular components of the marrow microenvironment.1-3 By

promoting cell- to- cell interactions, the expression of cytokines and growth factors and

secretion of extracellular matrix proteins this microenvironment provides the necessary

requirements for the localization, renewal and differentiation of hematopoietic stem cells.4

The growth and differentiation of hematopoietic stem cell progenitor cells rely on instructive

signals provided by a specialized micro-environment. MSC’s provide signaling by cell-cell

contact and release of soluble factors.5-7 They themselves are influenced by the developing

hematopoietic stem cell. 8,9 Infiltration of abnormal cell, as seen in malignant hematopoietic

conditions may dysregulate the fine control exhibited between the non hematopoietic stromal

compartment and regulation of hematopoiesis. It has been reported that normal

hematopoiesis relies on the functional integrity of mesenchymal stem cells within the bone

marrow.10

MSC’s can be isolated albeit in low numbers from bone marrow but are easily expanded ex

vivo for biological studies. At present no unique phenotype has been identified that allows the

reproducible isolation of MSC’s precursors with predictable developmental potential. 11

Restricted non-hematopoietic antigen expressions, phenotypic appearances and their ability

to differentiate into specific cell lineages readily characterize them.12

Standard conditions for expansion of MSC’s include the presence of serum, in most instances

fetal calf serum. Cell density is a critical factor affecting the growth of cells. Culture attempts

are usually unsuccessful below a critical cell density. The cells can be grown directly i.e.

unmanipulated, following collection or after density gradient separation.

The characterization of stromal cell function of MSC’s therefore, still relies primarily on their

ability to adhere to plastic and their expansion potential.

Treatments with MSC’s are presently being pursued in various investigational clinical phase I-

II protocols.


1.1.2. Myelodysplastic syndrome in children

Myelodysplastic syndrome (MDS) in children is a rare (<10% of all hematological

malignancies) yet diverse disease characterized by cytopenias, hematomorphological

dysplasia of the bone marrow and the risk of transformation into acute myeloid leukemia.13

The clinical course of MDS can be divided into several distinct phases related to the

percentage of leukemic blasts in the bone marrow. In the early indolent phase affected

patients exhibit only refractory cytopenia (RC). Increasing number of blasts signifies a pre-

leukemic phase and eventually the development of MDS related AML. Adult MDS is classified

according to specific morphological features (FAB classification). 14 This has been widely

accepted for adult type MDS but although in some ways similar to its adult counterpart,

childhood MDS differs in distribution (more advanced disease), progression and is associated

with specific chromosome re-arrangements e.g. monosomy 7.15,16 In children there is an

overlap with the myeloproliferative syndromes, many of which are unique to the pediatric age

group, the most common being Juvenile Myelomonocytic Leukemia (JMML).17

Presently, the European Working Group of MDS in Childhood (EWOG-MDS/JMML 2006

study) is conducting clinical and epidemiological studies into the diagnosis and outcome of

treatment of children with MDS and JMML. These patients routinely undergo bone marrow

evaluation around the time of diagnosis, before and following HSCT.

1.1.3 Treatment of childhood MDS/JMML

At present, the only curative treatment for these pediatric disorders is allogeneic HSCT.

HSCT can cure more than half of the affected children.16 In a recent study of outcome of

allogeneic bone marrow transplant for the treatment of pediatric MDS/JMML, the estimated 3-

year probabilities of survival; event free survival (EFS), non-relapse mortality and relapse

were 50, 41, 28 and 29%, respectively. Patients with RA/RARS had an estimated 3 year

survival of 74% compared to 68% in those with RAEB and 33% in patients with JMML. In

multivariate analysis patients with RAEBt or JMML were 3.9 and 3.7 times more likely to die

compared to those with RA/RARS and RAEB (P=0.005 and 0.004, respectively). Patients

with RAEBt were 5.5 times more likely to relapse (P=0.01).18

1.1.4 Phenotype and biological functions of MDS MSC’s

There is evidence to suggest that bone marrow stromal defects occur in adult MDS patients

and that stromal cell/hematopoietic stem cell interactions may influence the initiation and/or

progression of MDS.19 The post natal trans-germal plasticity of adult MSC's (i.e. the ability to


develop into cells phenotypically unrelated to the cell of origin) has been demonstrated in so

much that they can give rise to neural and muscle tissue as well as to myeloblasts.20

Various methodologies have been reported to investigate functional and phenotypic

characterization of normal MSC's21, but only limited information exists in adult MDS patients

in relation to phenotypic and cytogenetic characterization.22 Chromosomal abnormalities of

MSC’s in adult MDS patients however remain controversial 23, with only one sporadic report in

children.24 Thus further investigation in children with defined clinical and morphological

determinants is warranted.

Gene expression profiling has been reported in mainly adult MDS patients. Selection of an

appropriate hematopoietic fraction is important and most reports have included the

neutrophil25 or CD34+ hematopoietic stem cells26 fraction isolated from MDS patients.

Although several genes were commonly up or down regulated gene profiling has

demonstrated a subset of genes which were able to discriminate between the different

subtypes of MDS and AML.25-28

Gene expression profiling in pediatric MDS ha not been widely studied albeit attempts have

been made in the pediatric setting to determine gene stage specific expression in the

microenvironment.29 Using a cDNA microarray assay a clear difference in the gene

expression profile of the bone marrow stroma from children with MDS and AML compared to

normal donors was evident. Interestingly the profile between de-novo MDS and a MDS

induced AML was sufficiently different to be discriminatory.

There is no published data concerning homing functions as determined by chemokine

receptor expression in MSC's from patients with MDS although some limited investigation has

been undertaken in normal healthy donors to determine the receptors that can influence

homing to and positioning of MSC’s within the bone marrow.30,31 Several chemokine axes

have been elucidated in early passage of MSC cultures that may be important in stromal cell

biology. 31 This study lends itself to an unique opportunity to compare the chemokine axes in

pediatric MDS patients compared to de novo ANLL and normal pediatric bone marrow

expanded MSC’s. Whether or not this could be used as an expression of MSC's dysfunction

in MDS patients requires further investigation.

MSC’s are functionally immune modulatory32-35 and characterize normal stromal progenitors.

Investigation of immune modulatory function of MSC’s isolated from children with MDS is thus

an additionally relevant comparative determinant.

Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or

nonspecific mitogenic stimuli 32 and inhibit the response of naïve and memory antigen-


specific T-cells to their cognate peptide.32 Human MSCs altered the cytokine secretion profile

of dendritic cells (DCs), naive and effector T cells (T helper 1 [T(H)1] and T(H)2), and natural

killer (NK) cells to induce a more anti-inflammatory or tolerant phenotype. 34

At low NK-to-MSC ratios, MSCs alter the phenotype of NK cells and suppress proliferation,

cytokine secretion, and cyto-toxicity against HLA-class I– expressing targets. 35 Some of these

effects require cell-to-cell contact, whereas others are mediated by soluble factors, including

transforming growth factor–ß1 and prostaglandin E2, suggesting the existence of diverse

mechanisms for MSC-mediated NK-cell suppression. On the other hand, MSCs are

susceptible to lysis by activated NK cells.

Cytolytic function and survival of NK cells have been reported to be abnormal in

myelodysplastic syndrome.36 Although quantitatively normal numbers are found in the

peripheral blood of adult patients with MDS, they cytolytic function is abnormal. This is

despite expression of NKp46 and NKp30 as well as NKG2D (activating natural cytotoxicity

receptors). The proliferative capacities of MDS NK cells were also profoundly altered

compared to normal activated donor NK cells, with IL2 induced apoptosis being up-regulated.

Subtypes of MDS show a similar disturbed T cell repertoire as that seen in severe aplastic

anemia and increased apoptosis of hematopoietic stem cells.37,38 The number of CD3+ cells

in MDS bone marrow is significantly increased compared to normal controls both in relative

and absolute numbers. The percentage of CD34+ cells were also increased with no

significant differences in CD8+ or CD20+ cells.

Given the fact that adult MDS samples exhibit immunological disturbances both in T cell and

NK function and that MSC’s have demonstrated important interaction between these classes

of immunological cells, it would be interesting to investigate the properties of NK function in

pediatric MDS patients as well as the effect of MDS derived MSC’s on MDS and donor

derived NK cell and T cell function.

The role of indoleamine 2, 3-dioxygenase (IDO) production to inhibit T cell proliferation39 and

the interaction between IDO and MSC’s has been described.40 No data exists in MDS or

ANLL patients and identification of disturbances in this pathway would lend credence to the

hypothesis that MDS MSC’s functionally are different than normal derived cells.

To date there is scarce data regarding the phenotypic or biological properties of MSC’s that

are derived from pediatric patients with MDS.


There has however, been attempts to define abnormalities in a limited number of children

with MDS by isolating a trypsin resistant macrophage like “stroma” from children with MDS

and co-culturing with umbilical cord blood normal CD34+ cells.41 The study concluded that

because the MDS “stroma” induced pathological differentiation, this reflected alteration in

hemato-myelo supportive role. The latter as previously determined is the function of normal

MSC’s.10 The same group continued to define these abnormalities using cDNA microarray

technology in their attempts to determine gene stage specific expression in the

microenvironment.29

1.1.5 Stem cell transplantation in MDS

The treatment of MDS is allogeneic bone marrow transplantation. In general, there are few

reports of randomized studies between peripheral blood stem cell transplantation and bone

marrow as source of cells.42-45 Most are comparative retrospective analyses with occasional

attempts at case control studies, albeit in a small number of patients (n=42).46 In children,

small heterogeneous groups of childhood malignancy have been compared to historical

controls. 47 The largest comparison was undertaken by the Myelodysplastic Syndromes of the

chronic leukemia working party of the European Blood and Bone marrow transplantation

group, which include both adults and children.48 They concluded that PBSC’s may be

preferred for patients with high risk for relapse based upon advanced disease or high risk

cytogenetics as PBSC’s were associated with lower treatment failure and improved survival.

In a retrospective analysis by the EWOG-MDS group who analyzed data concerning children

transplanted for JMML, survival was influenced by the donor source.49 However, the majority

of patients had received bone marrow (n=79) with only 14 PBSC’s and 7 cord blood

transplants. In JMML, eradication of disease, irrespective of donor, source remains the

greatest challenge with a 5 year cumulative incidence of leukemia relapse of 35%.

All reports of multi center survival outcome are complicated by two main factors. Non –

relapse transplant related mortality, especially in the advances stages of MDS is

considerable.42,44,50 Infection is an important determinant of survival. Transplant with high

doses of peripheral blood stem cells reduced significantly the period of neutropenia following

SCT and has significant impact on survival. 42,44 Similarly, chronic GvHD is more evident

following PBSCT and is associated with a reduction on relapse rates.43,45,50,51 G-CSF not only

induced release of PBSC’s but has been shown to increase the number of MSC’s that can be

isolated from circulating blood.52 Given this and the fact that most children have received

bone marrow derived stem cells it is not possible to determine from clinical reports the

significance of stromal (marrow) transplant over highly purified PBSC’s in the outcome of

disease after HSCT.


In our unit, we have the unique capacity to undertake pre and post transplant analysis of cells

and in this study we will focus on functional aspects of MSC’s. Changes inherent to the

functional capacity of the MSC interactions with hematopoietic progenitors or modulatory

effector cells of the immune system have not been examined post SCT in MDS.

1.2 Rationale

Although clinical treatment protocols exist for other indications, any modulation of bone

marrow stromal interactions in MDS patients requires insights into the interactions between

MSC's and hematopoietic stem cells from children newly diagnosed with MDS and classified

according to standard diagnostic criteria.

The proposed series of experiments is designed to detail the comparative characterization of

populations of MSC's and HSC’s in both normal and MDS pediatric patients. Subsequently,

the ability of MDS MSC's to support normal hematopoiesis as well as normal MSC's to correct

abnormal MDS hematopoiesis pre and post transplant will be investigated.

1.3 Clinical relevance

To develop insights into the functional characteristics and interactions between MSC's and

HSC’s in pediatric MDS patients that could allow for potential new targets for therapy.

Should there be abnormalities observed for MDS derived MSC’s, both functionally and

related to their ability to support normal hematopoiesis, future strategies for the infusion of

allogeneic MSC’s to augment engraftment and reduce toxicities associated with bone marrow

transplantation could be contemplated in children treated for MDS.


2.0 OBJECTIVES

2.1 To determine the phenotype and biological characteristics of mesenchymal stem cells

derived from pediatric MDS patients as determined by flow cytometric analysis, cell

culture and differentiation abilities analyzed by subtype of MDS compared to de novo

ANLL and normal pediatric controls.

2.2 To analyze functional characteristics of MSC's isolated from pediatric MDS patients:

a. In relation to immune regulation of T and NK cell function

b. To compare cytokine and growth factor expression of MSC’s and the

expression of chemokine receptor profiles of MSC’s and HSC’s isolated

from children with MDS analyzed by subtype of MDS compared to de

novo ANLL and normal control marrows.

2.3 To compare chromosomal difference between patient HSC’s and MSC's in children

with different subtypes of MDS utilizing karyotyping and/or chromosome painting

techniques.

2.4 To analyze genetic profiles of cell proliferation, apoptosis and differentiation of MSC’s

isolated form children with MDS compared to MSC’s analyzed by subtype utilizing

gene array analysis compared to de novo ANLL and pediatric normal controls.

2.5 To determine whether or not normally functioning MSC’s impact on hematopoietic

growth in MDS patients and whether MDS derived MSC’s support hematopoietic

growth of normal CD34+ cells (pre and post allogeneic HSCT) by long-term culture

(Dexter type).


3.0 ELIGIBILITY CRITERIA

3.1 Patient inclusion criteria

3.1.1 Children aged 0-18 years.

3.1.2 Patients enrolled in EWOG-MDS/JMML 2006 study.

3.1.3 Informed written consent.

3.2 Patient exclusion criteria

3.2.1 Failure of bone marrow aspirate.

3.2.2 Failure of MSC expansion.

3.3 Study control inclusion criteria

3.3.1 Normal fully screened donor undergoing bone marrow harvest.

3.3.2 Sufficient cells for marrow recipient.

3.3.3 Pediatric patients with de novo ANLL

3.3.4 Informed written consent.

3.4 Donor exclusion criteria

3.4.1 Insufficient harvest (cell dose below target).

3.4.2 Failure of MSC expansion.


4.0 DESIGN AND CONDUCT OF THE STUDY

4.1 Trial design and sample size

The study is an open label design. Eligible, consented patients located in the Netherlands will

number 25 with 10 normal control subjects from Department of Pediatrics, Leiden University

Medical Center, (LUMC). Additional samples will be obtained if possible from FAB matched

de novo ANLL patients to act as abnormal hematopoietic controls (selected participating

centers). In addition patients undergoing subsequent HSCT will be tested pre and post

transplant from bone marrow samples obtained from collaborating centers. The expected

period of patient accrual is approximately 36 months. The study is designed to recruit one

patient per month. An interim report will be submitted to the Medical Ethical Committee,

LUMC and SKION onderzoekscommissie after the first fifteen patients have been recruited,

to be circulated to participating centers in the Netherlands.

4.2 Methodology

Any patient registered in the EWOG-MDS/JMML 2006 study either at or around diagnosis or

scheduled to undergo HSCT will be considered for the study.

A signed written consent form is required in order to participate in the study. Patients will be

informed by the treating physicians of the proposed study and given written detailed

information about the procedure. If in agreement, they must be informed of their right to

withdraw from the study at any time without impacting on their right to receive appropriate

care.

Normal pediatric bone marrow donors acting as controls will be given detailed information

about the study inclusive of the additional bone marrow volume (10-15cc) required during the

standard harvest. Donors must be informed of their right not to participate and or to withdraw

from the study at any time.

Parents or legal guardians will be informed in the same manner and receive detailed written

information about the study. If the child is > 12 years of age and capable of comprehending

the study they will also be asked to participate and sign a consent form after being given

written, age appropriate information. Younger children aged between 7 and 12 years will also

be given age appropriate information without requiring them to formally consent.

In the case of no participation or withdrawal of consent, the clinical treatment will not be

affected.

In consenting patients, arrangements will be made to undertake the routine bone marrow

diagnostic control samples (under general anesthetic) at which an additional 10-15cc of bone

marrow aspirate will be withdrawn purely for study purposes. Study marrow may alternatively

be taken at the time of insertion of a central line or at the time of bone marrow examination


immediately prior to hematopoietic stem cell transplantation when the patient is undergoing

general anesthetic. In children undergoing HSCT additional research material will be sampled

at the time of routine post transplant bone marrow examination up to 12 months post HSCT. 4.2.1 Isolation and expansion of MSC’s from bone marrow MSC’s will be isolated according to standard methodology. 10-15cc bone marrow aspirate will

be collected under general anesthesia into a standard sterile heparin-containing flask. The

bone marrow will be used as the source for ex vivo expansion of MSC’s and isolation of

CD34 positive cell populations. Bone marrow samples will be washed using human albumin /

0.9% sodium chloride mixture, filtered, and purified using Ficoll separation techniques.

Following further washing procedures, the centrifuged pellet will be re-suspended in MSC

culture medium [LG-D MEM/P/S and fetal calf serum (9:1 ratio)]. Phenotypic analysis by

FACS will be performed before expansion. Post-Ficoll separated MNC's will be suspended in

culture medium (FCS and DMEM-glucose /P/S) and plated onto tissue culture flasks. These

flasks will be incubated at 370C in a CO2 incubator and cultures examined at regular intervals.

Depending upon the growth kinetics and appearances of the cultured cells, the culture

medium will be refreshed until sufficient adherent cells (>90% confluence) are available for

trypsinization and re-plating. These adherent cells will be subsequently passaged to achieve

the desired cell numbers for further characterization. The MSC's if not immediately required

would be cryopreserved in DMSO. Viability assays will be undertaken as well as cell cycle

and proliferation assays using FACS analysis and immunocytochemistry (Ki-67).

Characteristics of the culture expanded MSC’s will be undertaken using morphological

identification of spindle shaped adherent cells. Immunophenotypical identification will

characterize the population using a panel of antibodies expressed on MSC's (HLA-I, CD73,

CD90 and CD105). Control immunophenotyping will be undertaken using a panel of non-

expressed antigens (CD34, CD45, HLA-II, CD80 and CD31). Uniformity of cultures will be

guaranteed by using the same batch of reagents and assaying at passage 3 of culture.

4.2.2 Isolation of CD34 positive stem cells from bone marrow

CD34+ cells will be isolated from the Ficoll fraction of fresh bone marrow using the MiniMACs

system according to the manufacture’s instructions (Miltenyi Biotech, Bisley, UK).12

4.2.3 Biological Characterization of MSC’s derived from childhood MDS

(Techniques are already operational)

4.2.3.1 Phase I

4.2.3.1.a) Differentiation


The ability of MSC’s from MDS patients to differentiate into specific cell lineages under certain

controlled conditions will be investigated and compared to normal controls as described in the literature

by Caplan.

BM-MSC of patients and controls will be tested for immunomodulatory capacity in vitro

functional assays. In short, variable numbers of irradiated MSC’s will be titrated into the

following culture systems to investigate their possible modulatory effect on the activity of

various effector cell populations:

4.2.3.1.b) T cell interactions

T-cell activation will be studied in cultures of PBMC stimulated with antigen (tetanus toxoid),

mitogen (phytohemagglutinin) or alloantigen (one-way mixed lymphocyte culture, MLC); read-

out: T-cell proliferation by 3H-thymidine incorporation, cytokine production in culture

supernatant (IFNgamma, TNFalpha, IL-4, IL-10) by ELISA. Similarly, the ability of MSC's to

inhibit in vitro T cell activation upon stimulation with viral antigens (adenovirus) will be

investigated

4.2.3.1.c) NK cell interactions

The interaction between MSC’s isolated from children with MDS and natural killer (NK) cell

will be explored if possible utilizing peripheral blood NK cells obtained from the patient or

normal pediatric donors using techniques adapted from those described by Sotiropoulou.30

We will aim to investigate the ability of MSC’s isolated from children with MDS to alter the

phenotype of NK cells, the effect on NK proliferation, cytokine expression and cytotoxicity

against viral infected and tumor cell lines as target cells. Activation of resting NK cells will be

studied in cultures of purified NK cells in the presence of IL-15; read-out: cytotoxicity of HLA

class I negative (K562) and positive target cells (51chromium release), IFN-gamma secretion

in culture supernatant (ELISA). 4.2.3.1.d) Antigen presenting cell interactions

Differentiation of purified monocytes to immature dendritic cells (iDC) in presence of GM-CSF

and IL-4, followed by further maturation into mature dendritic cells (mDC) after addition of

CD40L transfected cells will be analysed; read-out: immunophenotyping of DC (CD80, CD86,

HLA-DR, CD83) by flow cytometry, TNFalpha, IL-12, IL-10 secretion in supernatant (ELISA). 4.2.3.1.e) T regulatory cell interactions

Induction of regulatory T cells in alloantigen stimulated cultures of PBMC (one-way MLC) will

be investigated; read-out: immunophenotyping (CD4, CD25, CTLA-4) and cytokine

production (TGFbeta, IL-10).


4.2.3.1.f) Cytokine, chemokine and chemokine receptor analysis

Cytokine and growth factor expression will be analyzed using RT-PCR methodology as

described by Majumdar et al.21An inventory of pathways through which MSCs might modulate

immune effector functions will be made by quantification of various cytokines and

chemokines in culture supernatants, i.e. IL-6, IL-11, IL-7, IL-8, MCP-1, M-CSF, GM-CSF, HGF,

by ELISA. In addition, prostaglandin E2 (PGE2, ELISA)) and the tryptophan metabolite

kynurenine (HPLC) will be determined.

If relevant, PGE2 and kynurenine production will be selectively inhibited through addition of

inhibitors, i.e. indomethacin or NS-398, respectively 1-methyl tryptophan or norharmane, at

culture initiation.

Chemokine receptor analysis of MDS derived MSC’s will be undertaken using RT-PCR, flow

cytometric analysis together with cellular localization utilizing immune histocytochemistry and

Confocal microscopy. 4.2.3.1.g) Chromosomal characterization

Both standard karyotypic analysis as well as other techniques (e.g. chromosome painting –

cobra FISH technique) will be explored in MSC’s from patients with MDS obtained at

diagnosis and 3-12 months after HSCT and compared to the results obtained with isolated

HSC’s from patients and results of routine karyotypic analysis of patients’ diagnostic BM

material.

Further studies using gene profiling will be undertaken to investigate expression of specific

genes controlling cell signaling, proliferation, apoptosis, DNA excision repair, differentiation

and cytokine production.

4.2.3.1.h) Growth kinetics

Growth kinetics of MDS MSC’s will be compared to normal controls.

4.2.3.2 Phase II

(Techniques to be developed)

If sample size permits, based on the preliminary work, a second stage of experiments will be

planned which will consists of the long-term culture (Dexter type) CD34+ cells co-cultured

with MDS derived MSC’s and to determine their ability to support normal hematopoietic

growth. Cross-over experiments will be used to determine the effect of MDS derived MSC’s

on normal bone marrow derived CD34+ cells and similarly, MSC’s derived from normal donor


bone marrow will be co-cultured with CD34+ cells isolated from MDS bone marrow. These

experiments will help to determine whether or not normally functioning MSC’s impact on

hematopoietic growth in MDS patients,

and whether MDS derived MSC’s support hematopoietic growth of normal CD34+ cells

All experiments, depending upon available material will be undertaken pre and post

transplant in selected patients referred for allogeneic stem cell transplantation.

4.3 Additional materials required for the study

Patients and controls

4.3.1 Blood samples

Isolate PBMC from 10-20 cc EDTA blood (depending upon age and weight of child),

freeze cells vitally (i.e. 5 x106 PBMC per vial in DMSO containing medium; within 24

hrs after collection) and store in liquid nitrogen until transport to LUMC, Leiden, NL.

Store 5cc plasma or serum at -20o C until transport to LUMC, Leiden, NL.

4.3.2 Bone marrow samples

10-15cc (depending upon age and weight of child) will be obtained if possible at or

shortly after diagnosis or prior to HSCT: Collected into sterile heparin-containing flask

and transported to LUMC, Leiden, NL to arrive within (maximum) 48 hours of

aspiration.

In children undergoing SCT additional research bone marrow samples will be

obtained at the time of routine procedures between 3-12 months post HSCT.


5.0 ENDPOINTS

5.1 Study endpoints

5.1.1 Primary endpoints

5.1.1.1 Determination for each patient and control the phenotypical and

biological characteristics of MSC’s by flow cytometric analysis, cell

culture and differentiation abilities.

5.1.1.2 Determination of functional characteristics of MSC's isolated from

pediatric MDS patients and controls:

- immune regulation of T and NK cell function

- cytokine and growth factor expression of MSC’s and the

expression of chemokine receptor profiles

5.1.1.3 Determination of cytogenetic abnormalities of MSC’s and HSC’s in

MDS patients and controls.

5.1.1.4 Determination of cell cycle control, apoptosis and differentiation by

gene array of MSC’s and HSC’s from children with MDS and controls.

5.1.1.5 Determination of MSC and CD34+ve HSC’s from MDS patients to

support normal hematopoiesis compared to controls.

5.2 Patient’s discontinuation

Patients (and/or parents) can request at any time to withdraw from the study and their

reasons for doing so (if possible) will be documented.

The treating physician can withdraw any patient from the study if he/she feels it is in the

patient’s best interest, and his/her reasons for doing so will be documented.

5.3 End of study

An interim analysis of the data will be undertaken after the first 15 patient samples have been

investigated. A full analysis will be undertaken if 25 patients and 10 normal control groups

have been recruited or at a maximum of 48 months after the initiation of the study.


6.0 DATA and ADMINISTRATIVE ASPECTS

6.1 Data handling and record keeping Data will be recorded on data collection forms and laboratory work sheets, which will be

entered after validation into a computer system for subsequent tabulation and analyses. The

data will be handled confidentially and, as far as possible, anonymously. Each patient will be

assigned a study number. All samples and data generated from the study samples will be

collected and analyzed using this number. Only the principal investigator will have access to

the initials of patients and the study number assigned to each patient/control.

6.2 Data analysis

6.2.1 General and functional tests

The analysis will be exploratory and therefore depend on the data sets obtained.

Descriptive and summary measures will be used to characterize the study population.

6.3 Statistical analysis

6.3.1 Planned sample size

The study will be undertaken such that a minimum of 25 patients and 10 controls will

be included.

6.3.2 Power calculation

This is an exploratory study and as such no power calculations have been

undertaken. Statistical analysis, depending on the data sets obtained, will be

undertaken using univariate and multivariate analysis of the observations utilizing

SPSS package with assistance from the Department of Medical Statistics, University

of Leiden. P<0.05 is assumed to be statistically significant.

6.3.3 Missing, unused and spurious data

The data will be reviewed before analysis of the data and the results of this review

documented in the study.

6.3.4 Selection of subjects

All data that meaningfully contributes to the objectives of the study will be included.

6.4 Access to source data and documents All study data will be handled confidentially. The prinicpal investigator will retain the originals

of the source documents generated for a minimum of two years after the study is complete.

After this all documents will be archived according to GCP regulations. The results of the

study will be published in recognized medical journals if applicable. If so, the patient identity

will not be revealed and patients will be assigned for the purpose of the study and


publication, a UPN.

6.5 Budget considerations

The cost of the ex vivo expansion of MSC’s within this study are contained within the

Pediatric Immunology Laboratory as are the cytokine and chemokine receptor analysis and

NK and T cell studies. On the basis of the preliminary results external funding will be sought

to increase the number of patient samples analyzed and to financially support the work

related to karyotyping, gene profiling and the hemopoietic cultures planned in phase II

6.6 Quality control and quality assurance

This study will be conducted according to the GLP procedures of the Pediatric Immunology

Laboratory under the auspices of the Head of the Department (Maarten van Tol).

6.7 Address for biological samples

BIOLOGICAL SAMPLES

c/o Dr Lynne M Ball,

MDS EWOG add on Study,

Pediatric Immunology Laboratory P3 – P,

Leiden University Medical Center,

Albinusdreef 2,

2300 RC, Leiden,

the Netherlands


7.0 ETHICAL CONSIDERATIONS

7.1 Regulatory statement

The study will be conducted according to the principles of the declaration of Helsinki (as

amended in Tokyo, Venice and Hong Kong, Somerset West and Edinburgh) and in

accordance with the Guideline for Good Clinical Practice (CMPP/ICH/135/95 – 17th July

1996).

7.2 Recruitment and consent

The protocol will be submitted to the Medical Ethics Committee of Leiden University Medical

Center (LUMC) for national accreditation and will not begin until formal approval has been

granted. Patients enrolled in the study (and in the case of a minor: their parents or

guardians) will be given oral and written information before starting the protocol treatment.

For children below the age of 12 years written informed consent will be obtained from the

parents or guardians. Children aged between 12-16 years of age will be asked to give

informed written or verbal consent together with their parents or guardians written informed

consent. In case of conflict, an independent pediatrician will assess the child and if he feels

that the child fully understands the protocol and still their decision is in conflict with that of the

parents, the child’s wishes will be followed.

Only after written informed consent is obtained will the participants be entered into the study.

Children and parents will be informed of their right to not enter the study or withdraw from the

study at any time without this impacting upon the care provided to the patient. Although not

required the reasons for doing so, if possible will be documented. The physician in charge of

the patient may decide to withdraw the child from the study if he/she feels it is in the best

interest of the patient. Their reasons for doing so will be explained to the child and the

parents or guardians and documented.

7.3 Compensation for injury

The chance of injury as a result of this study is negligible. However, the participating center

must ensure that any pertinent legal requirements are met to cover injury or death as a result

of participation in the study.


8.0 LITERATURE

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14. Bennet J, Catovsky D, Daniel DT, et al. The French- American- British (FAB)

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migration to bone marrow. Blood 2004; 104: 2643-2645.

31. Honczarenko M, Le Y, Swierkowski M, et al. Human bone marrow stromal cells

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24: 1030-1041.

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suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic

stimuli. Blood. 2002; 99(10): 3838-3843.

33. Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit

the response of naïve and memory antigen-specific T-cells to their cognate peptide.

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with bone marrow transplantation in the management of hematologic malignancies: an

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blood stem cells compared to marrow for allogeneic transplantation. Bone Marrow

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single center comparison of mobilized peripheral blood stem cell with bone marrow

transplantation. Klin Paditr. 2005; 217: 135-141.

48. Guardiola P, RundeV, Bacigalupo A, et al. Retrospective comparison of bone marrow

and granulocyte-stimulating factor mobilized peripheral blood progenitor cells for

allogeneic stem cell transplantation in HLA identical sibling donors in myelodysplastic

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extensive chronic graft-versus-host disease after allogeneic peripheral blood stem cell

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APPENDIX A: Case Report Form

PATIENT INFORMATION FORM Patient details. EWOG-MDS number: STUDY number Surname: (initials only) Forename: (initials only) Date of birth: Gender: Male c Female c Primary disease: MDS c FAB ……………………………………………………. JMML …………………………… Date of diagnosis of primary disease: …………………………………………………… Diagnostic material c Pre SCT material c Cytogentic abnormal (if known) : Monosomy 7 c Trisomy 8 c Other (state)……………………………………………………………… Center Contact person and e-mail address: Remarks Form to be completed and be sent with study marrow and blood samples to LUMC (Address -

see page 171)


APPENDIX B: Patient Information Form and Informed Consent

Informatie brief voor ouders van kinderen die in aanmerking komen voor

deelnemen aan het wetenschappelijk onderzoek: ‘Bepalen van biologische

karakteristieken van mesenchymale stamcellen bij kinderen met

myelodysplastische ziekten’ - add-on EWOG MDS/JMML studie

Dr. ……………………………heeft u verteld dat uw kind is gediagnosticeerd met

myelodysplastisch syndroom (MDS) of juveniele myelomonocytaire leukemia (JMML). Uw

kind doet mee aan de EWOG (European Working Group) MDS/JMML studie. Recentelijk zijn

we begonnen met een hieraan verbonden experimentele studie om meer te weten te komen

over het ontstaan van MDS/JMML. We zouden graag uw toestemming willen vragen om uw

kind mee te laten doen aan deze studie, genaamd: ‘Bepalen van biologische karakteristieken

van mesenchymale stamcellen bij kinderen met myelodysplastische ziekten’. Lees de

volgende tekst nauwkeurig door en neem de tijd om de informatie met uw dokter te

bespreken zodat U inzicht krijgt in het wetenschappelijk onderzoek voor U en uw kind

betekent. Als u bereid bent om uw kind aan deze studie deel te nemen, willen we u vragen

een toestemmingsformulier te tekenen.

Wat is precies een onderzoeksstudie?

Een ondezoeksstudie is gericht op het vinden van antwoorden op belangrijke vragen zoals

waarom/hoe bepaalde ziektes ontstaan en hoe ze nog beter behandeld kunnen worden.

Waar gaat deze onderzoeksstudie over?

Het beenmerg, de fabriek voor bloedplaatjes, rode- en witte bloedcellen, is afwijkend bij

mensen met myelodysplastisch syndroom en JMML. Mesenchymale stamcellen, die zich ook

in het beenmerg bevinden, vormen een soort ondersteunend netwerk dat nodig is voor de

normale vorming van bloedcellen. We denken dat deze ondersteunende mesenchymale

stamcellen bij MDS/JMML patiënten mogelijk niet normaal functioneren en dat deze cellen

dus onderdeel vormen van de ziekte en mogelijk zelfs betrokken zijn bij de ontwikkeling van

leukemie.

We kunnen mesenchymale stamcellen en de bloed-vormende stamcellen uit het beenmerg

isoleren. Vervolgens kunnen we in het laboratorium verschillende testen doen om te kijken in

hoeverre de mesenchymale stamcellen van MDS patiënten verschillen van kinderen die deze

ziekte niet hebben en of de mesenchymale stamcellen van MDS patiënten in staat zijn de

benodigde ondersteunende functie uit te oefenen, die van belang is voor de aanmaak van

bloedcellen.


Uiteraard hopen we dat meer inzicht in de rol van mesenchymale stamcellen bij MDS zal

leiden tot nieuwe therapeutische opties in de toekomst.

Wat is er nodig voor deze onderzoeksstudie en wat is de extra belasting?

Op momenten in de behandeling dat uw kind toch onder narcose moet (beenmergpunctie

voor diagnose, plaatsen diepe lijn, controle voor beenmergtranplantatie) zullen we, met uw

toestemming, extra beenmerg afnemen (1 à 2 buisjes van 10 ml). Uw kind hoeft dus nooit

speciaal onder narcose voor deze studie, wel kan het zijn dat er tijdens de narcose voor iets

anders, speciaal een beenmergpunctie gedaan wordt om materiaal te krijgen voor de studie.

Naast beenmerg worden ook 1 à 2 buisjes bloed afgenomen (maximaal 25 ml, afhankelijk

van het gewicht van het kind) voor deze studie. Tot slot zullen we ook voor de studie

belangrijke informatie verzamelen zoals diagnose, leeftijd van het kind en de eerdere

uitslagen van onderzoek. Deze informatie wordt gecodeerd bewaard en alleen uw eigen

behandelde arts kan dit herleiden naar de naam van uw kind. De onderzoekers zullen de

analyse uitvoeren met behulp van een code en zij hebben dus geen toegang tot de

persoonlijke gegevens van uw kind.

De extra belasting van dit onderzoek voor uw kind bestaat maximaal uit de belasting van een

reguliere beenmergpunctie, zoals een beurs gevoel op de plek waar de punctie heeft

plaatsgevonden. In principe wordt geprobeerd de afname voor de studie te combineren met

een reguliere beenmergafname, maar er kan u gevraagd worden om toestemming voor een

afname tijdens een narcose waarbij niet standaard een beenmergpunctie gepland staat. Uw

kind zal altijd vóór een ingreep beoordeeld worden door een arts om te kijken of uw kind in

voldoende conditie is om een narcose en de daarbij behorende ingreep te ondergaan.

Mochten er medische redenen zijn waarom een beenmergpunctie niet uitgevoerd kan

worden, dan zal de punctie voor het wetenschappelijk onderzoek niet worden gedaan.

Hoewel deelname aan de studie voor uw kind in principe geen risico’s met zich meebrengt, is

het ziekenhuis wel verzekerd tegen onverwachte effecten, voortvloeiend uit afnames voor

wetenschappelijk onderzoek.


Verzekering

Er is door het ziekenhuis een verzekering afgesloten waaruit eventuele schade als gevolg

van de deelname van uw kind aan het wetenschappelijk onderzoek betaald kan worden.

Wanneer u vindt dat U schade heeft ondervonden als gevolg van deelname aan het

onderzoek kunt u contact opnemen met de arts-onderzoeker.

Informatie over de afgesloten verzekering treft U aan in de bijlage.

Wanneer worden de resultaten van dit onderzoek bekend?

Alle kinderen die deelnemen aan de EWOG MDS/JMML studie kunnen in principe meedoen.

Deze extra onderzoeksstudie wordt gecoördineerd en uitgevoerd binnen de afdeling

kindergeneeskunde van het Leids Universitair Medisch Centrum. We hopen dat tenminste 25

kinderen met MDS/JMML in de komende 2-3 jaar willen deelnemen. Mochten de resultaten

gepubliceerd worden in een medisch wetenschappelijk tijdschrift, dan zullen alle deelnemers

anoniem blijven. Verder zal er aan het eind van de studie een analyse gemaakt worden voor

de EWOG werkgroep, die naar alle deelnemende centra gestuurd kan worden.

Wat gebeurt er als ik niet wil dat mijn kind meedoet aan dit onderzoek?

Deelname aan dit onderzoek is volledig op vrijwillige basis en heeft dus geen enkele

(negatieve) invloed op de behandeling van uw kind. Uw kind krijgt gewoon de standaard

klinische behandeling volgens het EWOG MDS protocol, los van het feit of uw kind

deelneemt aan deze extra studie.

Als ik vragen heb over het onderzoek, bij wie moet ik dan zijn? Uw behandelend arts kan u verdere uitleg geven over deze studie. Mocht u behoefte hebben

aan informatie van één van de artsen, die direct betrokken is bij dit onderzoek of van een

onafhankelijke kinderarts, die het onderzoek kent maar niet direct hieraan verbonden is, dan

kunt u dit aangeven en wordt dit voor u geregeld.

Organisatoren van dit onderzoek zijn: …………………. arts onderzoeker tel contact

Onafhankelijke arts:

………………… kinderarts tel contact


Toestemmingsformulier ouders* voor deelname aan onderzoeksstudie:

Bepalen van biologische karakteristieken van mesenchymale stamcellen bij kinderen met myelodysplastische ziekten’; add-on EWOG MDS/JMML studie

- Ik ben goed geïnformeerd over deze onderzoeksstudie en heb de informatie gelezen, die ik gekregen heb over

deze studie.

- Ik heb de kans gekregen alle vragen te bespreken, die ik had over deze studie en ik heb duidelijke antwoorden

gekregen.

- Ik had genoeg tijd om na te denken of ik mijn kind mee wilde laten doen aan deze studie

- Ik begrijp dat ik altijd kan beslissen om toch niet mee te doen, ook al had ik eerst wel toegestemd, zonder dat

ik daarvoor een reden hoef te geven.

- Als er cellen over zijn, die niet meer nodig zijn voor deze studie, dan geef ik toestemming om die cellen te

gebruiken voor andere onderzoeksprojecten: Ja □ Nee □

Ik geef mijn toestemming om mijn kind deel te laten nemen aan deze onderzoeksstudie

Naam van uw kind :…………………………………………..

Geboortedatum van uw kind :............................................(dd/mm/jj)

Uw achternaam en initialen : ……………………………………………………………………..

Handtekening : ………………………………………….Datum: …………………. (dd/mm/jj)

- Ik verklaar dat de bovengenoemde persoon, zowel mondeling als schriftelijk, zo goed mogelijk op de hoogte is

gesteld van de implicaties van dit onderzoek. Hij/zij weet dat de toestemming om mee te doen aan dit onderzoek, op elk

gewenst moment ingetrokken mag worden, zonder dat het gevolgen heeft voor zijn/haar behandeling in de toekomst.

Volledige naam : ……………………………………………………

Functie : ……………………………………………………

Handtekening : …………………………………………………… Datum…………………… (dd/mm/jj) *Dit document is bedoeld voor onderzoek bij patienten < 12 jaar of patiënten die anders zins zelf juridisch geen toestemming kunnen geven voor deelname.


Informatie brief voor patiënten 7-12 jaar die deelnemen van het

wetenschappelijk onderzoek: ‘Bepalen van biologische karakteristieken van

mesenchymale stamcellen bij kinderen met myelodysplastische ziekten’· -add-

on EWOG MDS/JMML studie

Beste ……………………………………

Met je vader en moeder hebben we besproken dat we graag wat speciale testen zouden

doen om te kijken of we er achter kunnen komen, waarom je ziek

bent geworden. Dat betekent dat we graag willen kijken naar je

beenmerg. Je beenmerg is de fabriek van de bloedcellen en zit in

al je botten. Je hebt drie soorten bloedcellen: rode bloedcellen

zorgen dat je niet zo snel moe wordt, witte bloedcellen

beschermen je tegen ziektes en bloedplaatjes zorgen ervoor dat

als je stopt me bloeden, als je je gestoten hebt. De cellen waar wij speciaal in

geïnteresseerd zijn heten ‘mesenchymale stamcellen’. Dit zijn cellen, die helpen bij het

maken van bloedcellen.

Tijdens de behandeling voor je ziekte worden er soms onderzoeken gedaan, waarvoor je in

slaap wordt gebracht. Als je in slaap bent zullen we een prikje geven aan de zijkant van je

rug (merk je niets van, want je slaapt) om beenmerg eruit te halen

voor onderzoek. Als je weer wakker wordt weet je daar niets meer

van, soms kan er wel een blauwe plek zijn, waar je geprikt bent in

je slaap. Het beenmerg wordt naar het laboratorium gebracht, waar

we gaan testen wat de cellen precies te maken hebben met jouw

ziekte.

Aan alle kinderen met jouw ziekte vragen we om mee te doen aan dit onderzoek. Alle

gegevens worden dan verzameld, zodat we weten of die cellen bij iedereen hetzelfde

werken. De mensen die het onderzoek doen komen niet te weten hoe jij heet of wie jij bent,

dat weten alleen maar je eigen dokters en verpleegsters.

We hopen dat we in de toekomst, dankzij dit onderzoek, nog meer kinderen beter kunnen

maken die deze ziekte hebben.


Als je dit gelezen hebt en je hebt nog vragen, dan kan je je vragen aan je vader of moeder

stellen, maar ook natuurlijk aan je dokter of de verpleegster die voor je zorgt.

Mijn verpleegster is:………………………

Mijn dokter is:………………………


Informatie brief voor patiënten 12 t/m 16 jaar die in aanmerking komen voor

deelnemen van het wetenschappelijk onderzoek: ‘Bepalen van biologische


myelodysplastische ziekten’· -add-on EWOG MDS/JMML studie

Dr. ……………………………heeft je verteld dat je myelodysplastisch syndroom (MDS) of

junveniele myelomonocytaire leukemia (JMML) hebt. Je doet mee aan de EWOG (European

Working Group) MDS/JMML studie. Sinds kort zijn we ook begonnen aan een studie om

meer te weten te komen over het ontstaan van MDS/JMML. We zouden graag je

toestemming willen vragen om mee te doen aan deze studie, genaamd: ‘Bepalen van

biologische karakteristieken van mesenchymale stamcellen bij kinderen met

myelodysplastische ziekten’. Voordat jij en je ouders hierover beslissen, is het belangrijk om

eerst deze informatie te lezen. Natuurlijk kun je ook alles over deze studie vragen aan je arts.

Als je bereid bent aan deze studie mee te doen, willen we je vragen een

toestemmingsformulier te tekenen.

Wat is precies een studie?

Een onderzoeksstudie is gericht op het vinden van antwoorden op belangrijke vragen zoals


Waar gaat deze studie over?


mensen met MDS en JMML. We zouden graag weten waarom er dingen misgaan bij het

maken van bloedcellen bij MDS en JMML. In de beenmergfabriek zijn ook mesenchymale

stamcellen aanwezig, die de bloedvormende cellen helpen om bloedcellen te maken. Het zijn

dus eigenlijk ondersteunende cellen. We denken dat deze ondersteunende mesenchymale


dus onderdeel vormen van de ziekte.

.

We kunnen mesenchymale stamcellen en de bloed-vormende stamcellen uit het beenmerg

halen en bekijken in het laboratorium. Vervolgens kunnen we verschillende testen doen om

te kijken in hoeverre de mesenchymale stamcellen van MDS/JMML patiënten verschillen van

mensen die deze ziekte niet hebben. Ook willen we graag weten of de mesenchymale

stamcellen van MDS patiënten in staat zijn de ondersteunende functie uit te oefenen, die van

belang is voor de aanmaak van bloedcellen.


Natuurlijk hopen we dat we ook betere behandelingen kunnen ontwikkelen voor MDS/JMML

als we meer begrijpen over de rol van de mesenchymale stamcellen in het beenmerg.

Wat moet ik doen voor deze studie?

Op momenten in de behandeling dat je toch al onder narcose moet, zullen we extra

beenmerg afnemen (1 à 2 buisjes van 10 ml). Je hoeft dus nooit speciaal onder narcose voor

deze studie, wel kan het zijn dat er tijdens de narcose voor iets anders, speciaal een

beenmergpunctie gedaan wordt om materiaal te krijgen voor de studie. Naast beenmerg

worden ook 1-2 buisjes bloed afgenomen (maximum 25 ml) voor deze studie. Tot slot zullen

we ook voor de studie belangrijke informatie verzamelen zoals welke ziekte je hebt, je leeftijd

en de eerdere uitslagen van onderzoek. Deze informatie wordt gecodeerd bewaard en alleen

je eigen arts weet dat deze informatie van jou is. De onderzoekers zullen de analyse

uitvoeren met behulp van een code en zij hebben dus geen toegang tot je persoonlijke

gegevens.

En wat is de extra belasting voor mij?

De extra belasting van dit onderzoek voor je bestaat maximaal uit de belasting van een

beenmergpunctie, zoals een beurs gevoel op de plek waar de punctie heeft plaatsgevonden.

In principe wordt geprobeerd de afname voor de studie te combineren met een reguliere

beenmergafname, maar er kan je gevraagd worden om toestemming voor een afname

tijdens een narcose waarbij niet standaard een beenmergpunctie gepland staat. Je zal altijd

vóór een ingreep beoordeeld worden door een arts om te kijken of je in voldoende conditie

bent om een narcose en de daarbij behorende ingreep te ondergaan. Mochten er medische

redenen zijn waarom een beenmergpunctie niet uitgevoerd kan worden, dan zal de studie

punctie niet worden gedaan. Hoewel deelname aan de studie voor jou in principe geen

risico’s met zich meebrengt, is het wel verzekerd tegen onverwachte neveneffecten,

voortvloeiend uit afnames voor wetenschappelijk onderzoek.


Verzekering

Er is door het ziekehuis een verzekering afgesloten waaruit eventuele schade als gevolg van

het deelnemen van het wetenschappelijk onderzoek betaald kan worden. Wanneer je vindt

dat je schade heeft ondervonden als gevolg van deelname aan het onderzoek kunt je contact

opnemen met de arts-onderzoeker.

Informatie over de afgesloten verzekering tref je aan in de bijlage.


Alle patiënten die deelnemen aan de EWOG MDS/JMML studie kunnen in principe meedoen.

Deze extra studie wordt gecoördineerd en uitgevoerd binnen de afdeling kindergeneeskunde

van het Leids Universitair Medisch Centrum. We hopen dat tenminste 30 patiënten met

MDS/JMML in de komende 2-3 jaar willen deelnemen. Mochten de resultaten gepubliceerd

worden in een medisch wetenschappelijk tijdschrift, dan zullen alle deelnemers anoniem

blijven. Verder zal er aan het eind van de studie een analyse gemaakt worden voor de

EWOG werkgroep, wat naar alle deelnemende centra gestuurd kan worden.

Wat gebeurt er als ik niet meedoe aan dit onderzoek?


(negatieve) invloed op je behandeling. Je krijgt gewoon de standaard klinische behandeling

volgens het EWOG MDS protocol, los van het feit of je deelneemt aan deze extra studie.

Als ik vragen heb over het onderzoek, bij wie moet ik dan zijn?

Je behandelend arts kan je verdere uitleg geven over deze studie. Mocht je behoefte hebben

aan informatie van een van de artsen, die direct betrokken is bij dit onderzoek of van een

onafhankelijke kinderarts, die het onderzoek kent maar niet direct hieraan verbonden is, dan

kan je dit aangeven en wordt dit voor je geregeld.

Organisatoren van dit onderzoek zijn: ………………………… naam van arts contact tel


…………………… kinderarts contact tel


Toestemmingsformulier patient* voor deelname aan onderzoeksstudie:


Voor adolescenten (12-16 jaar):

• Ik ben goed geïnformeerd over deze onderzoeksstudie en heb de informatie gelezen, die ik gekregen heb over

deze studie. Ik heb de kans gekregen alle vragen te bespreken, die ik had over deze studie en ik heb duidelijke

antwoorden gekregen. Ik had genoeg tijd om na te denken of ik mee wilde doen aan deze studie. Ik begrijp dat ik

altijd kan beslissen om toch niet mee te doen, ook al had ik eerst wel toegestemd, zonder dat ik daarvoor een

reden hoef te geven.

• Als er cellen over zijn, die niet meer nodig zijn voor deze studie, dan geef ik toestemming om die cellen te


• Ik geef mijn toestemming om deel te nemen aan deze onderzoeksstudie

Achternaam en initialen : ………………………………………..………………………………………..

Geboortedatum : ………………………………………………………………………………….(dd/mm/jj)

Handtekening : ………………………………………………….Datum: ……………………..(dd/mm/jj)

Voor ouders/voogd:

• Ik ben volledig geïnformeerd over deze onderzoeksstudie en heb de schriftelijke informatie gelezen. Ik heb de

mogelijkheid gehad om deze studie te bespreken met een arts en mijn vragen zijn naar tevredenheid beantwoord.

Ik had genoeg tijd om na te denken over deze studie. Ik begrijp dat ik mijn toestemming zonder opgaaf van

redenen, altijd weer kan intrekken.

• Ik geef toestemming dat mijn kind deelneemt aan deze onderzoeksstudie

Achternaam en initialen : ………………………………………………………………

Handtekening : …………………………………………………Datum: ………………………(dd/mm/jj)

Ik verklaar dat de bovengenoemde persoon, zowel mondeling als schriftelijk, zo goed mogelijk op de hoogte is gesteld

van de implicaties van dit onderzoek. Hij/zij weet dat de toestemming om mee te doen aan dit onderzoek, op elk


Volledige naam : ……………………………………………………

Functie : ……………………………………………………

Handtekening : ………………………………………………………….. Datum ……………………… (dd/mm/jj) *Dit document is bedoeld voor onderzoek bij patienten > 12 jaar, die in staat zijn toestemming te geven. Het is in deze studie noodzakelijk dat deze patiënten zelf en hun ouders, schriftelijk toestemming geven.


Patiënten informatie en toestemmingsformulier voor patiënten ouder dan16 jaar Informatie brief voor patiënten >16 jaar die in aanmerking komen voor

deelnemen van het wetenschappelijk onderzoek: ‘Bepalen van biologische


myelodysplastische ziekten’· -add-on EWOG MDS/JMML studie

Dr. ……………………………heeft je verteld dat je myelodysplastisch syndroom (MDS) of

juveniele myelomonocytaire leukemie (JMML) hebt. Je doet mee aan de EWOG (European

Working Group) MDS/JMML studie. Recentelijk zijn we begonnen met een hieraan

verbonden experimentele onderzoek om meer te weten te komen over het ontstaan van

MDS/JMML. We zouden graag je toestemming willen vragen om mee te doen aan dit

onderzoek, genaamd: ‘Bepalen van biologische karakteristieken van mesenchymale

stamcellen bij kinderen met myelodysplastische ziekten’. Lees de volgende tekst nauwkeurig

door en neem de tijd om de informatie met je dokter te bespreken. Voordat je hierover beslist

is het belangrijk dat je onderstaande informatie leest zodat je inzicht krijgt in wat het

wetenschappelijk onderzoek voor je betekent. Als je bereid bent aan deze studie deel te

nemen, willen we je vragen een toestemmingsformulier te tekenen.

Wat is precies een onderzoek?

Een onderzoeksstudie is gericht op het vinden van antwoorden op belangrijke vragen zoals


Waar gaat dit onderzoek over?


mensen met myelodysplastisch syndroom en JMML. Mesenchymale stamcellen, die zich ook

in het beenmerg bevinden, vormen een soort ondersteunend netwerk dat nodig is voor de

normale vorming van bloedcellen. We denken dat deze ondersteunende mesenchymale


dus onderdeel vormen van de ziekte en mogelijk zelfs betrokken zijn bij de ontwikkeling van

leukemie.

We kunnen mesenchymale stamcellen en de bloedvormende stamcellen uit het beenmerg

isoleren. Vervolgens kunnen we in het laboratorium verschillende testen doen om te kijken in

hoeverre de mesenchymale stamcellen van MDS patiënten verschillen van mensen die deze

ziekte niet hebben en of de mesenchymale stamcellen van MDS patiënten in staat zijn de

benodigde ondersteunende functie uit te oefenen, die van belang is voor de aanmaak van

bloedcellen.


Uiteraard hopen we dat meer inzicht in de rol van mesenchymale stamcellen bij MDS zal

leiden tot nieuwe therapeutische opties in de toekomst.

Wat is er nodig voor dit onderzoek en wat is de extra belasting?

Op momenten in de behandeling dat je toch onder narcose moet (beenmergpunctie voor

diagnose, plaatsen diepe lijn, controle voor beenmergtransplantatie) zullen we, met jouw

toestemming, extra beenmerg afnemen (1 à 2 buisjes van 10 ml). Je hoeft dus nooit speciaal

onder narcose voor deze studie, wel kan het zijn dat er tijdens de narcose voor iets anders,

speciaal een beenmergpunctie gedaan wordt om materiaal te krijgen voor de studie. Naast

beenmerg worden ook 1-2 buisjes bloed afgenomen (maximum 25 ml) voor deze studie. Tot

slot zullen we ook voor de studie belangrijke informatie verzamelen zoals diagnose, je leeftijd

en de eerdere uitslagen van onderzoek. Deze informatie wordt gecodeerd bewaard en alleen

je eigen arts kan dit herleiden naar jouw naam. De onderzoekers zullen de analyse uitvoeren

met behulp van een code en zij hebben dus geen toegang tot je persoonlijke gegevens.

De extra belasting van dit onderzoek voor je bestaat maximaal uit de belasting van een

reguliere beenmergpunctie, zoals een beurs gevoel op de plek waar de punctie heeft

plaatsgevonden. In principe wordt geprobeerd de afname voor de studie te combineren met

een reguliere beenmergafname, maar er kan je gevraagd worden om toestemming voor een

afname tijdens een narcose waarbij niet standaard een beenmergpunctie gepland staat. Je

zult altijd vóór een ingreep beoordeeld worden door een arts om te kijken of je in voldoende

conditie bent om een narcose en de daarbij behorende ingreep te ondergaan. Mochten er

medische redenen zijn waarom een beenmergpunctie niet uitgevoerd kan worden, dan zal de

punctie voor het wetenschappelijk onderzoek niet worden gedaan. Hoewel deelname aan het

onderzoek voor jou in principe geen risico’s met zich meebrengt, is het ziekenhuis wel

verzekerd tegen onverwachte effecten, voortvloeiend uit afnamen voor wetenschappelijk

onderzoek.

Hoewel deelname aan de studie voor uw kind in principe geen risico’s met zich meebrengt,


Verzekering

Er is door het ziekenhuis een verzekering afgesloten waaruit eventuele schade als gevolg

van het deelnemen van de wetenschappelijke onderzoeksstudie betaald kan worden.

Wanneer je vindt dat je schade heeft ondervonden als gevolg van deelname aan het

onderzoek kunt je contact opnemen met de arts-onderzoeker.

Informatie over de afgesloten verzekering tref je aan in de bijlage.


Alle patiënten die deelnemen aan de EWOG MDS/JMML studie kunnen in principe meedoen.

Dit extra onderzoek wordt gecoördineerd en uitgevoerd binnen de afdeling

kindergeneeskunde van het Leids Universitair Medisch Centrum. We hopen dat tenminste 25

patiënten met MDS/JMML in de komende 2-3 jaar willen deelnemen. Mochten de resultaten

gepubliceerd worden in een medisch wetenschappelijk tijdschrift, dan zullen alle deelnemers

anoniem blijven. Verder zal er aan het eind van de studie een analyse gemaakt worden voor

de EWOG werkgroep, die naar alle deelnemende centra gestuurd kan worden.

Wat gebeurt er als ik niet meedoe aan dit onderzoek?


(negatieve) invloed op je behandeling. Je krijgt gewoon de standaard klinische behandeling

volgens het EWOG MDS protocol, los van het feit of je deelneemt aan dit extra

wetenschappelijk onderzoek.

Als ik vragen heb over het onderzoek, bij wie moet ik dan zijn? Je behandelend arts kan je verdere uitleg geven over deze studie. Mocht je behoefte hebben

aan informatie van één van de artsen, die direct betrokken is bij dit onderzoek of van een

onafhankelijke kinderarts, die het onderzoek kent maat niet direct hieraan verbonden is, dan

kan je dit aangeven en wordt dit voor je geregeld.

Organisatoren van dit onderzoek zijn: ………………………….. arts onderzoeker tel contact


………………………….. kinderarts tel contact


Toestemmingsformulier patiënt* voor deelname aan onderzoeksstudie:


- Ik ben goed geïnformeerd over deze onderzoeksstudie en heb de informatie gelezen, die ik gekregen heb over

deze studie.

- Ik heb de kans gekregen alle vragen te bespreken, die ik had over deze studie en ik heb duidelijke antwoorden

gekregen.

- Ik had genoeg tijd om na te denken of ik mee wilde doen aan deze studie

- Ik begrijp dat ik altijd kan beslissen om toch niet mee te doen, ook al had ik eerst wel toegestemd, zonder dat

ik daarvoor een reden hoef te geven.

- Als er cellen over zijn, die niet meer nodig zijn voor deze studie, dan geef ik toestemming om die cellen te


- Ik geen mijn toestemming om deel te nemen aan deze onderzoeksstudie

Achternaam en initialen : …………………………………………………………………………………

Geboortedatum : …………………………………………………………………………………. (dd/mm/jj)

Handtekening : …………………………………………………. Datum: ……………………. (dd/mm/jj)

- Ik verklaar dat de bovengenoemde persoon, zowel mondeling als schriftelijk, zo goed mogelijk op de hoogte is

gesteld van de implicaties van dit onderzoek. Hij/zij weet dat de toestemming om mee te doen aan dit onderzoek, op elk


Volledige naam : ……………………………………………………

Functie : ……………………………………………………

Handtekening : ………………………………………………… Datum ……………………… (dd/mm/jj)

*Dit document is bedoeld voor onderzoek bij patiënten > 16 jaar, die in staat zijn toestemming te geven. Het is in deze studie noodzakelijk dat deze patiënten zelf schriftelijk toestemming geven.


Appendix 12 Invoice Form

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