EURO-LB 02. Treatment Protocol for Lymphoblastic Lymphoma. of the European Inter-group Co-operation on Childhood Non-Hodgkin-Lymphoma (EICNHL)

Transcription

1 EURO-LB 02 Treatment Protocol for Lymphoblastic Lymphoma of the European Inter-group Co-operation on Childhood Non-Hodgkin-Lymphoma (EICNHL) Final Draft November 2002 Participating Goups: AIEOP: Associazione Italiana di Ematologia ed Oncologia Pediatrica BFM Austria, Czechia, Germany and Switzerland CoALL Germany EORTC: European Organisation for Research and Treatment of Cancer SFCE: Société Française des Cancers de l'enfant UKCCSG: United Kingdom Children Cancer Study Group PPLLSG: Polish Paediatric Leukaemia/Lymphoma Study Group SHOP: Spanish Leukemia Group DCOG: Dutch Childhood Oncology Group NOPHO: Nordic Society of Pediatric Haematology and Oncology

2 Treatment Plan EURO-LB 02 for T-Cell Lymphoblastic Lymphoma Induction I/a Prednisone Stage I + II Maintenance 6-Mercaptopurine / MTX Random 2 Maintenance 6-Mercaptopurine / MTX Induction Cytoreductive Prephase Random 1 Induction I/b Protocol M Induction I/a Dexamethasone Stage III + IV Re-Induction II/a + II/b Maintenance 6-Mercaptopurine / MTX Random 2 Maintenance 6-Mercaptopurine / MTX CRT only for CNS positive months Treatment Plan EURO-LB 02 for non T-Cell Lymphoblastic Lymphoma Stage I + II Maintenance 6-Mercaptopurine / MTX Induction Cytoreductive Prephase Induction I/a Prednisone Induction I/b Protocol M Stage III + IV Re-Induction II/a + II/b Maintenance 6-Mercaptopurine / MTX CRT only for CNS positive

3 EURO-LB 02 3 Members of the International Study Committee Paediatric Oncology AIEOP BFM BFM Austria CoALL EORTC SFCE UKCCSG PPLLSG Spain DCOG NOPHO Angelo Rosolen Alfred Reiter Georg Mann Gritta Janka-Schaub Anne Uyttebroeck Christophe Bergeron Robert Wynn Grazyna Wróbel Rafael Delgado Auke Beishuizen Ildikó Márky

4 4 EURO-LB 02 Members of the International Steering Committee and of the Data Safety and Monitoring Committee DMC Dr. P. Brice, oncologist, Paris, France Dr. D. Hasenclever, statistics, Leipzig M. Pamar, PhD, statistics, London, UK D. Poplack, MD, professor of pediatric oncology, Houston, USA

5 EURO-LB 02 Important Note 5 Important Note This document is intended to describe collaborative studies in lymphoblastic lymphomas and to provide information for entering patients. The International Study Committee does not intend it to serve as a guide for the treatment of unregistered patients. The present study Protocol does not represent recommendations for the standard treatment and is solely for the purpose of the current study. Whether the objectives of the study will be reached remains open. Treatment of patients according to the present Protocol without consultation of the International Study Committee is not justifiable. Before entering patients into one of the study clinicians must ensure that the study Protocol has received clearance from their ethical committee. The completion of the Protocol has been done with great care. Amendments may be necessary; these will be circulated to known participants in the trial, but institutions entering patients are advised to contact the appropriate Study Centres to confirm the correctness of the Protocol in their possession. Despite our best efforts the possibility of errors within this document cannot be entirely discounted. We therefore remind investigators that the responsibility for any therapy given lies with the attending physician alone and the authors of this Protocol do not take responsibility for any adverse consequences arising from application of this treatment. The content of the Protocol is confidential and may not be divulged in any way to centres not involved in the study, without the approval of the International Study Committee. For centres treating paediatric Non-Hodgkin-Lymphoma (NHL), but not affiliated to the study, a special reference Protocol without randomisation will be prepared and is available by request at the study centre. Protected labels are marked with the symbol ; the absence of does not imply that the term is free.

6 6 Table of Contents EURO-LB 02 TABLE OF CONTENTS 1 Introduction: Results of Different European Studies Goals and Rationale of Study EURO-LB Conclusions from Previous Studies Study Objectives Type of Study Participating Groups Patient Eligibility Eligibility to the Study Patients not evaluable for Treatment Response/Trial s Results Eligibility for Randomisation Emergency Situations Large Mediastinal Tumour Oliguria/Anuria/Hyperpotassemia Incipient Paraplegia and Cranial Nerve Palsy in Epidural/Paraspinal Lymphoma Surgery Diagnostics Diagnostic Procedures Classification of Lymphoma Processing of Tumour Material Initial Staging Observation of Tumour Extension Definition of Organ Involvement Staging System Differentiation of NHL Stage IV Disease versus Acute Lymphoblastic Leukaemia Treatment Plan Treatment Plan for T-cell Lymphoblastic Lymphoma Treatment Plan for Patients with Precursor-B-cell Lymphoblastic Lymphoma and Patients with Lymphoblastic Lymphoma with Immunophenotype not available Initial CNS Involvement Initial Testicular Involvement Incomplete Tumour Regression Non-Response Treatment Elements and Guidelines General Guidelines Guidelines for Dose Calculation Induction Protocol I Protocol M Reinduction Protocol II... 56

7 EURO-LB 02 Table of Contents Maintenance Therapy Radiotherapy Drugs Supportive Therapy Acute Cell-Lysis Syndrome Complication of Asparaginase Therapy Substitution of Blood Products Infection Prophylaxis und Therapy Osteoporosis-Prophylaxis Bisphosphonates Gastritis-Prophylaxis Extravasation of administered drugs Sagittal Sinus Thrombosis Evaluation of the Kinetics of Treatment Response Progressive Disease Follow-Up Studies of Disease Status Monitoring of late Effects Research Projects Organisational Aspects and Documentation Status of Study The Protocol International Study Committee Data Safety and Monitoring Committee (DMC) The Master Database Registration Randomisation Study Forms and Data Collection Follow-Up Reporting of Severe Adverse Events (SAE) Statistical Considerations Study questions Criteria of Assessment Number of Subjects required Analysis Interim Analysis of Event Free Survival Stopping Rule (Monitoring Toxicity) Information of Patient and Consent Publication Rules/Presentation of Results Declaration of Helsinki... 90

8 8 Table of Contents EURO-LB Reference List Index of Abbreviations Appendix...99 Appendix. Imaging Guidelines for mediastinal Disease.. Therapy schema of High-Dose Methotrexate Schema Processing of Tumour Material.... Reference Laboratories: Address and Dispatch Note Consent Forms.. Patient Informed Consent Form for Data Exchange.... Patient Informed Consent Form for Participation in the EURO-LB 02 Study Patients Information Protocol... Patient Informed Consent Form for 1 st Randomisation Patient Informed Consent Form for 2 nd Randomisation... Patient Informed Consent Form for Participation in Research Projects and Tumour Banking Forms... Flow Sheet for Data Flow and Collection... Registration.. Initial Observation... 1 st Randomisation... 2 nd Randomisation.. Events... Severe Adverse Events (SAE)

9 EURO-LB 02 Table of Contents 9 Late Effects.. Maintenance Therapy.... End of Treatment... Toxicity Form... Kinetics of Treatment Response.. Histopathological and Immunohistochemical Review.. Genetics... Immunological Report Participating Groups and Centres... Contract of Participation of a Clinical Institution Principal Investigators of the Study Groups/National Reference Centres. Participating Centres of Paediatric Haematology and Oncology Index. 14 1

10 10 EURO-LB 02 National Study Coordination Centre Version of the Protocol Start of the Pilot-Phase End of the Pilot-Phase Start of the Main-Study End of Patient Recruitment Prospective End of Study Address Study chairman Coordination Documentation Statistics Cytomorphology Radiology Vice Study Chairman

11 EURO-LB 02 National Study Coordination Centre 11 Reference Pathology Biology Diagnostic Radiology Radiotherapy

12 12 1 Introduction: Results of Different European Studies EURO-LB 02 1 Introduction: Results of Different European Studies Lymphoblastic lymphoma (LBL) accounts for % of the Non Hodgkin lymphoma of childhood and adolescence. The vast majority are T-cell-lymphoblastic lymphoma (T-LBL). With contemporary treatment Protocols event-free survival rates in the range of 70% to 80% have been reported 1-9. A major problem is the dismal prognosis of individuals with T-LBL failing first line therapy. Therefore, efforts have to be undertaken to induce and maintain disease response without early relapse. Although the majorities of failures are local recurrences, the contribution that local therapy modalities can make to the cure of patients is uncertain. Local radiotherapy (LRT) is certainly an effective local therapy 10. However, the contribution of LRT to failure-free survival in addition to chemotherapy may depend on the efficacy of the chemotherapy applied. Furthermore, mediastinal irradiation in particular carries serious late risks Criteria to identify those patients that are at risk for local relapse and who might therefore benefit from LRT are currently lacking 14. Because of the relatively low incidence of the disease a number of national European cooperative study groups decided to co-operate in optimising the treatment and investigating the disease. The participating groups in this European Inter-Group Co-operation on Childhood Non- Hodgkin`s Lymphoma are: AIEOP (Italy), BFM (Austria, Czechia, Germany, Switzerland), CoALL (Germany), DCOG (Netherlands), EORTC (France, Belgium), NOPHO (Norway, Sweden, Finland, Denmark, Iceland), PPLLSG (Poland), Spain, SFCE (France) and UKCCSG (United Kingdom). AIEOP Studies From 1992 two different Protocols for lymphoblastic lymphomas were conducted. The AIEOP LNH92 Protocol was based on a modified LSA2L2 strategy with a 4-week Induction phase including Prednisone, Cyclophosphamide, Daunomycin and High-Dose (HD) Methotrexate; a consolidation phase with HD-Methotrexate, low dose Cytarabine and Etoposide succeeded this Induction phase and was followed by a Maintenance treatment according to LSA2L2 regimen in order to complete a 24 months therapy in total. A total of 56 patients were enrolled, 5 stage I or II and 51 stage III or IV. The 8-year probability of event-free survival (pefs) was 71% (confidence interval (CI) 59.6%-83.3%) and the probability of overall survival (pos) 74.9% (CI 63.5%-86.2%). There were 16 recorded events, all of them resistant disease or relapses and occurring predominantly during early stages of maintenance treatment. The AIEOP LNH97 Protocol was designed with the aim of intensifying the Induction phase of LNH92 (with the inclusion of early Cyclophosphamide and multiple doses of L-Asparaginase) and the treatment preceding Maintenance, with the addition of a Reinduction phase, following the strategy of the AIEOP acute lymphatic leukemia (ALL) Protocol. Total therapy duration was 12 months for localised disease and 24 months for stage III and IV. 42 patients have been enrolled so far, 2 stage I or II and 40 stage III or IV. Estimated pefs at 3 years is currently 87% (CI 74.9%- 98.9%) with a total of 4 events, all of which were relapses occurring during the Maintenance treatment, within the first 12 months from diagnosis. Three year pos is 74.7% (CI 45%-100%). Events were 4; 3 of them early relapses. A longer follow-up is needed to have a reliable comparative analysis of these two Protocols, in order to ascertain whether we could obtain an improvement in outcome and/or to define whether the pattern of relapses has significantly changed from the LNH92 to the LNH97 Protocol. BFM Studies In the study NHL-BFM 90, patients were stratified according to stage 15. All patients received an 8-drug Induction including Prednisone over 9-weeks followed by an 8-week consolidation including Methotrexate 4 x 5g/m². Patients with stage I and II disease continued with maintenance therapy (6-Mercaptopurine daily/methotrexate weekly, both orally) for a total therapy duration of 24 months. Patients with stage III and IV disease received, after consolidation additionally an 8-drug Reinduction over 7 weeks and cranial radiotherapy (12 Gray (Gy) for prophylaxis). Patients received intensified chemotherapy if tumour regression on day 33 of Induction was <70% or when vital residual tumour was present after the Induction, but no local radiotherapy. With a median follow-up of 4.5 years, the estimated event-free survival at 5 years for 137 patients with

13 EURO-LB 02 1 Introduction: Results of Different European Studies 13 lymphoblastic lymphoma (T-cell: 106; precursor B-cell: 24; immunophenotype not available: 7) was 82+5%. For 105 evaluable patients with T-LBL pefs at 5 years is 90% (CI 82%-100%). Among these 105 T-LBL patients events were one early death, 8 tumour failures (within the first year from diagnosis) and one secondary AML. All eight T-LBL patients who failed therapy died. Tumour regression in patients with lymphoblastic lymphoma is frequently incomplete. Therefore the evaluation of the prognostic impact of the degree of tumour regression at defined points in time during Induction was one objective of that study. 101 T-LBL patients were evaluable for the speed of tumour response. Two patients received intensified therapy due to <70% tumour regression on day 33. Of 19 patients with tumour residues after Induction, 2 relapsed as compared to 4 of 80 patients with complete tumour regression. Except B-symptoms there was no prognostic factor identified that was predictive for increased risk for failure 14. The main question of the subsequent study NHL-BFM 95 is whether prophylactic cranial irradiation (CRT) for Central Nervous System (CNS) negative patients can be omitted. Although the final analyses of that trial is still pending, in two of two planned interim analysis there was no indication that omission of prophylactic CRT will worsen the outcome of these patients. The main toxicity of this treatment program is haematotoxicity, while non-haematological toxicity is moderate. By now, the results of the toxicity monitoring according to GPOH-classification (modified NCI-CTC) of toxicity are available. The results are shown in the table below. Percentage of Patients with Grad 3 and Grade 4 Toxicity in Study NHL-BFM 95 Treatment Phase Induction phase I/a Induction phase I/b Prot. M Reinduction phase II/a Reinduction phase II/b N=139* N=139 N=141 N=128 N=125 % % % % % general condition Haematology haemoglobin leukocytes granulocytes thrombocytes Infection infection fever Mucosa stomatitis diarrhoea Skin skin lesions Kidney: same percentage, less or no grade 3/4 toxicity for creatinine, haematuria and creatinine-clearance proteinuria Liver bilirubin SGOT/SGPT Cor: no grade 3 or 4 toxicity at arrhythmia, action of heart or Echo-CG Neurology central neurotoxicity peripheral neurotoxicity *) The number of patients is slightly reduced for some scales because of missing values. CoALL Studies Since 1982 children with T-NHL have been treated in 5 therapy trials COALL 82,85,89,92 and 97. Patients with T NHL stage III + IV received the high risk Protocol for ALL patients. In COALL 82 patients received one additional course of Cyclophosphamide before Induction was started and local radiotherapy was performed in case of residual tumour at the end of intensive therapy. In COALL 85 local irradiation and Cyclophosphamide in front of Induction was omitted. Only 1 patient in study COALL 89 with T-NHL stage 1 was treated with a six-week Induction therapy consisting Vincristine/Methotrexate/ Cyclophosphamide and Prednisone and then continued with maintenance therapy. Since COALL 92 patients with stage I + II have been treated according to the low risk ALL Protocol.

14 14 1 Introduction: Results of Different European Studies EURO-LB 02 From 1999 the cranial irradiation for CNS negative patients was omitted. For all patients with T NHL treated in the COALL Protocol the estimated event free survival was 79% (60 patients/11 events), patients with stage III + IV T NHL had an EFS of 77% (56 patients/11 events) and patients with stage I + II had an EFS of 100% (4 patients no event). DCOG The DCLSG (currently the Dutch Childhood Oncology Group: DCOG) treats children with non B- NHL since 1994 according to the BFM-90 strategy without profylactic CNS radiation. 37 children are included in the protocol: 34 (92%) T-cell, 2 (5%) pre-b cell, 1 (3%) unknown immunophenotyping. 1 child is stage 2, 28 children stage 3, 8 stage 4. Four children did not receive CR: one child died before starting treatment, 1 early death, 2 nonresponders. Thirty three (92%) achieved CR; 4 relapses were monitored, all during therapy. The EFS at 5 years is 73,8%, the overall survival 74,7%. NOPHO Studies Since 1995 the five Nordic countries (Denmark, Finland, Iceland, Norway and Sweden), use a common Protocol for the diagnostic work-up, classification, staging and treatment of children with NHL: the NOPHO - NHL. 95. Treatment is based on immune phenotypic subgroup: Non- B-NHL, B-NHL and LACL and the stage of the disease. Patients with non-b-nhl are treated with a modified NOPHO ALL 92 Protocol while B-NHL and LCAL cases were treated with the BFM 90 Protocol but without prophylactic CNS irradiation. During the period of , 230 children under the age of 15 years were diagnosed with NHL. The pefs was % at 5 years. The 5 year pefs-values were 90 % for B-cell, 87% for pre-b, 84 % for LCAL and 82% for T-cell NHL. Patients with CNS involvement at diagnosis had a significant worse prognosis among the Stage IV patients with a pefs of 49% compared to children without CNS involvement (pefs =90 %); (p<0.01). Patients without CNS-disease at diagnosis had a 5 -year pefs of 89 %. PPLLSG Studies From 1993 to patients 0.9 to 17 years of age with non-b LBL were treated in 11 centres of PPLLSG according to the NHL-BFM-90 Protocol. According to the extent of primary disease, patients were sub-staged into 4 groups; stage I: 1 (1.9%); II: 5 (9.3%); III: 30 (55.5%); IV: 18 (33.3%). 47 children entered a complete remission (87%), 3 partial remission (5.6%). After median follow-up of 2.8 years the estimated EFS was 65% for all stages and 60% for those with stage III disease and 63% for those with stage IV disease. EFS for patients with residual tumour less than 70% on day 33 was 68% compared with 84% for those patients with complete tumour response on this day. Events were: 1 toxic death (1.9%), 3 progression during therapy and eight relapses during and after therapy. Most relapses were local and occurred up to 16 months from diagnosis. We did not identify the significant prognostic factors, which may have had an impact on outcome in these patients. SFCE Studies Since 1981 SFOP (currently the Société Française des Cancers de l'enfant; SFCE ) has organised three consecutive therapy trials. In study LMT 81 (84 pts) the former LSA2L2 strategy was complemented by 10 courses of high dose Methotrexate (3g/m 2 i.v. over 3 hours). Local radiotherapy was performed in case of residual tumour. The estimated event-free survival at 5 years is 75% (standard deviation (SE): 2.5). Events were 13 tumour failures (2 to 29 months from diagnosis) and 4 deaths in first complete remission. In study LMT 89 (88 pts) the LMT 81 Protocol was complemented by an Induction Protocol containing 1 course of COP (Cyclophosphamide, Vincristine, Prednisone) and 2 courses COPADM (Cyclophosphamide, Vincristine, Prednisone, Adriamycin and Methotrexate). 1 year therapy duration was given for localised lymphoma and 2 years for stage IV. The pefs at 5 years is 69% (SE: 9.0). 24 events occurred: 2 partial remissions, 1 toxic death and 21 relapses.

15 EURO-LB 02 1 Introduction: Results of Different European Studies 15 In conclusion, the pulse therapy during Induction did not improve the results of study LMT81 and strategy was re-focussed on sequential therapy. Study LMT 96 is a modified BFM Protocol: After a classic prephase with Prednisone alone, Cyclophosphamide (1g/m 2 ) and HD-Methotrexate (3g/m² i.v. over 3 hours) are given in Induction phase. After consolidation with 6 courses of HD-Methotrexate with Cytarabine or Asparaginase, a classic BFM Protocol II is given and, in addition, 6 monthly Reinduction with HD-Methotrexate, Cytarabine and Prednisone, followed by the classic maintenance therapy (6-Mercaptopurine, Methotrexate) for a total therapy duration of 24 months. pefs at 20 months is 78%. Spain The Spanish group for the treatment of lymphoblastic lymphomas registers and treats children within the Protocol LNHnoB98 (based on LMT96 of the SFOP without modifications) since The OS is 72% and the EFS is 69% with a median follow-up of 16 months. UKCCSG Studies NHL 9004 This trial ran between 1 st July 1990 and 30 th April 1995 and followed identically the Medical research Council UKALL XI protocol. Between 1990 and March 1992 patients received a 5 drug Induction regimen consisting of daunorubicin 45 mg/m 2 daily on the first two days of treatment, weekly intravenous vincristine X 5, daily oral prednisolone for 28 days, 9 injections of Erwinia asparaginase over the first three weeks of therapy and intrathecal methotrexate injections X 3 over one month followed by randomisation to either a late intensification pulse at week 20 or both an early and late intensification identical module at week 5 and week 20. Central nervous system directed therapy was with high dose methotrexate 6-8 g/m 2 X 3 doses. After 1992 all patients received 2 intensification modules but the Induction daunorubicin was dropped to keep the total dose below 200mg/m 2 in view of the worries about late cardiac toxicity. 98 patients were recruited into the study; Stage I - 10, II - 4, III - 63, IV Of the Stage I patients there was just one event, a secondary leukaemia at 5 years, whilst in Stage II there was a single relapse at 4 years 2 months from diagnosis but the patient was rescued and remains alive and well. Of the Stage III patients there have been 17 relapses of whom 8 are still alive. The median time to relapse was 8.7 months. In Stage IV 13 out of the 21 patients have relapsed at a median time of 8.0 months and of them 11 have died. For all patients in the study 5 year overall survival was 81.6% (95% CI %) and at 9 years 77.1% ( %). Corresponding event free survival figures were 69.4% and 66.7% at 5 and 9 years respectively. For Stage III the overall survival was 86% and event free 73%. For Stage IV 48% and 38% respectively at 5 years. Conclusions Overall in 9004 the event free survival at 5 years was comparable to that seen in 8503, possibly a little improved. However the most disappointing feature was the poor survival overall and of events for Stage IV patients. Clearly the low stage patients in general faired well although the secondary leukaemia in the Stage I patient in a worrying feature. These results however are disappointing when set alongside the results of the recently published BFM 90 protocol. NHL 9503 Between May 1995 and February 2000 UKCCSG registered 109 patients on to the 9503 protocol. However 12 patients have been excluded from the analysis for the following reasons: 5 had peripheral T cell disease, 2 had previous treatment, 2 had no pathology review and 3 were removed for various other reasons. The breakdown by stage of the residual 97 patients was 4 Stage I, 5 Stage II, 73 Stage III and 15 Stage IV. A recent review of staging and histology has identified that 3 of the patients formerly of stage II did in fact have Stage III disease making a total number of Stage II patients of 76. Patients analysed have T or precursor B cell lymphoblastic lymphoma or lymphoblastic lymphoma of determinant cell type where immunophenotyping is absent. Between 1995 and June 1998 patients received a leukaemia protocol identical to the then current Medical Research Council protocol UKALL XI and ALL 97 in which they received

16 16 1 Introduction: Results of Different European Studies EURO-LB 02 standard Induction without daunorubicin, 2 intensification pulses, high dose methotrexate as CNS therapy and were then randomised to receive a third intensification module or not. After June 1998 until the close of the study in February 2000 all patients received the same basic protocol plus a third intensification. 15 patients were randomised to a third block of chemotherapy, 18 to no further treatment and after June 1998 all remaining patients received a third intensification. Median follow-up for all 97 patients is 50 months with the range from 4 to 85 months off treatment. 21 patients have died since registration, 5 from other causes with no previous relapse and 24 have relapsed of whom 8 remain alive. In view of the doubts about staging of patients in Stage I and II they will be further analysed in due course but for the whole study 5 year event free survival is 66.7% and overall survival 77.3%. The values for Stage III disease are 70.2% event free and 80.1 overall survival, for Stage IV 77.9% and 76.6% respectively. There appears in this trial to be a better survival for Stage IV patients with almost identical therapy to that given in Conclusion There seems to be little evidence of improvement in event free or overall survival for advanced stage lymphoblastic lymphoma over the last decade in the UKCCSG studies. Variations between the protocols in terms of outcome despite almost identical therapy is delivered probably due to small numbers and individual adverse prognostic features which we are very poorly able to define. It is too early to predict long term survival on the patients from the protocol 9503 but there is no doubt that the results are still inferior to BFM 90 giving greater strength to our decision to move towards usage of that protocol in the proposed new European wide lymphoblastic lymphoma protocol.

17 EURO-LB 02 2 Goals and Rationale 17 2 Goals and Rationale of Study EURO-LB Conclusions from Previous Studies The NHL-BFM 90 study produced favourable results, especially for patients with T-LBL. The participating national study groups have agreed to use NHL-BFM 90 as a reference arm with the exception, that prophylactic cranial irradiation should not be applied in CNS-negative patients. In the NHL-BFM 90 study in T-LBL patients all therapy failures occurred within the first year of diagnosis, whilst relapses in patients with Precursor-B-Cell-Lymphoblastic Lymphoma (pb-lbl) were observed later, as late as the 3 rd and 4 th year after diagnosis. Whilst all patients with T-LBL who failed to the front-line therapy died, 3 of 5 patients with pb-lbl who relapsed entered long term second remission. The overall survival of patients with pb-lbl of study NHL-BFM 90 is 91% at 10 years (SE 6%). Based on this observation, patients with pb-lbl will be treated according to the reference arm without randomisation. Patients with lymphoblastic lymphoma in whom immunophenotype is not available are also to be treated according to the reference arm without randomisation. Patients with T-LBL will enter a randomised trial aimed at optimisation of treatment. Based upon in vitro and in vivo data, which demonstrated that compared to Prednisone, Dexamethasone is associated with increased anti-lymphoblastic activity and enhanced CNS penetration, the effect of Dexamethasone during Induction will be tested in a randomised study against the reference treatment strategy of study NHL-BFM It is reported that the substitution of Dexamethasone for Prednisone during remission Induction may be associated with a higher incidence of septic episodes and deaths and, in post-remission therapy, neurocognitive late effects 23,24. In some studies the incidence of osteopathology is reported to be increased in patients receiving Dexamethasone Therefore, it has to be ensured that the introduction of Dexamethasone during Induction does not lead to an unacceptable excess of toxicity. Thus toxicity will be closely monitored (see chapter 21.6, page 86). In the MRC ALL97/99 Study in UK there was an equal distribution of avascular necrosis of bone for Dexamethasone and Prednisolone (Mitchell C, Kinsey S, Vora A, Richards S, Eden T; MRC ALL 97/99 A Randomized Comparison of Dexamethasone versus Prednisolone and Thioguanine versus Mercaptopurine in the Treatment of Childhood Acute Lymphoblastic Leukemia. Abstract. Blood. Volume 100, Number 11, November 2002). Based on the observation that in patients with T-LBL treatment failure occurred almost exclusively in the first 12 to 15 months after the beginning of therapy the Protocol will further test whether the duration of maintenance treatment for these patients can be reduced from 24 months to 18 months total therapy duration (counted from the first day of cytoreductive prephase). Incomplete tumour regression during Induction is a frequent observation in patients with lymphoblastic lymphoma. In previous BFM studies, especially in T-LBL patients with mediastinal tumours, absence or presence of tumour remnants after Induction phase I/a and I/b were not predictive for the subsequent course of the patients. Therefore, no change in therapy is recommended in case of incomplete tumour regression. An exception may be those rare cases of virtually no tumour shrinking at all. Even in these patients, however, there is currently no alternative therapeutic strategy proven to be efficacious. The vast majority of tumour failures, especially in T-LBL patients, occurred within the first year of treatment, and all these relapsed T-LBL patients died. This observation suggests a major biological difference of the tumour in these patients. None of the currently available clinical and biological parameters was predictive for the risk for failure. However, important candidate parameters such as kinetics of treatment response and genetics are not available for the majority of patients. Therefore, an important component of EURO-LB 02 is the assessment of these parameters in as many patients as possible so that they may be assessed as candidate

18 18 2 Goals and Rationale EURO-LB 02 markers for risk of treatment failure. The identification of the patients at risk for this kind of early failure is an essential step in designing risk-based therapy in future trials. The kinetics of response are the most important prognostic factor in childhood T-ALL 28. Monitoring the decrease of leukemic blasts in the bone marrow by means of clone specific probes technology (minimal residual disease monitoring) resulted in an even higher predictive power 29,30. Due to shortness of diagnostic tumour material, clone specific probes are not always available; this method is difficult to apply in lymphoma patients. More recently, immunological methods have become available which allow the detection of rare residual lymphoblasts in blood or bone marrow by phenotype at a detection level of 10 3 to 10 4 even during the course of treatment 31,32. In an international cooperation, a consensus panel of antibodies useful for minimal residual disease (MRD) monitoring in particular in T-cell acute lymphoblastic leukemia has recently been accepted. According to pilot observations, this panel can also be adopted for T-LBL. The technique of MRD monitoring could therefore also be used to estimate the presence of minimally disseminated disease (MDD) in blood and bone marrow of patients with clinically localised T-LBL. There is a currently ongoing pilot-study concerning Minimal Residual Disease Monitoring of children with lymphoblastic lymphoma. One aim of this pilot study is to find out about the feasibility of MRD Monitoring for these patients. If the study will show, that MRD Monitoring is feasible, an accompanying research project of the EURO-LB 02 Protocol with MRD Monitoring will be started. 2.2 Study Objectives The first aim of the study is to test the impact of replacing Prednisone by Dexamethasone during Induction treatment (Protocol I) on the improvement of the event free survival of patients with newly diagnosed T-LBL. The second question is to test whether for patients with T-LBL the standard maintenance therapy of 24 months total therapy duration can safely be reduced to 18 months total therapy duration (calculated from the first day of treatment). The first main question of the study will be able to be answered after a recruiting period of 3.5 to 4 years, according to the expected amount of patients of the participating groups. See chapter 21.3, page 84. For answering the second aim, it will be necessary to continue the second randomisation in the subsequent inter-group study on lymphoblastic lymphoma. Further aims of the study are: to test if the treatment results of study NHL-BFM 90 can be reproduced in an international study including most European study groups. The omission of prophylactic cranial irradiation for patients with advanced stage disease in study EURO-LB 02 has to be considered in this regard. to evaluate prognostic factors highly predictive for treatment failure. to study the epidemiology and biology of the disease.

19 EURO-LB 02 3 Type of Study 19 3 Type of Study EURO-LB 02 is an international multicentre cooperative study. The study includes: registration of all patients of participating centres with newly diagnosed lymphoblastic lymphoma trial with two consecutive randomisations using a factorial design for patients with T-LBL reference treatment arm for patients with pb-lbl and patients with LBL in whom immunophenotype is not available program of investigations to identify prognostic factors highly predictive for failure of current treatment The first randomisation concerns the glucocorticoide hormone given in Induction phase I/a, namely Prednisone versus Dexamethasone. The second randomisation concerns the total therapy duration of 24 months versus 18 months. After second randomisation, there will be four arms: Arm 1 (reference arm): strategy NHL-BFM 90 without prophylactic CRT Arm 2: NHL-BFM 90 without prophylactic CRT, but Dexamethasone 10 mg/m²/d instead of Prednisone 60 mg/m²/d in Induction phase I/a Arm 3: NHL-BFM 90 without prophylactic CRT, but with a duration of maintenance treatment until 18 months instead of 24 months total therapy duration calculated from the first day of therapy Arm 4: NHL-BFM 90 without prophylactic CRT, but with Dexamethasone 10 mg/m²/d instead of Prednisone 60 mg/m²/d in Induction phase I/a and duration of maintenance treatment until 18 months instead of 24 months total therapy duration calculated from the first day of therapy The programs to identify prognostic factors include: 1. Kinetics of early tumour response to treatment 2. Cytogenetics of diagnostic material 3. Gene expression profile of tumour cells 4. Matrix comparative genomic hybridisation (Matrix-CGH) 5. SELDI (Surface Enhanced Laser Desorption/Ionisation) 6. Tumour cell/dna/rna-banking to ascertain appropriate material for additional projects, which may become available during the course of study. A biological committee will be responsible for approval of research projects that should have access to this material. It is agreed that research projects approved by the biological committee will have access to the material of the patients from all participating groups and to the clinical data of the study in an anonymous way. In turn, data generated in research projects will be made available to the database of the study. The rules for publication of data are described in chapter 23, page 89.

20 20 4 Participating Groups EURO-LB 02 4 Participating Groups Participating groups are: AIEOP: Associazione Italiana di Ematologia ed Oncologia Pediatrica BFM Germany - Switzerland - Czechia BFM Austria CoALL Germany EORTC for France and Belgium SFCE: Société Française des Cancers de l'enfant UKCCSG: United Kingdom Children s Cancer Study Group PPLLSG Poland Spain DCOG: Dutch Childhood Oncology Group NOPHO for Scandinavian group These groups form the core collaborative group. Other national groups may join this study after discussion with the core group. Participating groups are expected to register all patients with lymphoblastic lymphoma diagnosed within the clinics of that groups in order to avoid patient selection bias. to provide diagnostic material for central review and research projects. All centres are expected to obtain approval for the study from their local research ethical committee according to their national policy and to control whether the Protocol matches with GCP-guidelines and local law/requirements. Further more all participating centres have to agree to the contract of participation and all centres have to declare to adhere to the EURO-LB 02 Protocol. For the participating clinics see Appendix Participating Centres of Paediatric Haematology and Oncology page 140.

21 EURO-LB 02 5 Patient Eligibility 21 5 Patient Eligibility 5.1 Eligibility to the Study Patients meeting the following criteria are admitted to the study: lymphoblastic lymphoma diagnosed either by histomorphological or cytomorphological characterisation age < 22 years (i.e. up to the 22 nd birthday, not including the 22 nd birthday) signed informed consent of patient/guardians for participation in the study EURO-LB 02 no evidence of pregnancy or lactation period no participation in another clinical study All patients fulfilling these eligibility criteria will be registered in the study. A registration fax must be sent to the responsible coordination centre within 7 days after the beginning of the prephase. For the Registration Form see Appendix, page 119. In return the treating hospital will receive a confirmation of registration. A subsequent exclusion is allowed only if the diagnosis of a lymphoblastic lymphoma has been incorrectly made. 5.2 Patients not evaluable for Treatment Response/Trial s Results Patients who fulfil the eligibility criteria for the study and who meet one of the following criteria will not be randomised and are not evaluable for treatment response and trial s results. Nevertheless, they should be registered and documented in the study. The national study coordinator should be contacted for potential therapy modifications before the start of treatment. HIV infection or AIDS severe immunodeficiency previous organ transplantation previous malignancy pre-existing disease prohibiting chemotherapy as per instructions of the Protocol previous chemotherapy or radiotherapy previous systemic corticosteroid treatment for more than 8 days within two months before the beginning of therapy according to the Protocol EURO-LB Eligibility for Randomisation Only patients with T-LBL will be randomised. Patients with pb-lbl are to be treated according to the reference arm without randomisation. Patients with lymphoblastic lymphoma in whom immunophenotype is not available are also to be treated according to the reference arm without randomisation First Randomisation (Prednisone versus Dexamethasone in Induction phase I/a) Eligibility for Randomisation: eligibility for the study (see chapter 5.1, page 21) evaluable for treatment response (see chapter 5.2, page 21) diagnosis of T-LBL prephase started slides available for national pathological and/or cytomorphology review potential follow-up of at least 36 months signed informed consent of patient/guardians Randomisation should be obtained for eligible patients within the first seven days of prephase.

22 22 5 Patient Eligibility EURO-LB Second Randomisation (total therapy duration 18 versus 24 months) Eligibility for Randomisation: eligibility for the study (see chapter 5.1, page 21) evaluable for treatment response (see chapter 5.2, page 21) diagnosis of T-LBL confirmed by the national reference laboratory (pathology and/or cytomorphology and immunophenotyping) signed informed consent of patient/guardians no progressive disease Randomisation should be obtained within 21 days after the beginning of the maintenance treatment. In the case of refusal of either one of these randomisations the patients can still be included in the other randomisation.

23 EURO-LB 02 6 Emergency Situations 23 6 Emergency Situations Patient management in emergency situations is primarily the responsibility of the physician in charge. In this chapter only recommendations are given for certain situations. The national study coordinators are available for consultation. 6.1 Large Mediastinal Tumour If there is considerable respiratory impairment, e.g. orthopnea, then, except for a blood count, all invasive diagnostic procedures including lumbar puncture should be postponed. If at the same time a pleural effusion exists, it should be carefully relieved under local anaesthesia with a 16 G Teflon needle. Replace plasma! No long-term drainage, if possible. For critical pericardial effusion drainage is required. The drained fluid is used for diagnosis (see chapter 8, page 26). A cytoreductive therapy with Prednisone/Prednisolone 60 mg/m²/d and Cyclophosphamide 100 mg/m 2 /d should be started immediately. The dosage can be increased depending on clinical progress. Guidance for the prevention and treatment of tumour lysis syndrome is given in chapter 14.1, page 65. After stabilisation of the clinical condition, normally after 1 or 2 days, diagnostic procedures may be undertaken. If there is slight, but noticeable respiratory congestion, the next step is to try to make a diagnosis by examining the bone marrow and any available pleural effusion. Following intubation life-threatening respiratory failure can occur due to tracheal edema and tracheal compression post-operatively. If intubation is performed in such patients, it is recommended to electively continue ventilation after surgery and to start cytoreductive therapy immediately. Extubation should be postponed until a significant shrinking of the tumour is achieved by cytoreductive therapy. If a thoracotomy is necessary for the sample excision, then a debulking of the tumour should be performed in order to reduce the risk of tracheal compression post-operatively. 6.2 Oliguria/Anuria/Hyperpotassemia (see also chapter 14.1, page 65) Definition Urine excretion < 50 ml/m²/h in spite of: Furosemide 10 mg/kg/d i.v. hydration ml/m²/h The usual definition of < 5 ml/m²/h is not useful in this situation: Because of the rapid accumulation of potassium, in this situation it is more useful to evaluate the urine output in relation to the fluid input. Differential Diagnosis of reduced urine output: Four reasons for oligo-/anuria are most frequent in these patients and require different therapy procedures: - urinary tract obstruction by lymphoma - direct kidney infiltration - uric acid nephropathy due to pre-existing cell lysis - calcium-phosphate nephropathy due to pre-existing cell lysis It is possible to make a differential diagnosis with ultrasound and by determining the serum concentration of creatinine, urea and phosphate. Ultrasound: Biochem: Urine: * obstruction of the urinary tract * kidney infiltration * potassium * uric acid * phosphate * calcium * uric acid crystals * calcium-phosphate crystals

24 24 6 Emergency Situations EURO-LB 02 Therapy - If hyperpotassemia already exists (potassium > 5.6 mmol /l), Resonium A is given orally 1 g/kg (Caution: constipation), glucose 1 g/kg plus soluble insulin 0.3 IU/kg infusion is used for 30 minutes pending institution of haemodialysis, and NaHCO 3 2 mmol/kg i.v. should be given. If there are electrocardiographic (ECG) changes, then calcium gluconate 10% (-2) ml/kg is injected slowly i.v. (Caution: bradycardia). Begin haemodialysis immediately. If this is not possible, then the patient has to be moved as soon as possible to an oncology centre that can provide haemodialysis. See chapter , page If it is probable that a kidney infiltration and/or a urinary tract obstruction is responsible for the oliguria/anuria, then a cytoreductive therapy with Prednisone 30 mg/m 2 /day is added to the haemodialysis. Prednisone is given 3 times a day and if Cyclophosphamide is necessary additionally, Cyclophosphamide is administered in between two dialysis sessions (it is low protein binding and 30% to 60% is eliminated with haemodialysis). - If oligo-/anuria is most probably caused by an already existing acute cell lysis syndrome (hyperuricaemia, hyperphosphataemia, no kidney infiltration and no obstruction of the ureter), then cytoreductive therapy should be postponed until acute cell lysis syndrome has been treated (see chapter 14.1, page 65). However, the cytoreductive therapy should not be postponed much beyond 48 hours. 6.3 Incipient Paraplegia and Cranial Nerve Palsy in Epidural/Paraspinal Lymphoma Caution: Lumbar puncture may carry the risk of brainstem herniation and incarceration. If the diagnosis of lymphoblastic lymphoma can be established by other manifestations, a cytoreductive therapy with Cyclophosphamide 200 mg/m²/d and Prednisone 60 mg/m²/d should be started immediately. The dosage of both medications can be increased, if necessary. In case of no other manifestations useful for diagnostic procedures beside an epidural manifestation, a surgery should be performed with a decompression of the cord. In case of a cranial nerve palsy caused by lymphoma localised at epipharynx, paranasal sinus or skull base, surgery should be limited and used for gaining utilisable biopsy, if decompressing resection would cause mutilation or a loss of function. Cytoreductive therapy should be started immediately.

25 EURO-LB 02 7 Surgery 25 7 Surgery Surgery is firstly for diagnosis. In case of suspected lymphoma all other options to establish the diagnosis should be considered before surgery is performed: examination of blood and bone marrow in case of pleural effusion/ascites: puncture and cytological and immunophenotypic examination Only if the diagnosis cannot be established using these simple procedures surgery should be performed. Any surgery is primarily to establish the diagnosis. Complete resection should not be intended except if it is possible without any risk and functional loss for the patient. The most peripheral lesion should be chosen for biopsy, e.g. in case of mediastinal tumour, extra-thoracic manifestations which can be used for a biopsy should be carefully sought. A complete primary resection should only be performed under the following three circumstances: 1. It is carefully shown (including bone marrow aspiration (BMP)) that the lymphoma to be resected is the only one existing (stage I). 2. An unusual or incalculable operation risk is not foreseen, and it is possible to begin chemotherapy within 5 days of the operation. 3. The resection does not lead to any substantial loss of function. Substantial loss of function is: Loss of a large portion of the gastrointestinal tract, which is more than in normal resections and which could lead to a decrease in resorption capability. Loss or partial loss of the kidney, spleen, pancreas, adrenal gland, uterus, both ovaries, both testicles, loss of an eye. Mediastinal Tumour Important: For a mediastinal tumour, all other diagnostic procedures (BMP, pleura puncture, etc.) should be used before surgery. If there is no pleural effusion and no lymphoblasts in the Bone Marrow (BM), then a thorough search for extrathoracic manifestations, which can be used for a biopsy, should be carried out. Note that with intubation respiratory failure can occur due to tracheal edema and tracheal compression post-operatively. If intubation is performed in such patients, it is recommended to electively continue ventilation after surgery and to start cytoreductive therapy immediately. Extubation should be postponed until a significant shrinking of the tumour is achieved by cytoreductive therapy. If a thoracotomy is necessary for the sample excision, then a debulking of the tumour should be performed in order to reduce the risk of tracheal compression post-operatively. Under no circumstances should a critically large mediastinal tumour with clinical symptoms of respiratory distress be treated surgically. In such a situation, a cytoreductive therapy with Prednisone and eventually with Cyclophosphamide should be started immediately. Surgery should be postponed until clinical stabilisation is achieved (see chapter 6, page 23). Most important: Although surgery should be a minimal invasive process, in most cases, an important contribution of surgery is to gain enough material for comprehensive characterisation of the disease. In no case the biopsy material may be fixed completely in Formol, because most important biological investigations of the tumour cells, e.g. Cytogenetics, would then be prevented. The optimal way is to the surgeon and the paediatric oncologist and pathologist to co-operate in planning surgery. Ideally, the biopsy material should be taken over by the paediatric oncologist or the pathologist already in the operation room for further appropriate processing (see chapter 8, page 26).

26 26 8 Diagnostics EURO-LB 02 8 Diagnostics Important notice: If there is either severe respiratory insufficiency and/or vena cava compression syndrome due to a mediastinal tumour, then with the exception of blood tests all further diagnostic and especially invasive diagnostic procedures should be postponed. These procedures (Bone Marrow Puncture (BMP)/Liquor Puncture (LP) etc.) can all be safely completed once the clinical condition has been stabilised. Cytoreductive therapy with Prednisone and eventually with Cyclophosphamide should be started immediately; the surgical intervention should be postponed until clinical stabilisation has taken place (see chapter 6, page 23). 8.1 Diagnostic Procedures To establish the diagnosis of Non-Hodgkin s-lymphoma, the least invasive procedure should be preferred. It is important to notice, however, that fine-needle or trocar needle biopsy is usually not recommended. Tissue specimen gained by needle biopsy is mostly insufficient for complete characterisation of the disease. Therefore, needle biopsy should be restricted to those where more invasive intervention poses undue risk. In case of suspected lymphoma all other options to establish the diagnosis should be considered before surgery is performed: examination of blood and bone marrow in case of pleural effusion/ascites: puncture and cytological and immunophenotypic examination In case of malignant effusions and/or significant bone marrow infiltration (>20% blasts) the diagnosis can be established by means of cytomorphology on cytospin preparations and immunophenotyping of cell suspensions. Only if the diagnosis cannot be established using these simple techniques surgery should be performed. Surgery is primarily performed so that a diagnosis is achieved. Complete resection should not be intended, except if possible, without any risk and functional loss for the patient. The most peripheral lesion should be chosen for biopsy, e.g. in case of a mediastinal tumour, extra-thoracic manifestations should be carefully sought, which can be used for a biopsy (see also chapter 7, page 25). Material has to be ascertained in the following way: Tumour touch imprints Formol fixated material for histology Fresh material in cell culture media or, if not available, NaCl 0.9% for: 1. Cytogenetics 2. Immunophenotyping 3. Gene expression profiling 4. Matrix comparative genomic hybridisation 5. Proteomics studies 6. Tumour cell banking

27 EURO-LB 02 8 Diagnostics 27 If only limited biopsy material is available for carrying out all the diagnostic and research studies described above, tumour processing must follow a priority algorithm. 1. Assurance of diagnosis In case of tumour biopsy assurance of diagnosis by histology, immunohistochemistry and cytomorphology of tumour touch imprints In case of malignant effusions or bone marrow infiltration assurance of diagnosis by cytomorphology and immunophenotyping 2. Cytogenetics 3. Gene expression profiling 4. Matrix-CGH 5. SELDI 6. Tumour cell banking The following procedure is recommended: Ideally, the paediatric oncologist or the pathologist should take over the biopsy specimen directly in the surgery room for appropriate processing. As a first step, the biopsy should be subdivided into one-third to two-thirds of volume. The twothird portion is kept sterile. Prepare touch preparations from the one-third proportion. If cytomorphology shows NHL, use the one-third proportion for pathology, if sufficient according to the agreement of the pathologist. Use the two-thirds proportion for further biological studies according to the priority list above. Ideally, the tissue for further biological studies should be sent as one sterile piece within 24 hours to a national central laboratory for further processing (see Appendix: Processing of tumour material page 105). Important note: Central histological review is necessary to enter in the study. A block of paraffin embedded material or 16 unstained slides will be requested for each patient. The processing and shipment of the tumour material (biopsy, effusions, bone marrow, blood) is described in details (see chapter 9, page 30). 8.2 Classification of Lymphoma Cytomorphology Cytomorphological classification will be performed according to the French-American-British (FAB) classification for haematological malignancies. FAB-L1 or FAB-L2 type cytomorphology defines lymphoblastic lymphoma.

28 28 8 Diagnostics EURO-LB Immunological Classification by FACS-Analysis Immunological classification will be performed according to the Criteria of the European Group for Immunophenotyping of Leukemias (EGIL) Lymphoblastic Lymphoma of the T-cell lineage Subtype all: pro T pre-t intermediate T mature α/β + T δ/γ + T Marker cytoplasmic or membrane-bound CD3 pos., mostly TdT pos., HLA-DR neg. and CD34 neg. CD7 pos. CD2 pos. and/or CD5 pos. and/or CD8 pos. CD1a pos. membrane-bound CD3 pos., CD1a neg. anti TCR α/β pos. anti TCR δ/γ pos. 2. Lymphoblastic Lymphoma of precursor-b-cell-lineage Subtype all: pro-b common-all type pre-b Marker CD19 pos. and/or CD79a pos. and/or CD22 pos. (at least 2 of 3 pos.) TdT pos. and HLA-DR pos. no further antigens for differentiation CD10 pos. cytoplasmic IgM pos., surface-ig negative Criteria for Marker Positivity: For cytoplasmic or nuclear markers: Found in at least 10% of the lymphoma cells. For surface markers: Found on at least 20% of the lymphoma cells. Early-B-Cell-Lymphoma Early B-Cell-Lymphoma is defined as lymphoma, where the morphology is FAB L1 or L2 but surface immunoglobulin (sig) is identified by immunophenotype. These patients should be treated as lymphoblastic lymphoma. The complete immunophenotypic and genetic characterisation of these cases is especially important, since these constellations are not yet confirmed entities. In any case, it is requested to consult the Study Centre. Lymphoblastic lymphoma with co-expression of myeloid antigens Expression of CD13, CD33, CDs65 on 20% of the lymphoblasts Histopathological and Immunohistochemical Classification Histopathological classification will be performed according to the WHO classification for haematological malignancies The WHO classification divides lymphoblastic lymphoma into two subtypes: Precursor-B-cell-lymphoblastic Lymphoma T-cell-lymphoblastic-Lymphoma Precursor-B-lymphoblastic lymphoma (WHO: precursor B-lymphoblastic lymphoma; ICD-0: 9728/3) Precursor-B-lymphoblastic lymphoma express TdT, CD79a, HLA-DR, CD19, CD22 and are usually negative for surface immunoglobulin. In addition, most express CD10, and some of them express CD34. T-lymphoblastic lymphoma (WHO: precursor T-lymphoblastic lymphoma; ICD-0: 9729/3) T-lymphoblastic lymphoma express TdT and cytoplasmic CD3. In addition, there is a variable expression of membrane-bound CD3, CD1a, CD4, CD8, CD2, CD5, CD7, ßF1 and CD34. Only CD3 and anti TCR antibodies are to be regarded as line-specific.

29 EURO-LB 02 8 Diagnostics 29 In case of uncertain differential diagnosis of physiological thymic tissue or a thymoma (very rare during childhood), a pan-cyto-ceratin marker may be employed to identify thymic epithelium. Precursor lymphoma/leukemia with uncertain line-specification 1) stem-cell neoplasia/undifferentiated leukemia (WHO: undifferentiated acute leukemia/lymphoma; ICD /3) variable expression of line-un-specific markers /stem-cell-markers: HLA-DR, TdT, CD7, CD34, CD38 absence of markers defining specific lineage commitment 2) bilineage neoplasia (WHO: bilineage acute leukemia/lymphoma; ICD /3) two populations of blasts with expression of myeloid/lymphoid or B-/T- markers 3) biphenotypic neoplasia (WHO: biphenotypic acute leukemia/lymphoma; ICD /3) blast with expression of markers of myeloid/lymphoid lines resp. B-/T-markers according to EGIL score-system The panel of mandatory and optional antibodies for a comprehensive characterisation of the cases by the reference pathological laboratories are described on the histopathological Review form (see Appendix, page 131).

30 30 9 Processing of Tumour Material EURO-LB 02 9 Processing of Tumour Material Material Processing Investigation send to Formol fixated Histology Immunohistochemistry National Reference-Pathology Lymphoma in culture media or, if not available, 0,9% NaCl Cytogenetics Molecular genetics Immunology Cell banking Tumour touch imprints preparations, unstained Morphology Bone marrow ml with heparin** Cytogenetics* Molecular genetics Immunology* Cell banking Liquor 10 slides, unstained Cytomorphology 2 cytospin preparations, unstained Cytomorphology Study Centre 10 slides, unstained Cytomorphology Blood 10 ml with heparin** Molecular genetics Cell banking Serum Pleural effusion/ascites 2 ml 10 cytospin preparations, unstained as much as possible with heparin** Cytomorphology Cytogenetics Molecular genetics Immunology Cell banking *) Only if there are at least 10% Lymphoma-cells in BM. **) Use heparin without stabilizer. Study Centre Cytomorphology / Genetics National Reference Pathology National Reference Immunology

31 EURO-LB Initial Staging Initial Staging 10.1 Observation of Tumour Extension Important note: The evaluation of the prognostic impact of the kinetics of tumour response to treatment is a major goal of this study. Therefore, the primary (largest) tumour usually a mediastinal tumour should be documented by CT-scan for accurate calculation of tumour volume and to gain reproducible and comparable data for all patients in all countries. See chapter 15, page 73. A Protocol for accurate calculation of tumour volume is given in Appendix page 100. Observation of tumour extension Detailed clinical examination with careful attention to o general condition o fever over 38 C for at least 7 days o loss of weight >10% over 6 month o night sweats Performance Status (Karnofsky) o able to carry on normal activity, no or minor symptoms o normal activity with effort, cares for self, requires no occasional assistance o requires occasional assistance, limited normal activity o bedridden, requires special care and assistance o very sick, in need of intensive care, bedridden x-ray of chest (p.a. and lateral) and, in case of a mediastinal mass, chest CT (computerized tomography). Please note renal function before administration of contrast agent Ultrasound scan of abdomen/pelvis, thorax (pleural effusion, pericardial effusion), testes BM aspirate from iliac crest (one site for patients with stage III or IV disease and 2 sites for patients with stage I or II disease) CSF (cerebrospinal fluid) cytospin and cell number Recommendations for cytospin preparations cells in CSF filling quantity revolutions/min time < 100/µl 0.5 ml 1 000/min 5 min /µl 0.2 ml 1 000/min 5 min. >1 000/µl 0.05 ml 1 100/min 3 min. Even if there is no apparent infiltrate, an initial cytospin preparation should be done. Cytology of pleural fluid/ascites (cytospin preparations) abdominal MRI (magnetic resonance imaging) or CT in case of equivocal results in ultrasound cranial MRI in case of neurological signs and/or blasts in CSF and/or lymphoma manifestations at head/neck spinal MRI in case of neurological signs local x-ray in case of local bone symptoms Further Examinations: full blood count and film Electrolytes, urea, creatinine, uric acid, calcium, phosphate, alkaline phosphatase GFR (glomerular filtration rate), if there is any evidence of renal dysfunction LDH (lactate dehydrogenase) clotting tests for inherited thrombophilia Echocardiography

32 32 10 Initial Staging EURO-LB Definition of Organ Involvement Bone Marrow Involvement Bone Marrow involvement is diagnosed if there are 5% and < 25% lymphoblasts in the BM aspiration smears. If there are 25 % lymphoblasts in the BM the diagnosis of Acute Lymphoblastic Leukemia (ALL) should be made. There is one exception: patients with a focal bone lesion and a bone marrow infiltration of more than 25% of blasts in this lesion, but less than 25% blasts in all other puncture sites. These patients are to be treated according to the EURO-LB 02 Protocol CNS Involvement Three patterns of involvement may be distinguished: Type 1: CNS-negative: No identifiable blasts in the CSF on cytospin-preparations (independent of cell number) no cerebral/medullary infiltrates on cranial/spinal MRI no cranial nerve palsy that cannot be explained by extradural lesions Type 2: CNS-positive: > 5 cells/µl CSF and morphologically identifiable blasts in the CSF on cytospin-preparations and/or cerebral/medullary infiltrates on cranial/spinal MRI and/or a cranial nerve palsy that cannot be explained by extradural lesions Type 3: CSF blasts low cell number < 5 cells/µl CSF, but definite blasts in cytospin-preparation of uncontaminated CSF, initial CNS-disease is not to be diagnosed, but these patients should receive additional intrathecal MTX administrations at day 18 and 27 of Protocol I. Traumatic lumbar puncture (blood contamination of CSF) Blood-contamination of CSF is to be diagnosed, when the ratio erythrocytes/leukocytes in CSF cytospin-preparation exceed 100:1. In case of blood contamination and blasts in the CSF, counting of slide cells in the CSF and differentiation of nucleated cells is necessary. If the absolute number of blasts exceeds 5/µl the patient is to be considered CNS positive. If the absolute number of blasts is < 5/µl, the patient is to be considered CNS-type Mediastinal Involvement Mediastinal involvement should be confirmed by x-ray or CT-scan. If the histopathological diagnosis can be performed by biopsy of other organs, such as peripheral lymph nodes, invasive methods, such as mediastinoscopy or thoracotomy, should not be carried out, because biopsy of these other organs is likely to be less hazardous to the patient Lung Involvement Lung involvement should be confirmed by CT-scan. If the histopathological diagnosis can be performed by biopsy of other organs, such as peripheral lymph nodes, a lung biopsy should not be carried out since it might compromise the patient Testicular Involvement Testicular involvement is diagnosed clinically as the presence of painless enlargement of one or both testicles, provided the diagnosis of NHL is established otherwise. If painless enlargement of one or both testicles is the only detectable lesion, biopsy must be performed.

33 EURO-LB Initial Staging Bone Involvement Bone involvement is diagnosed if there are bone lesions on x-ray, provided the diagnosis of NHL is already histologically established. If the bone lesion is the only manifestation of a suspected NHL, biopsy must be performed Staging System St. Jude's Classification 37 Stage I II III IV Criteria for extent of disease A single tumour (extra nodal) or single anatomic area (nodal) with the exclusion of mediastinum or abdomen or epidural. A single tumour (extra nodal) with regional node involvement. Two or more nodal areas on the same side of the diaphragm. Two single (extra nodal) tumours with or without regional node involvement on the same side of the diaphragm. A primary gastrointestinal tumour usually in the ileocaecal area with or without involvement of associated mesenteric nodes only, grossly completely resected. Two single tumours (extra nodal) on opposite sides of the diaphragm. Two or more nodal areas above and below the diaphragm. All primary intra-thoracic tumours (mediastinal, pleural, thymic). All extensive primary intra-abdominal disease. All paraspinal or epidural tumours regardless of other tumour site(s). Any of the above with initial CNS and/or bone marrow involvement Differentiation of NHL Stage IV Disease versus Acute Lymphoblastic Leukaemia Bone Marrow involvement is diagnosed, if there are 5% and < 25% lymphoblasts in the BM. This is equivalent to stage IV disease, according to St. Jude s Classification. If there are 25% lymphoblasts in the BM the diagnosis of ALL should be made. There is one exception: patients with a focal bone lesion and a bone marrow infiltration of more than 25% of blasts in this lesion, but less than 25% blasts in all other puncture sites. These patients are to be treated according to the EURO-LB 02 Protocol.

34 34 11 Treatment Plan EURO-LB Treatment Plan Note: Only patients with proven T-cell lymphoblastic lymphoma are eligible for the randomised trial. Patients with pb-lbl are to be treated according to the reference arm without randomisation. Patients with lymphoblastic lymphoma in whom an immunophenotype is not available are also to be treated according to the reference arm without randomisation Treatment Plan for T-cell Lymphoblastic Lymphoma Details of all therapy elements, including guidelines for application, are given in chapter 12, page 36 ff. All patients receive a cytoreductive prephase consisting of one intrathecal dose of Methotrexate on day 1 and a 7-day therapy with Prednisone alone. This prephase is followed by Induction phase I/a and Induction phase I/b. During the 7-day cytoreductive prephase, patients are to be randomised either to receive Prednisone or to receive Dexamethasone in Induction phase I/a. Two weeks after completion of Induction phase I/b, patients receive Protocol M consisting of 6-Mercaptopurine and 4 cycles of Methotrexate 5g/m 2. After Protocol M patients are stratified according to stage of disease. Stage I, II disease Two weeks after completion of Protocol M, patients with stage I or II disease start with maintenance treatment consisting of daily 6-Mercaptopurine orally and Methotrexate orally once a week. All patients that are still free of progressive disease at the beginning of maintenance treatment are to be randomised at the beginning of maintenance treatment to receive maintenance treatment with a total therapy duration (beginning with the first day of cytoreductive prephase) of either 24 months or 18 months. Stage III, IV disease Two weeks after completion of Protocol M patients with stage III or IV disease start with the 8-drug Reinduction Protocol II. Two weeks after completion of Reinduction Protocol II patients receive maintenance treatment consisting of daily 6-Mercaptopurin orally and Methotrexate orally once a week. All patients that are still free of progressive disease at the beginning of maintenance treatment are to be randomised at the beginning of maintenance treatment to receive maintenance treatment until a total therapy duration (beginning with the first day of cytoreductive prephase) of either 24 months or 18 months Treatment Plan for Patients with Precursor-B-cell Lymphoblastic Lymphoma and Patients with Lymphoblastic Lymphoma with Immunophenotype not available These patients are to be treated according to the reference arm of the trial. They receive Prednisone in the Induction phase I/a and maintenance treatment continuing for a total therapy duration (beginning with the cytoreductive prephase) of 24 months Initial CNS Involvement For Definition of CNS involvement see chapter , page 32. Patients with initial CNS involvement receive two additional intrathecal Methotrexate-doses in Induction phase I/a (days 18 and 27) and cranial irradiation of 12 Gy (ages 1 - < 2 years) or 18 Gy (ages 2 years) after Reinduction. Cranial irradiation may be performed right after

35 EURO-LB Treatment Plan 35 Reinduction, provided the patient is in good clinical condition. Children less than 1 year of age receive no cranial irradiation, even in case of initial CNS involvement. Patients with a type 3 CNS status (CSF-blasts, but CSF-cell number < 5/µl, see chapter , page 32) receive the two additional i.th. Methotrexate-doses during Induction phase I/a at day 18 and 27. Cranial irradiation is not to be performed in these patients, however Initial Testicular Involvement In the case of testicular involvement, no orchidectomy is planned. If, after Protocol M, a complete normalisation of testicle size, according to both clinical examination and ultrasound appearances has occurred then testicular irradiation is not undertaken. If after Protocol M doubtful clinical findings remain, a biopsy should be performed. If vital lymphoma tissue is detected, testicular irradiation of 20 Gy is applied (see chapter , page 60) Incomplete Tumour Regression Tumour regression in patients with lymphoblastic lymphoma is frequently incomplete. The ability to diagnose residual tumour depends on the modality of investigation (plain x-ray or CT, for example). In previous BFM studies, especially in T-LBL patients with mediastinal tumours, the absence or presence of residual tumour after Induction phase I/a and I/b was not predictive for the subsequent course of the patients. Therefore, no therapeutic consequences are recommended, in case of incomplete tumour-regression. Exceptions are those few patients where there is virtually no tumour regression, see below (chapter 11.6, page 35) Non-Response Non-responders are patients with less than 35% volume response/regression at day 33, and/or persistence of >5% blasts in the bone marrow and/or persistence of blasts in the CSF at day 33. In such cases the national study coordinator should be contacted, and diagnostic material should be sent to the national study centre. If non-response is confirmed, these patients are to be counted as treatment failures. They may be treated according to national Protocols for high risk ALL. Local irradiation can also be considered.

36 36 12 Treatment Elements EURO-LB Treatment Elements and Guidelines 12.1 General Guidelines Adhere to the Protocol as close as possible. Interruptions in a therapy element which has already begun should be avoided, except in case of a severe infection or serious toxicity. There are no planned adjustments to drug doses (except after consultation with the national study coordinator). If necessary, a postponement is preferable. Special guidelines are given within the description of each treatment element Guidelines for Dose Calculation Systemic Therapy Dosage is based on body surface area (BSA), which is determined before every therapy element. Infants receive reduced chemotherapy dosage according to age: Age Dosage according to BSA birth - 6 months 2/3 of the calculated dose 7-12 months 3/4 of the calculated dose 1 year full dose Intrathecal Therapy Dose according to age. The age at the time of administration is the determining factor. Age Methotrexate i.th. (mg) <1 year years years 10 3 years 12

37 Induction Protocol I EURO-LB 02 Prednisone 60 mg/m 2 /d Corticosteroid i.v. or p.o. Prednisone 60 mg/m 2 /d Randomisation Dexamethasone 10 mg/m 2 /d Vincristine 1.5 mg/m 2 /d (max. 2mg) Daunorubicin 30 mg/m 2 /d E. coli Asparaginase U/m 2 /d Cyclophosphamide mg/m 2 /d Cytarabine 75 mg/m 2 /d 6-Mercaptopurine 60 mg/m 2 /d Methotrexate * only in CNS positive patients i.v. i.v. (1h) i.v. (1h) i.v. (1h) i.v. p.o. i.th. * * Day

38 38 12 Treatment Elements EURO-LB Induction Protocol I Induction Protocol I consists of cytoreductive prephase phase I/a (for T-LBL, randomisation I/a-Prednisone versus I/a-Dexamethasone during the cytoreductive prephase) phase I/b

39 Cytoreductive Prephase EURO-LB 02 Name (or initials): Date of birth:.. Registration number: (dd mm yy) (if known) Weight:. kg Height: cm Body Surface:. m² Day Date The grey fields have to be filled in, in any case! Prednisone 60 mg/m²/d, i.v. or p.o. in 3 single doses mg mg mg Methotrexate i.th. age-adjusted dose mg Rasburicase 0.2 mg/kg/d, i.v. (30min). mg ( ) ( ) ( ) ( ) Creatinine x UNL* x UNL x UNL Initial complications during cytoreductive prephase: Uric acid x UNL* x UNL x UNL Phosphate x UNL* x UNL x UNL Calcium x UNL* x UNL x UNL LDH x UNL* * before start of therapy UNL = Upper normal limit no yes Cell lysis syndrome Reduced renal function Reduced cardial function Paraplegia/paresis Life-threatening sepsis Life-threatening bleeding Complication(s) of primary surgery no yes Complication(s) caused by a mediastinal tumour: Respiratory impairment Superior vena cava syndrome Complication(s) caused by effusions: Respiratory impairment Superior vena cava syndrome Other complication(s) Please describe the complication(s): Dose modification? Cytostatic agents added or omitted? no yes Description of modification(s) and reason(s): In case of dose modification, please fill in the cumulative doses! Prednisone: mg; MTX i.th.: mg; Rasburicase:. mg Remarks: Date (dd mm yy) Physician: Name (in block letters) Physician: Signature Hospital stamp Please fill in a toxicity form for this therapy element! Please send this form to the responsible study centre!

40 40 12 Treatment Elements EURO-LB Cytoreductive Prephase Prednisone 60 mg/m²/d, i.v. or p.o. (in three single doses), days 1 7 Methotrexate intrathecal Rasburicase Hydration in an age-adjusted dose on day 1: Age Methotrexate i.th. (mg) <1 year years years 10 3 years 12 lowered-head position for at least 2 hours after intrathecal Methotrexate application Cave: postpone i.th. therapy and diagnostic lumbar puncture in the case of either upper vena cava syndrome or respiratory distress until the period after stabilisation of the patient! 0.2 mg/kg/d, i.v. over 30 min., days 1, 2, 3 treatment may be prolonged where clinically indicated ml/m²/d Prevention/Treatment of acute cell lysis syndrome: complete details see chapter 14.1, page 65.

41 Induction Phase I/a Prednisone EURO-LB 02 Name (or initials): Date of birth:.. Registration number: (dd mm yy) (if known) Weight:. kg Height: cm Body Surface:. m² Day * Date The grey fields have to be filled in, in any case! Prednisone 60 mg/m²/d, i.v. or p.o. in 3 single doses mg mg mg Vincristine 1.5 mg/m²/d (max. 2 mg), i.v.. mg Daunorubicin 30 mg/m²/d, i.v. (1h) mg E. coli Asparaginase U/m²/d, i.v. (1h) U Methotrexate i.th. age-adjusted dose mg 30 mg/m²/d 15 mg/m²/d ( ) only if CNS pos. ( ) only if CNS pos. *At day 33: In case of blasts in CSF at diagnosis, please send slides to the national study centre! In case of bone marrow infiltration at diagnosis, please send slides to the national study centre! In case of Non-Response please fill in the event-form and send it to the responsible study centre (see Protocol chapter 11.6, page 34)! Dose modification? Cytostatic agents added or omitted? no yes Description of modification(s) and reason(s): In case of dose modification, please fill in the cumulative doses! Asparaginase: E. coli Asparaginase U; (Erwinase U); (PEG-Asparaginase U) Prednisone (incl. tapering): mg; Vincristine:. mg; Daunorubicin: mg; MTX i.th.: mg Remarks: * 7.5 mg/m²/d Date (dd mm yy) Physician: Name (in block letters) Physician: Signature Hospital stamp Please fill in a toxicity form for this therapy element! Space for address of the responsible study centre

42 42 12 Treatment Elements EURO-LB Induction Phase I/a Prednisone Non-T-LBL patients and T-LBL patients in randomisation arm Prednisone. Therapy guidelines during Induction phase I/a-Prednisone The schedule of treatment during phase I/a, is to be modified only in exceptional cases, e.g. high fever or documented significant toxicity. Prednisone 60 mg/m²/d, i.v. or p.o. (in 3 single doses(sd)), days 8 28 from day 29: decrease over 3x3 days, each time with half the dosage Recommendation for prevention of gastritis/ulceration: Ranitidine 2 mg/kg/d, i.v. in 2 SDor 4 mg/kg/d, p.o. in 2 SD; dose-reduction in case of restricted GFR In case of persistent abdominal pain: Omeprazole 0.25 mg/kg/d, i.v. over 30 min or mg/kg/d, p.o. in 2 SD Vincristine 1.5 mg/m 2 /d, i.v. (maximum single dose 2 mg), days 8, 15, 22, 29 Main toxicity: peripheral neuropathy, paresis, myopathy Daunorubicin 30 mg/m 2 /d, i.v. over one hour, days 8, 15, 22, 29 Main toxicity: cardiomyopathy Echocardiogram before first and third Daunorubicin-dose In case of significant and reproducible decrease in ejection fraction compared to the initial value, omission of Daunorubicin is to be considered. E. coli Asparaginase Methotrexate intrathecal U/m 2 /d (KYOWA ), i.v. over one hour, days 12, 15, 18, 21, 24, 27, 30, 33 Main toxicity: allergy, hyperglycaemia, pancreatitis, thrombosis, bleeding A test dose of U could be given In the case of hypersensitivity to native E. coli Asparaginase: PEG-Asparaginase U/m²/SD, i.v. over 2 hours recommended: If hypersensitivity to native E. coli Asparaginase occurs during the first four doses, give two doses PEG-Asparaginase 10 days apart. If hypersensitivity to native E. coli Asparaginase occurs during doses Nr. 5-7, give one dose PEG-Asparaginase. In the case of hypersensitivity to PEG-Asparaginase, Erwinia Asparaginase should be given: U/m 2 i.m. every second day (replace 3 doses Erwinia Asparaginase for 2 doses E. coli). in an age-adjusted dose on days 12 and 33 for initial CNS involvement: additional Methotrexate i.th. on days 18 and 27 Age Methotrexate i.th. (mg) <1 year years years 10 3 years 12 lowered-head position for at least 2 hours after intrathecal Methotrexate application

43 Induction Phase I/a Dexamethasone EURO-LB 02 Name (or initials): Date of birth:.. Registration number: (dd mm yy) (if known) Weight:. kg Height: cm Body Surface:. m² Day * Date The grey fields have to be filled in, in any case! Dexamethasone 10 mg/m²/d, i.v. or p.o. in 3 single doses mg mg mg Vincristine 1.5 mg/m²/d (max. 2 mg), i.v.. mg Daunorubicin 30 mg/m²/d, i.v. (1h) mg E. coli Asparaginase U/m²/d, i.v. (1h) U Methotrexate i.th. age-adjusted dose mg 5 mg/m²/d 2.5 mg/m²/d ( ) only if CNS pos. *At day 33: In case of blasts in CSF at diagnosis, please send slides to the national study centre! In case of bone marrow infiltration at diagnosis, please send slides to the national study centre! In case of Non-Response please fill in the event-form and send it to the responsible study centre (see Protocol chapter 11.6, page 34)! Dose modification? Cytostatic agents added or omitted? no yes Description of modification(s) and reason(s): In case of dose modification, please fill in the cumulative doses! Asparaginase: E. coli Asparaginase U; (Erwinase U); (PEG-Asparaginase U) Dexamethasone (incl. tapering): mg; Vincristine:. mg; Daunorubicin: mg; MTX i.th.: mg Remarks: ( ) only if CNS pos. * 1.25 mg/m²/d Date (dd mm yy) Physician: Name (in block letters) Physician: Signature Hospital stamp Please fill in a toxicity form for this therapy element! Space for address of the responsible study centre

44 44 12 Treatment Elements EURO-LB Induction Phase I/a Dexamethasone T-LBL-patients in randomisation arm Dexamethasone only! Therapy guidelines during Induction phase I/a-Dexamethasone The schedule of treatment during phase I/a, is to be modified only in exceptional cases, e.g. high fever or documented significant toxicity Dexamethasone 10 mg/m²/d, i.v. or p.o. (in 3 divided doses), days 8 28 from day 29: decrease over 3x3 days, each time with half the dosage Recommendation for prevention of gastritis/ulceration: Ranitidine 2 mg/kg/d, i.v. in 2 SD or 4 mg/kg/d, p.o. in 2 SD; dose-reduction in case of restricted GFR In case of persistent abdominal pain: Omeprazole 0.25 mg/kg/d, i.v. over 30 min or mg/kg/d, p.o. in 2 SD Vincristine 1.5 mg/m 2 /d, i.v. (maximum single dose 2 mg), days 8, 15, 22, 29 Main toxicity: peripheral neuropathy, paresis, myopathy Daunorubicin 30 mg/m 2 /d, i.v. over one hour, days 8, 15, 22, 29 Main toxicity: cardiomyopathy Echocardiogram before first and third Daunorubicin-dose In case of significant and reproducible decrease in ejection fraction compared to the initial value, omission of Daunorubicin is to be considered. E. coli Asparaginase Methotrexate intrathecal U/m 2 /d (KYOWA ), i.v. over one hour, days 12, 15, 18, 21, 24, 27, 30, 33 Main toxicity: allergy, hyperglycaemia, pancreatitis, thrombosis, bleeding A test dose of U could be given In the case of hypersensitivity to native E. coli Asparaginase: PEG-Asparaginase U/m²/SD, i.v. over 2 hours recommended: If hypersensitivity to native E. coli Asparaginase occurs during the first four doses, give two doses PEG-Asparaginase 10 days apart. If hypersensitivity to native E. coli Asparaginase occurs during doses Nr. 5-7, give one dose PEG-Asparaginase. In the case of hypersensitivity to PEG-Asparaginase, Erwinia Asparaginase should be given: U/m 2 i.m. every second day (replace 3 doses Erwinia Asparaginase for 2 doses E. coli). in an age-adjusted dose on days 12 and 33 for initial CNS involvement: additional Methotrexate i.th. on days 18 and 27 Age Methotrexate i.th. (mg) <1 year years years 10 3 years 12 lowered-head position for at least 2 hours after intrathecal Methotrexate application

45 Induction Phase I/b EURO-LB 02 Name (or initials): Date of birth:.. Registration number: (dd mm yy) (if known) Weight:. kg Height: cm Body Surface:. m² Day Date The grey fields have to be filled in, in any case! Prednisone or Dexamethasone (see Prot. I/a), i.v. or p.o. in 3 single doses mg Cyclophosphamide mg/m²/d, i.v. (1h) mg Cytarabine 75 mg/m²/d, i.v. mg 6-Mercaptopurine 60 mg/m²/d, p.o. mg Methotrexate i.th. age-adjusted dose mg 7.5 mg/m²/d or 1.25 mg/m²/d Dose modification? Cytostatic agents added or omitted? no yes Description of modification(s) and reason(s): In case of dose modification, please fill in the cumulative doses! Prednisone / Dexamethasone: see documentation form "Phase I/a" Cyclophosphamide: mg; Cytarabine: mg; 6-Mercaptopurine: mg; MTX i.th.: mg Remarks: Date (dd mm yy) Physician: Name (in block letters) Physician: Signature Hospital stamp Please fill in a toxicity form for this therapy element! Space for address of the responsible study centre

46 46 12 Treatment Elements EURO-LB Induction Phase I/b Requirements for beginning phase I/b in case of initial BM and/or CSF blasts: < 5% BM-blasts and no CSF-blasts at day 33* at least 35% of tumour volume regression* good general condition without serious infections creatinine within normal limits according to age WBC 2 x 10 9 /l platelets 50 x 10 9 /l *) If these criteria are not fulfilled see chapter 11.6, page 35. Therapy guidelines during phase I/b Requirements for beginning an Cytarabine block - WBC > 0.5 x 10 9 /l - platelets > 30 x 10 9 /l Requirements for the second Cyclophosphamide-dose at day 64 - WBC 1 x 10 9 /l - granulocytes 0.3 x 10 9 /l - platelets 50 x 10 9 /l If possible the Cytarabine blocks should not be interrupted. If nevertheless a Cytarabine block has to be postponed or interrupted, the 6-Mercaptopurine should also be interrupted. Omitted 6-Mercaptopurine doses should be made up until the planned cumulative total dose of mg/m 2 (28 60 mg/m 2 ) is reached. Cyclophosphamide mg/m 2 /d, i.v. over one hour, days 36 and 64 Main toxicity: haemorrhagic cystitis, suppression of myelon Hydration and haemorrhagic cystitis-prophylaxis: ml/m 2 fluid/24 h MESNA (Uromitexan ) 400 mg/m 2, i.v., before and 4 and 8 hours after the beginning of each Cyclophosphamide-infusion Furosemide 0.5 mg/kg, i.v., 6 hours and 12 hours after Cyclophosphamide, if required check the fluid balance Cytarabine 6-Mercaptopurine Methotrexate intrathecal 75 mg/m 2 /d, i.v. in 4 blocks spread over 4 days each, days: 38, 39, 40, 41; 45, 46, 47, 48; 52, 53, 54, 55; 59, 60, 61, mg/m²/d, p.o., days (28 days total) Omitted 6-Mercaptopurine doses should be made up until the planned cumulative total dose of mg/m 2 (28 60 mg/m 2 ) is reached Administration: with empty stomach, in the evening, not with milk in an age-adjusted dose parallel to the first Cytarabine dose in block 2 (day 45) and 4 (day 59) Age Methotrexate i.th. (mg) <1 year years years 10 3 years 12 lowered-head position for at least 2 hours after intrathecal Methotrexate application

47 Protocol M EURO-LB 02 6-Mercaptopurine 25 mg/m 2 /d p.o. Methotrexate i.v. (24h) 5 g/m² Folinic Acid i.v. 15 mg/m² at h 42, 48, 54 after start of MTX infusion Methotrexate i.th. Day

48

49 Protocol M EURO-LB 02 Name (or initials): Date of birth:.. Registration number: (dd mm yy) (if known) Weight:. kg Height: cm Body Surface:. m² Day Date The grey fields have to be filled in, in any case! 6-Mercaptopurine 25 mg/m²/d, p.o., in the evening. mg HD - Methotrexate 5 g/m², i.v. (24h). g Folinic Acid Rescue 15 mg/m², i.v. at h 42, 48, 54 after the start of MTX infusion Please fill in the white fields in the table below! Methotrexate i.th. age-adjusted dose mg Dose modification? Cytostatic agents added or omitted? no yes Description of modification(s) and reason(s): In case of dose modification, please fill in the cumulative doses! 6-Mercaptopurine:. mg; HD-MTX:. g; MTX i.th.: mg hour after start of MTX infusion 0 24 (36) Remarks: MTX level (µmol/l) 1 st HD-MTX 2 nd HD-MTX 3 rd HD-MTX 4 th HD-MTX Creatinine Folinic Acid Rescue MTX level Creatinine Folinic Acid Rescue MTX level Creatinine Folinic Acid Rescue MTX level Creatinine times of upper normal limit (mg) (µmol/l) times of upper normal limit (mg) (µmol/l) times of upper normal limit (mg) (µmol/l) times of upper normal limit Folinic Acid Rescue (mg) later Folinic Acid Rescue given after hour 54: no yes Folinic Acid Rescue given after hour 54: no yes Folinic Acid Rescue given after hour 54: no yes Folinic Acid Rescue given after hour 54: no yes Date (dd mm yy) Physician: Name (in block letters) Physician: Signature Hospital stamp Please fill in a toxicity form for this therapy element! Space for address of the responsible study centre

50 50 12 Treatment Elements EURO-LB Protocol M Protocol M begins 2 weeks after the completion of Protocol I. Requirements for beginning Protocol M good general condition without severe infections renal function within the normal age-adapted limits GOT/GPT 5 upper normal limit (UNL), bilirubin normal WBC 1.5 x 10 9 /l platelets 50 x 10 9 /l Requirements for high-dose Methotrexate 5 g/m² no infection, no diarrhoea, no mucositis renal function within normal limits corrected for age no effusions SGOT/SGPT 5 upper normal limit for age, bilirubin normal limit corrected for age If transaminases are between 5x maximum normal range and 20x maximum normal range, wait hours and check to ensure that the levels are decreasing. If SGOT and/or SGPT are 20 times of normal upper limits, contact the national study coordinator for further recommendations. In case of seizures, which may be due to Methotrexate, contact the national study coordinator for drug modification. Drug interactions Drugs which compromise renal function, e.g. aminoglycosides, can decrease clearance of Methotrexate and lead to systemic toxicity. Due to Methotrexate-metabolism interactions, Cotrimoxazole should not be given for at least 6 days prior to beginning Methotrexate-therapy, and should only be resumed after ending Methotrexate-therapy. 6-Mercaptopurine high-dose Methotrexate Folinic Acid Methotrexate intrathecal 25 mg/m 2 /d, p.o., for 56 days Administration: with empty stomach, in the evening, not with milk 5 g/m 2 /d, i.v., over 24 hours, days 8, 22, 36, 50 Main toxicity: mucositis, dermatitis, nephrotoxicity Schedule of Methotrexate administration, see chapters to , page 51 and Appendix page mg/m², i.v., 42, 48, 54 hours after beginning of Methotrexate infusion in an age-adjusted dose 2 hours after the start of the Methotrexateinfusion (see also above) on days 8, 22, 36, 50 Age Methotrexate i.th. (mg) <1 year years years 10 3 years 12 lowered-head position for at least 2 hours after intrathecal Methotrexate application

51 EURO-LB Treatment Elements Method of Administration of High Dose Methotrexate 5 g/m² Detailed description plan see Appendix Therapy schema of High-Dose Methotrexate page 104. Pre-Hydration 2 ml/kg/1 hour NaHCO 3 8.4% 2 ml/kg/1 hour aqua dest. followed by: 500 ml NaCl 0.45% / Dex 5% + 40 ml NaHCO 3 8.4% + 10 ml KCl 7.45% over a minimum of 2 hours in order to achieve a ph of 7 and a urine output of 100 ml/m 2 /h Methotrexate Infusion 10% of Methotrexate dose in 5% Dex loading dose continuous infusion over 30 minutes 90 % of Methotrexate dose continuous intravenous infusion over 23 ½ hours ml/m² NaCl 0.45% / Dex 5% ml/m² NaHCO 3 8.4% + 90 ml/m² KCl 7.45% Hydration after HD-Methotrexate infusion, continue with hydration for another 48 hours: ml/m²/24 hours NaCl 0,45% / Dex 5% 180 ml/m²/24 hours NaHCO 3 8.4% 90 ml/m²/24 hours KCl 7.45% check fluid balance every 12 hours check for every miction urine ph with test strips if urine ph< 7.0, administer 2 ml/kg NaHCO 3 and 2 ml/kg aqua dest., i.v. (30 min.) Racemic Folinic Acid (Leucovorin) Rescue It is important to note that for this schedule the Folinic Acid rescue starts very late and is short (3 doses in cases of normal Methotrexate excretion). Therefore the Folinic Acid should be administered intravenously, so that the rescue is optimal. Methotrexate plasma concentrations are measured at hours 24, (36), 42, 48 and 54 from start of the Methotrexate infusion. The expected Methotrexate plasma concentrations are given in the following table. Folinic acid rescue begins 42 hours from the start of Methotrexate infusion. 15 mg/m² Folinic Acid should be given intravenously at 42, 48, and 54 hours from the start of the Methotrexate infusion as shown in the table below. Time from start of Methotrexate Serum creatinine measurement Methotrexate-level measurement 24 hours yes yes < 150 µmol/l 36 hours (yes) < 3 µmol/l Methotrexate-plasma concentration expected Folinic acid dose 42 hours yes < 1 µmol/l 15 mg/m² i.v. 48 hours yes yes < 0.4 µmol/l 15 mg/m² i.v. 54 hours yes < 0.25 µmol/l 15 mg/m² i.v. Folinic Acid rescue is stopped after 54 hours and no further measurements of plasma Methotrexate concentrations are needed, if the plasma Methotrexate concentration at 54 hours from start of Methotrexate infusion is <0.25 µmol/l.

52 52 12 Treatment Elements EURO-LB 02 In the case that the serum Methotrexate concentration at the end of HD-Methotrexate infusion (hour 24) exceeds 150 µmol/l and/or there is a significant increase of serum creatinine level compared to baseline, determination of the serum Methotrexate level at 36 hours is recommend. If the serum Methotrexate concentration at 36 hours exceeds 3 µmol/l give 30 mg/m² i.v. Folinic Acid immediately ensure that 42 hours Methotrexate concentration measurement is promptly processed so that the Folinic Acid rescue dose may be adjusted in the case of persistently increased Methotrexate levels according to the table below Impaired Methotrexate Excretion Impaired Methotrexate excretion is to be expected in case of diarrhoea starting during Methotrexate infusion severe vomiting during Methotrexate infusion significant increase of serum creatinine concentration 24 hours after the start of Methotrexate infusion compared to the baseline Methotrexate-serum concentration > 150 µmol/l at the end of Methotrexate-infusion (24 hours from the start of Methotrexate infusion) In cases of impaired Methotrexate excretion increase hydration to ml/m²/24 h carry out alkalisation of urine (ph >7.0) care for strict balance of fluid intake and output determine serum Methotrexate level at 36 hours. If the serum Methotrexate concentration at 36 hours exceeds 3 µmol/l, give 30 mg/m² Folinic Acid i.v. immediately. Ensure that 42 hours Methotrexate concentration measurement is promptly processed so that the Folinic Acid rescue dose may be adjusted in the case of persistently increased Methotrexate levels. If the plasma Methotrexate concentration at hours 42, 48 or 54 from the start of Methotrexate exceeds the normal range for time, increase the Folinic Acid rescue according the schedule below. Continue with Folinic Acid rescue and measurement of plasma Methotrexate concentration every 6 hours until plasma Methotrexate concentration is <0.25 µmol/l. Methotrexate plasma concentration µmol/l >1-2 >2 3 >3-4 >4-5 >5 mol/l 2.5x x10-6 >1x10-2x10-6 >2x10-3x10-6 >3x10-4x10-6 >4x10-5x10-6 >5x10 mol Folinic acid rescue 15 mg/m² i.v. q6h 30 mg/m² i.v. q6h 45 mg/m² i.v. q6h 60 mg/m² i.v. q6h 75 mg/m² i.v. q6h mg Folinic Acid i.v. q6h = plasma Methotrexate concentration [µmol/l] x body weight [kg] * Consider use of carboxipeptidase- G 2 if available. Contact national study coordinator. *) Caution: Use continuous infusion of Folinic Acid preparation over 1 hour in order to avoid hypercalcaemia.

53 Re-Induction Protocol II EURO-LB 02 Dexamethasone 10 mg/m 2 /d Vincristine 1.5 mg/m 2 /d(max. 2 mg) Doxorubicin 30 mg/m 2 /d E. coli Asparaginase U/m 2 /d Cyclophosphamide mg/m 2 /d Cytarabine 75 mg/m 2 /d 6-Thioguanine 60 mg/m 2 /d Methotrexate p.o. i.v. i.v. (1h) i.v. (1h) i.v. (1h) i.v. p.o. i.th. Day

54 54 12 Treatment Elements EURO-LB 02

55 EURO-LB Treatment Elements 55 Re - Inductions Phase II/a EURO-LB 02 Name (or initials): Date of birth:.. Registration number: (dd mm yy) (if known) Weight:. kg Height: cm Body Surface:. m² Day Date The grey fields have to be filled in, in any case! Dexamethasone 10 mg/m²/d, i.v. or p.o. in 3 single doses mg mg mg Vincristine 1.5 mg/m²/d (max. 2 mg), i.v.. mg Doxorubicin 30 mg/m²/d, i.v. (1h). mg E. coli Asparaginase U/m²/d, i.v. (1h) U Dose modification? Cytostatic agents added or omitted? 5 mg/m²/d 2.5 mg/m²/d 1.25 mg/m²/d no yes Description of modification(s) and reason(s): In case of dose modification, please fill in the cumulative doses! Asparaginase: E. coli Asparaginase U; (Erwinase U); (PEG-Asparaginase U) Dexamethasone (incl. tapering): mg; Vincristine:. mg; Doxorubicin:. mg Remarks: Date (dd mm yy) Physician: Name (in block letters) Physician: Signature Hospital stamp Please fill in a toxicity form for this therapy element! Space for address of the responsible study centre

56 56 12 Treatment Elements EURO-LB Reinduction Protocol II Protocol II begins 2 weeks after the end of Protocol M and consists of phase II/a and phase II/b Phase II/a Requirements for beginning Protocol II/a good general condition without severe infections WBC 2.5 x 10 9 /l granulocytes 1 x 10 9 /l platelets 100 x 10 9 /l Therapy guidelines in phase II/a In the case of severe neuropathy it is permissible to omit Vincristine. In the case of insufficient WBC recovery (WBC < 0.5 x 10 9 /l or granulocytes < 0.2 x 10 9 /l), the Doxorubicin/Vincristine-doses may be postponed until count recovery. Dexamethasone 10 mg/m 2 /d, i.v. or p.o. (in 3 single doses), days 1-21 from day 22: decrease over 3x3 days, each time with half the dosage Recommendation for prevention of gastritis/ulceration: Ranitidine 2 mg/kg/d, i.v. in 2 SD or 4 mg/kg/d, p.o. in 2 SD; dose-reduction in case of restricted GFR In case of persistent abdominal pain: Omeprazole 0.25 mg/kg/d, i.v. over 30 min or mg/kg/d, p.o. in 2 SD Vincristine 1.5 mg/m 2 / d, i.v. (maximum single dose 2 mg), days 8, 15, 22, 29 Doxorubicin 30 mg/m 2 /d, i.v. over one hour, days 8, 15, 22, 29 Main toxicity: cardiomyopathy Echocardiogram before first and third Doxorubicin-dose In case significant and reproducible decrease in ejection fraction compared to the initial value, omission of Daunorubicin is to be considered. E. coli Asparaginase U/m 2 /d (KYOWA ), i.v. over one hour, days 8, 11, 15, 18 Main toxicity: allergy, hyperglycaemia, pancreatitis, thrombosis, bleeding A test dose of U should be given In the case of hypersensitivity to native E. coli Asparaginase: One dose of PEG-Asparaginase U/m²/SD, i.v. over 2 hours is recommended. In the case of hypersensitivity to PEG-Asparaginase, Erwinia Asparaginase should be given: U/m 2 i.m. every second day (replace 3 doses Erwinia Asparaginase for 2 doses E. coli).

57 EURO-LB Treatment Elements 57 Re - Inductions Phase II/b EURO-LB 02 Name (or initials): Date of birth:.. Registration number: (dd mm yy) (if known) Weight:. kg Height: cm Body Surface:. m² Day Date The grey fields have to be filled in, in any case! Cyclophosphamide mg/m²/d, i.v. (1h) mg Cytarabine 75 mg/m²/d, i.v. mg 6-Thioguanine 60 mg/m²/d, p.o.. mg Methotrexate i.th. age-adjusted dose mg Dose modification? Cytostatic agents added or omitted? no yes Description of modification(s) and reason(s): In case of dose modification, please fill in the cumulative doses! Cyclophosphamid: mg; Cytarabine: mg; 6-Thioguanine:. mg; MTX i.th.: mg Remarks: Date (dd mm yy) Physician: Name (in block letters) Physician: Signature Hospital stamp Please fill in a toxicity form for this therapy element! Space for address of the responsible study centre

58 58 12 Treatment Elements EURO-LB Phase II/b Requirements for beginning phase II/b good general condition without acute infections creatinine within normal limits (corrected for age) WBC 2 x 10 9 /l granulocytes 0.5 x 10 9 /l platelets 50 x 10 9 /l Therapy guidelines during phase II/b Requirements for beginning a Cytarabine block are: WBC 0.5 x 10 9 /l platelets 30 x 10 9 /l If possible, the Cytarabine blocks should not be interrupted. If nevertheless a Cytarabine block has to be postponed or interrupted, 6-Thioguanine should also be interrupted. Missed 6-Thioguanine doses should be administered until the planned cumulative dose of 840 mg/m 2 is reached. Cyclophosphamide mg/m²/d, i.v. over one hour, day 36 Main toxicity: haemorrhagic cystitis, suppression of myelon Hydration and Haemorrhagic cystitis-prophylaxis: ml/m 2 fluid/24 h MESNA (Uromitexan ) 400 mg/m 2 i.v., before and 4 and 8 hours after the beginning of the Cyclophosphamide-infusion Furosemide 0.5 mg/kg, i.v., 6 hours and 12 hours after Cyclophosphamide, if required check the fluid balance Cytarabine 6-Thioguanine Methotrexate intrathecal 75 mg/m 2 /d, i.v. (in 2 blocks spread over 4 days each) days 38, 39, 40, 41 days 45, 46, 47, mg/m 2 /d, p.o., days 36-49, a total duration of 14 days Administration: with empty stomach, in the evening, not with milk in an age-adjusted dose, at the same time as the first dose of Cytarabine in block 1 (day 38) and block 2 (day 45) Age Methotrexate i.th. (mg) <1 year years years 10 3 years 12 lowered-head position for at least 2 hours after intrathecal Methotrexate application

59 EURO-LB Treatment Elements Maintenance Therapy Maintenance therapy begins for patients with stage I or II disease 2 weeks after the end of Protocol M and for patients with stage III or IV disease 2 weeks after completion of Reinduction Protocol II. Patients with pb-lbl and patients with LBL, in whom immunophenotype is not available receive maintenance therapy until 24 months total therapy duration calculated from the first day of the cytoreductive prephase. Randomisation of Patients with T-LBL: Patients are to be randomised to receive maintenance therapy consisting of a total duration of either 24 months or 18 months, calculated from the first day of the cytoreductive prephase. Patients need to be free from progressive disease to be eligible for the randomisation. The randomisation has to be performed within the first 21 days of maintenance treatment. Requirements for beginning of maintenance therapy Freedom from progressive disease good general condition without acute infections WBC 1 x 10 9 /l granulocytes 0.2 x 10 9 /l platelets 50 x 10 9 /l (increasing tendency) Therapy Management 6-Mercaptopurine 50 mg/m 2 /d, p.o., once a day Administration: with empty stomach, in the evening, not with milk Methotrexate Complete Blood Count, once a week WBC determines therapy management Guidelines for dose calculation WBC x 10 9 /l < mg/m 2, p.o., once a week Administration: in the evening, not with milk > 3 up to 150 lymphocytes < % of 6-Mercaptupurine/Methotrexate dose Pneumocystis carinii prophylaxis with Co-trimoxazole (TMP-SMZ): 5 mg/kg/d TMP, p.o. in 2 single doses for two consecutive days, e.g. at weekend, with the largest possible interval to Methotrexate dose Disruption of Maintenance Therapy Maintenance therapy should be disrupted in case of infections liver toxicity grade 3: bilirubin > 3 x upper normal limit chronic diarrhoea radiological pulmonary changes (Methotrexate-pneumonitis)

60 60 12 Treatment Elements EURO-LB Radiotherapy Cranial Irradiation Indication for cranial irradiation and dose Cranial irradiation is performed only for patients with initial CNS disease. For criteria for CNSpositivity see chapter page 32. CNS positive patients receive cranial irradiation in agedependent dose. Age < 1 year 1 and < 2 years 2 years dose of cranial irradiation no cranial irradiation 12 Gy 18 Gy Note: Children less than 1 year of age do not receive cranial irradiation, even if they have initial CNS disease. Timing of Radiation Cranial irradiation may be performed straight after Reinduction, provided the patient is in good clinical condition. At the start of cranial irradiation, no signs of a central nervous system disorder should be present. Radiotherapy Technique Cranial irradiation is performed with a high-voltage Telecobalt-60 apparatus or linear accelerator. The exact reproducibility of the daily setting, e.g. with a mask-technique, must be possible. CNS irradiation must include the complete neurocranium, including the first two cervical vertebrae (C1 and C2), the retro bulbar space, and the complete skull base, including the middle cranial fossa. This implies the use of individual screens and the performance of a field-verification shooting. The dose-distribution during radiation therapy should be homogeneous. All fields must be irradiated in each sitting. The daily single dose is 1.5 Gy. This is given in 5 sittings a week until the total dose has been applied. For children with 1 to 2 years of age, a hyperfractionated irradiation (2 x 0,8 Gy or 2 x 1.0 Gy) may be considered. Dexamethasone (15 mg/m 2 /d) is recommended for radiation-induced headaches Testicular Irradiation Testicular irradiation is performed only in patients with vital Lymphoma tissue detected by biopsy after chemotherapy (see chapter 11.4, page 35). A total dose of 20 Gy has to be applied in single daily doses of 2.0 Gy five times a week. It is recommended to use fast electrons of an energy which takes into account the size and location of the testicles. Testicular Irradiation may be performed after diagnosis of vital Lymphoma tissue Quality management Individual documentations of shootings for field-verification have to be made available to the reference radiologist of the study. The documentation will be analysed. Naturally this will be done after completion of radiation treatment. It is assumed that there is a shooting for fieldverification at first adjustment and at least at one interim time point during therapy.

61 EURO-LB Drugs Drugs This chapter deals with the most important specific side effects of active substances. The side effects are related to the dosage given in the Protocol and are according to present knowledge and experience. The list also includes important interactions of these agents with other medications, which might augment or lessen the effect of the drug. The following are guidelines only and it is the responsibility of the attending physician to be thoroughly conversant with the full toxicity profile of each of the drugs. Only some of the possible interactions are stated below. Further information may be available from the manufacturers. Data about stability of the drugs are based on to manufacturers information but in some cases it is exceeded provided the drug is prepared in strictly aseptic conditions. The attending physician is responsible for all administered substances. Asparaginase (KYOWA ) Formulation U for reconstitution. Storage At room temperature. Stability 2 years. Reconstituted solution 6 hours. Administration See chapter 12, page 36 ff. Interaction Enhancement of effects of Vincristine. Toxicity Allergy, hyperglycaemia, pancreatitis, thrombosis, bleeding, hepatic toxicity, diarrhoea, encephalopathy, changes in EEG, vigilance disorder, kidney defects, transient hypothyroidism and hypoparathyroidism. Test dose: a test dose of U is recommended. Cyclophosphamide Formulation 100 mg, 200 mg, 500 mg and 1 g vials for reconstitution. Storage At room temperature. Stability Unreconstituted vials are stable for 5 years at room temperature. A solution of Cyclophosphamide appears to be chemically stable for at least 28 days when stored at 4 C. Reconstituted solution (20 mg/ml) should be used within 8 hours when stored at room temperature. Administration See chapter 12, page 36 ff. Interaction Cyclophosphamide plus Amphotericin B: hypotension, bronchospasm Cyclophosphamide plus insulin: enhancement of effects of insulin Cyclophosphamide plus narcotics: enhancement of effects of narcotics Toxicity Haemorrhagic cystitis, myelosuppression, nausea, vomiting, alopecia, fluid retention, kidney defects, sterility, second malignancies including leukaemia or bladder cancer, cardiotoxicity, gustatory disorders, SIADH, anaphylaxis. Cytarabine Formulation Storage Stability Administration Toxicity Vials containing freeze dried powder of 100 mg Cytarabine. Other preparations available. ARA-cell : Solution containing 20 mg/ml, 50 mg/ml, 100 mg/ml. At room temperature. Alexan is stable for 3 years below 15 C. Cytosar/ARA-cell vials are stable for 3 years at room temperature. Reconstituted solution (5% dextrose or 0.9% saline) is stable for 7 days. See chapter 12, page 36 ff. Bone marrow suppression, mucosal membrane inflammation, nausea, vomiting, oral ulceration, fever, arthralgia, diarrhoea, ulceration and bleeding, alopecia and flu-like syndrome. At higher doses (3 000 mg/m 2 ) cerebellar toxicity may occur. Gastrointestinal toxicity with diarrhoea, mucositis and vomiting may also be more severe. Pulmonary toxicity is uncommon, but may present with unexplained breathlessness. Conjuctivitis can be distressing, but it may be prevented by the regular use of Prednisolone eye drops.

62 62 13 Drugs EURO-LB 02 Daunorubicin Formulation Storage Stability Administration Interaction Toxicity Vials containing 20 mg as powder. Room temperature. Powder 3 years at room temperature. Reconstituted solution stable 24 hours at room temperature and 48 hours when stored in the refrigerator. Solutions should be protected from light during storage! See chapter 12, page 36 ff. Reduced effects of some antibiotics. Acute and chronic cardiotoxicity with cardiomyopathy, local necrosis if extravasation occurs, bone marrow suppression, mucosal ulceration, nausea, vomiting, alopecia. Dexamethasone Formulation 5 mg tablets. Intravenous preparation also available. Storage Room temperature. Administration See chapter 12, page 36 ff. Toxicity Obesity, hirsutism, fluid and salt retention, hypertension, irritability, glycosuria and hyperglycaemia, pancreatitis, seizures and mental instability. Doxorubicin Formulation Storage Stability Administration Interaction Toxicity Vials containing 10 mg or 50 mg in solution (2 mg/ml). Vials containing 10 mg, 50 mg and 150 mg as powder. Solution at 2-8 C in refrigerator. Powder can be stored at room temperature. Powder 4 years at room temperature. Solution 18 months at 2-6 C in refrigerator. Reconstituted solution (100 µg/ml) in 5% dextrose or 0,9% saline is stable for 24 hours at room temperature and 48 hours when stored in refrigerator 2-8 C, but providing it is manipulated in aseptic conditions, it is stable for 28 days at 2-6 C in refrigerator (protected from light). Solutions should be protected from light during storage and administration unless concentration is > 500 µg/ml and freshly prepared. Photodegradation may be substantial at concentrations below 100 µg/ml if solution is exposed to light. See chapter 12, page 36 ff. The drug should be mixed with 5% dextrose. Prolonged contact with solutions of alkaline ph should be avoided, as this will result in hydrolysis of the drug. Doxorubicin should therefore be infused via a separate lumen from alkaline solutions following Methotrexate administration. Doxorubicin plus Amphotericin B: enhancement of effects of Doxorubicin. Acute and chronic cardiotoxicity with cardiomyopathy, local necrosis if extravasation occurs, bone marrow suppression, mucosal ulceration, nausea, vomiting, alopecia. Folinic Acid (Leucovorin) Formulation Lyophilized powder, 3 mg, 5 mg, 25 mg, 50 mg and 100 mg per vial. Tablets of 5 mg, 10 mg, 15 mg, and 25 mg. Storage Room temperature. Reconstitution Reconstitute each vial with sterile water for injection, to achieve a final concentration of 10 mg/ml. No data is available concerning compatibility with KCl; incompatibility with NaHCO 3. Stability Reconstituted solution should be discarded after eight hours. Administration See chapter 12, page 36 ff. Toxicities Allergic reactions (rash, pruritus and erythema).

63 EURO-LB Drugs 63 6-Mercaptopurin Formulation 50 mg tablets. Storage Room temperature. Stability 5 years. Administration See chapter 12, page 36 ff. Interaction Allopurinol: enhancement of bioavailability. Toxicity Myelosuppression, nausea, vomiting, stomatitis, diarrhoea, hepatotoxicity, hyperuricaemia with nephropathy, drug fever, exanthema, pancreatitis. Methotrexate Formulation Storage Stability Administration Interaction Toxicity Ready mixed vials in following strengths: 2.5 mg in 1 ml 200 mg in 8 ml 5 mg in 2 ml 500 mg in 20 ml 25 mg in 1 ml 1 g in 40 ml 50 mg in 2 ml 5 g in 200 ml 100 mg in 4 ml 1 g in 10 ml and 5 g in 50 ml (hypertonic requires dilution) The vials contain sodium chloride and sodium hydroxide adjusted to a ph of approx 8.5; there is no preservative present. Room temperature. 25 mg/ml solution 3 years, other strengths 2 years at room temperature. The manufacturers do not recommend re-use of Methotrexate injection, but providing it is manipulated in aseptic conditions it is stable for 28 days at 2-6 C in refrigerator (protected from light). Solutions of 1-10 mg/ml in 5% dextrose or mg/ml in 0.9% saline are stable for 28 days at 4 C in e.g. PVC, PP, PE, and Polyisopren bags. See chapter 12, page 36 ff. Enhancement of effects of Methotrexate: non-steroidal antirheumatics, some antibiotics, Probenecid, Phenytoin, Barbiturats, Vitamin A and C, Theophyllin, Asparaginase (doubtful), salicylates. Reduction of effects of Methotrexate: Folinic Acid, Asparaginase (doubtful), Allopurinol, glucocorticoide hormones, Penicillin G, vinca alkaloids, thymilidat-infusion. Neurotoxicity, mucositis, liver dysfunction, bone marrow depression, renal failure, mucosal membrane inflammation, ulceration, and bleeding. In addition to the side effects associated with this agent, the effects of intrathecal administration include headache, stiff neck, lethargy, nausea and vomiting, confusion, and seizures. PEG-Asparaginase Formulation U/ml (concentration: 750 U/ml) Storage 4 years in the refrigerator 2-8 C. Do not shake, do not freeze (loss of activity). Reconstituted solution (100 ml NaCl 0,9% or dextrose 5%) prepared in strictly aseptic and validated conditions are stable for four hours at room temperature and up to 3 days when stored in the refrigerator (cave: no interruption of cooling; more favourable handling: storage up to four hours at room temperature). Do not use reconstituted solution if it was stored for more than 48 hours at room temperature, if solution is turbid or precipitated. Administration See chapter 12, page 36 ff. Toxicity Allergy, hyperglycaemia, pancreatitis, thrombosis, bleeding, hepatic toxicity, diarrhoea, encephalopathy, changes in EEG, vigilance disorder, kidney defects, transient hypothyroidism and hypoparathyroidism. Prednisone Formulation Storage 1 mg, 5mg, 20 mg, 50 mg tablets Room temperature.

64 64 13 Drugs EURO-LB 02 Administration Toxicity Thioguanin Formulation Storage Stability Administration Toxicity Vincristine Formulation Storage Stability Administration Toxicity See chapter 12, page 36 ff. Obesity, hirsutism, fluid and salt retention, hypertension, irritability, glycosuria and hyperglycaemia, pancreatitis, seizures and mental instability. 40 mg tablets Room temperature. 5 years. See chapter 12, page 36 ff. Myelosuppression, nausea, vomiting, stomatitis, diarrhoea, hepatotoxicity, hyperuricaemia with nephropathy, drug fever, exanthema, pancreatitis. Vials containing 1 mg, 2 mg, 5mg in solution (1 mg/ml). At 2-8 C in the refrigerator. 2 years. See chapter 12, page 36 ff. peripheral neuropathy, paresis, myopathy, fever, neuralgic pain, constipation, paralytic ileus, syndrome of inappropriate ADH, cerebral convulsion, myelosuppression, alopecia, cardiovascular complications, photosensitisation, headache, dysphagia, polyuria, dysuria, dysfunction of cranial nerves, rare: atrophy of optic nerve with blindness. Intrathecal drugs Please note details of the suitable solvent for the drug for intrathecal use. Methotrexate Formulation Storage Stability Administration Toxicity Ready mixed vials 5mg/2ml or 25mg/ml without preservative. The vials contain sodium chloride and sodium hydroxide adjusted to a ph of approx 8.5; there is no preservative present. Room temperature. 25 mg/ml solution 3 years, other strengths 2 years at room temperature. Dose is age dependent. See chapter 12, page 36 ff. The effects of intrathecal administration include headache, stiff neck, lethargy, nausea and vomiting, confusion and seizures.

65 EURO-LB Supportive Therapy Supportive Therapy Supportive Therapy is primarily the responsibility of the physician in charge. In this chapter only recommendations are given for the patient management in certain situations. The national study coordinators are available for consultation Acute Cell-Lysis Syndrome When lymphoma cells die, at least five major substances are released, which are eliminated only by the kidneys: purine metabolites (xanthine, hypoxanthine), uric acid, potassium and phosphate. If the product exceeds its solubility, then xanthine, hypoxanthine and uric acid can crystalise out. This takes place in the kidney tubules and collecting tubules. Phosphate can precipitate with calcium as calcium-phosphate in both the kidney tubules and in the tissue. The results are oliguria/anuria, tissue necrosis and hypocalcemia. The solubility of xanthine and uric acid is much higher in an alkaline milieu than in an acidic one, but the precipitation of phosphate with calcium is favored in an alkaline milieu. Hypoxanthine can also crystalise at a ph > 7.5. Alkalisation of the urine can, therefore, also favor the precipitation of cell lysis products. If the uric acid, potassium, phosphate and/or creatinine levels are already increased before the start of cytoreductive therapy, then measures for controlling these substances should be started first, before active cytoreductive therapy is begun. The start of cytoreductive therapy, however, should not be postponed much longer than 24 hours. The most important measure is the initiation and maintenance of a high urine output ( ml/m²/h). If this is working well, then metabolic imbalances which require intervention are rare. If, in spite of sufficient hydration and diuretics, it is not possible to initiate and maintain a satisfactory urine output, then early haemodialysis should be instituted. This situation is probably due to either direct infiltration of the kidneys, an obstruction of the urinary tract due to lymphomatous compression or an established urate-phosphate or calcium-phosphate nephropathy, or a combination of these pathological conditions. Hyperpotassemia is the most frequent, immediately life-threatening complication of acute cell-lysis syndrome. If potassium levels rise above normal or, in the case of an existing hyperpotassemia, do not fall quickly after starting measures for preventing respectively therapy of acute cell-lysis syndrome, a life-threatening hyperpotassemia can evolve within a few hours Prevention of acute Cell-Lysis Syndrom hydration: ml/m²/d (0.45% NaCl-solution within 5% glucose, i.v.) specific gravity of the urine fluid balancing: output = input insensible losses bodyweight measurement twice a day for insufficient output: Furosemide 1-10 mg/kg/d initially, no extra potassium in infusion; slight hypokalemia is without problem Rasburicase 0.2 mg/kg/d i.v. over 30 minutes, minimum for 3-5 days, depending on tumour size. Alkalisation of urine is not necessary and might even be of disadvantage, because it might enhance the precipitation of calcium-phosphate in both the kidney tubuli and tissues such as pancreas.

66 66 14 Supportive Therapy EURO-LB 02 If Rasburicase is not available Allopurinol 10 mg/kg/d p.o. in 2-3 single doses for 3-8 days alkalisation of urine NaHCO mmol/l added to infusion solution (or mmol/m²/d parallel infusion) control of NaHCO 3 -supply according to urine-ph optimal is ph 7.0 specific gravity of the urine Lab controls: blood count, Na, K, Cl, Ca, phosphate, uric acid, creatinine every hours or, where abnormal, even more frequently Hyperpotassemia Important: pseudo-hyperpotassemia due to leakage from red or white cells during or after sampling is to be excluded first serum-potassium plasma-potassium ECG-changes pseudo-hyperpotassemia increased normal none real hyperpotassemia increased increased QRS broad, T high potassium 6 mmol/l: potassium 7 mmol/l: prepare for haemodialysis (or move patient) immediate haemodialysis if technically possible, transvenous heart pacemaker Acute Measures: Medication Dosage Caution 1. Poly(styrol, 0.5-1g/kg divinylbenzol)sulfonic acid (Resonium A ) 2. glucose i.v. + soluble insulin i.v. 1 g/kg U/kg, i.v. in ½ h This causes only the redistribution of extracellular K to intracellular, K floods back after 2-4 hours. Therefore, this is only a temporary measure until dialysis is initiated. if CHANGES in the ECG occur, additionally: 3. Calcium-gluconate 10% i.v ml/kg slowly Bradycardia 4. NaHCO 3 i.v. 2 mmol/kg Hyperphosphataemia increase fluid administration to upper range (to ml/m²/24 h) urine-ph not > 7.0! stop enteral nutrition and parenteral nutrition, in order to reduce exogenous and endogenous phosphate generation, alternatively: aluminium hydroxide 0.1 g/kg p.o. in order to bind the phosphate in the food Problem Phosphate > 10 mg/100 ml (5 mmol/l) and/or product Ca x phosphate > 6.4 mmol/l Measure Haemodialysis Caution Hypocalcemia with symptoms Calcium-gluconate 10% 0,5-1 (- 2) ml/kg, i.v., slowly Bradycardia ECG monitoring,

67 EURO-LB Supportive Therapy Hypocalcemia Correct hypocalcemia only when phosphate is normal or when symptoms of hypocalcemia arise (Caution: precipitation with phosphate!) Calcium-gluconate 10%, (- 2) ml/kg, slowly i.v. (ECG monitoring, Caution: Bradycardia) check magnesium concentration! hypomagnesiemia: magnesium-sulfate mval/kg i.v Oliguria/Anuria Definition Urine excretion < 50 ml/m²/h in spite of: Furosemide 10 mg/kg/d i.v. hydration ml/m²/h The often used definition of < 5 ml/m²/h is not useful in this situation: The quick accumulation of potassium will not allow waiting until this definition is met. Therefore, it is more useful to evaluate the urine output in relation to the input. Diagnostics Ultrasound: Biochem: Urine: * obstruction of the urinary tract * kidney infiltration * potassium * uric acid * phosphate * calcium * uric acid crystals * calcium-phosphate crystals Indications for haemodialysis potassium > 7 mmol/l or > 6 mmol/l and increasing, in spite of increased hydration and diuretics phosphate > 10 mg/100 ml (5 mmol/l) or product Ca x P > 6.4 mmol/l urine excretion: < 50 ml/m²/1 h in spite of Furosemide 10 mg/kg/d i.v. and fluid input ml/m²/h high-grade or complete urinary tract obstruction on both sides 14.2 Complication of Asparaginase Therapy Anaphylactic Reactions to E. coli Asparaginase Test doses of U (Units) over 15 min are recommended before the full infusion is begun. In case of hypersensitivity reaction: Clemastinhydrogenfumarat 0.03 mg/kg, i.v. Methylprednisolone 2.5 mg/kg, i.v. In case of hypersensitivity reaction to E. coli Asparaginase replace by PEG-Asparaginase or Erwinia Asparaginase as described in chapter , page Coagulation Disorder During Asparaginase Therapy The incidence of thromboembolic complications or haemorrhage in the resembling Protocol ALL-BFM 90 accounted for 2.8% 38. As a cause, basically therapy-associated and/or congenital risk factors come into consideration. For Induction (Protocol I) parallel administration of Asparaginase and glucocorticoide hormones (acquired lack of protein C/S and lack of AT III), as well as a central venous catheter appear to be significant risk factors for thromboembolic complications. The biggest danger seems to be an imbalance between the pro- and anticoagulatory systems, and this most often appears between the 3 rd and the 8 th administration of Asparaginase 39,40.

68 68 14 Supportive Therapy EURO-LB 02 Congenital risk factors are also described including: factor V (FV) G1691A mutation, methylentetrahydrofolatreductase (MTHFR-) mutation, prothrombin G20210A-mutation, congenital lack of protein C, protein S and AT III, as well as raised lipoprotein(a) 41. In the ALL-BFM studies 90/ patients were analysed. 55 of them showed at least one of the congenital risk factors. Half of these 55 patients developed thromboembolic complications (p<0.0001). There was no information concerning central venous line placement in these children in this publication 42. Diagnostics of Coagulation Disorder During Asparaginase Therapy An initial screening (before hydration and steroids) of every patient should take place: APTT, PT, fibrinogen, D-Dimer, AT III, protein S, protein C, APC-resistance (activated protein C resistance (APCR, Factor V Leiden mutation)), prothrombin G20210A-mutation and lipoprotein (a), in order to find out about congenital risk factors for thromboembolic complications or haemorrhage. In addition, a clotting test with APTT, PT, AT III, fibrinogen and D-Dimer is recommended before every Asparaginase-administration. The presence of contaminating heparin should be actively excluded in samples drawn from central venous catheters. Therapy of Coagulation Disorders During Asparaginase Therapy In ALL-BFM studies during Asparaginase/glucocorticoide therapy in patients without congenital risk factors haemorrhage occurred in individual cases. Therefore a Protocol of empirical replacement with clotting factors is not recommended. A prophylactic substitution therapy with anti-coagulant proteins (e.g. AT III) or heparinisation without hints of activated coagulation is not recommended as a standard procedure 43. If possible (e.g. older children), the implantation of a central venous catheter should be performed at the beginning of phase I/b in Protocol I. In case of a congenital prothrombotic tendency, the risk of thromboembolic complications is significantly elevated. For these patients and in individual cases, the national study coordinators are available for consultation. In the case of documented activation of coagulation, the following therapy is recommended: Problem Therapy 44,45 distinct asynchronous decrease of AT III compared to fibrinogen parallel increase of D-Dimer overt bleeding massive haemorrhage disseminated intravascular coagulation (DIC) sepsis 14.3 Substitution of Blood Products AT-III as continuous infusion Dose: AT III calculated (80-100%) - AT III actual = U/kg substitution of specific factors according to results of coagulation analysis Fresh Frozen Plasma (FFP) ml/kg AT-III as continuous infusion Dose: see above additional vitamin K if necessary low dose heparin ( U/kg/d) Substitution of blood products should be performed according to local/national standards. Preventing GVHD (graft versus host disease): Irradiation of all blood products with a minimum of 30 Gy Infection Prophylaxis und Therapy The responsibility for infection prophylaxis is the attending physician's. The following is to be viewed as a general reference and are not considered binding guidelines.

69 EURO-LB Supportive Therapy 69 At the time of diagnosis and before beginning chemotherapy, the following basic tests should be performed: cultures of blood, stool and body cavity fluids swabs of skin lesions and mucous membrane lesions antibody titer: varicella-zoster, herpes simplex, cytomegaly, measles, EBV (Epstein-Barr virus), hepatitis A, B, C, HIV-1 (permission from patient/parents is required in some countries) C-reactive protein The most important infection prophylaxis is a thorough washing of hands before and after every contact with a patient, as well as detailed patient and parent education about neutropenia and risk of infection. The administration of non-absorbable antibiotics for the purpose of complete or selective decontamination of the digestive tract may select for resistant bacteria and has not yet clearly been shown to be effective 46,47. An oral antimycotic chemoprophylaxis with Amphotericin B-suspension or Fluconazole inhibits the colonisation by most Candida species, but does not inhibit the incidence of systemic Candida and Aspergillus mycoses 48. Constipation and ileus favour the growth of bacteria and fungi in the intestinal lumen and the invasion into the mucous membrane, especially if damage due to cytostatic agents and/or infiltration has occurred. It is therefore important to assure a daily bowel movement, for instance through administration of lactulose p.o. If this treatment is not successful, then stimulant laxatives might be indicated. Medication Fluconazole alternatively: Amphotericin-B Dosage 4-6 mg/kg/d, p.o. as SD Age < 3 years 400 mg/d, p.o. in 4 doses = 4 x 1ml Age 3 years 800 mg/d, p.o. in 4 doses = 4 x 2ml suspension washed in mouth, then swallowed Infection prophylaxis is necessary from the beginning until approximately 4 weeks after the end of intensive therapy Pneumocystis-carinii Prophylaxis All patients should receive a prophylaxis with Trimethoprim-Sulfamethoxazole (Cotrim) during the entire phase of chemotherapy to prevent a Pneumocystis carinii-pneumonia. In the case of a TMP-SMZ intolerance it is possible to treat alternatively with pentamidine inhalation 49. Medication TMP-SMZ alternatively: Pentamidine Aerosol Dosage 5 mg TMP/kg/d, p.o. in two doses on two consecutive days, e.g. on the weekend < 4 years: 150 mg/month in 5 ml aq. dest. inhaled over min 4 years: 300 mg/ month in 5 ml aq. dest. inhaled over min Pneumocystis carinii Pneumonia Medication TMP-SMZ i.v. for intolerance: Pentamidine i.v. Dosage TMP 20 mg/smz 100 mg/kg/d i.v. in 4 SD 4 mg/kg/d, i.v. (over 3h) Varicella Exposure Prophylaxis Contact between NHL patients during chemotherapy and individuals with varicella or varicella zoster must be avoided (parent instruction!). If exposure does occur, there is a risk of illness for at least 28 days, regardless of serological status, although sero-positive patients do have a

70 70 14 Supportive Therapy EURO-LB 02 markedly decreased risk 50. In individual cases, the degree of immunosuppression at the time of exposure decides the therapeutic measures Generally: Patient status has had Varicella (history, scars, titer) not yet had varicella ± serious immunosuppression Varicella appearance Recommendation observation Aciclovir 80 mg/kg/d, p.o. in 4 SD for d or varicella-zoster hyperimmunoglobin within 48 hours after exposition see below: varicellas, herpes zoster (manifested disease), chapter , page 70. Active immunisation of contact persons against Varicella (if they have not had natural exposure) can reduce the risk of Varicella infection of the patient Varicella, Herpes zoster und Herpes simplex (manifested illness) Medication Aciclovir i.v. Dosage mg/m 2 /d, i.v. (over 1 h) in 3 SD until all efflorescences have dried up (minimum 5 days) Fever with Neutropenia Definition Temperature oral/rectal 38.5 C Neutrophils < 0.5 x 10 9 /l Diagnostics Implementation/supplementation of tests according to clinical situation cultures: blood (every catheter channel), stool (incl. Clostridium difficile-toxin), urine swabs from throat, skin and mucous membrane lesions, anus chest x-ray if pulmonary symptoms if the x-rays of the lungs are reported abnormal, and the patient does not respond to the antibiotic therapy, a diagnostic bronchial lavage is recommended ultrasound of abdomen Therapy Broad Spectrum Antibiotic Therapy: The antibiotic therapy must be tuned to the unique situation of each patient and each clinic's bacterial spectrum. start with a combination covering gram positive and gram negativ bacteria if β-lactam-resistant Staphyloccocus aureus/staphylococcus mitis species or other virulent gram-positive bacteria (mucositis, catheter, abdominal symptoms) are known or suspected: additional initial therapy with vancomycin 40 mg/kg/d in 4 SD expansion of antibiotic therapy, if fever does not decrease after 2-3 days if fever is persistent > 5-7 d, or returns after i.v. antibiotics: additional i.v. therapy with Amphotericin B for suspected anaerobic infection: additional Clindamycin/Metronidazol antibiotics are continued until granulocytes > 0.5 x 10 9 /l, even if no explicit focus of infection is located!

71 EURO-LB Supportive Therapy Systemic (invasive) Fungal Infection For well-founded suspicion or proof of a systemic fungal infection: Medication Amphotericin B Dosage initially mg/kg/d increase to max. 1 (-1.5) mg/kg/d, i.v. (4h) no contact with NaCl minimal interval to granulocyte transfusion 8-10 h Caution: hypokalemia, hyponatremia Alternatively or in case of pre-existing/newly developing nephrotoxicity: Medication liposomal Amphotericin B Dosage (6) mg/kg/d, i.v. (4h) Serious systemic Cytomegalovirus (CMV)-infection (CMV-Pneumonitis) Medication Ganciclovir i.v. Dosage 10 mg/kg/d, i.v. (1h) in 2 doses 14.5 Osteoporosis-Prophylaxis Bisphosphonates A common problem for long-term-survivors after NHL-therapy is the occurrence of reduced bone density, respectively osteoporosis compared to healthy control groups 55. There is a controversial discussion about the application of bisphosphonates to these patients. Current paediatric practice limits the use of these drugs to hypercalcaemia, osteogenesis imperfecta and idiopathic osteoporosis and in some cases recommended But there are few reports about the long-term-effects of this therapy and for both short term and long term effects there are no well conducted studies. 59,60. Side effects of the normally intravenously administered therapy are grippal symptoms, hypocalcaemia and acute renal failure in individual cases. In consideration of these side effects up to now, a general indication for the use of bisphosphonates after NHL-therapy of adolescents cannot be recommended Gastritis-Prophylaxis On treatment with Prednisone or Dexamethasone there is a risk for the development of a gastric or duodenal ulcer. Recommendation for prophylaxis: Medication Ranitidine i.v. in case of persistent abdominal pain: Omeprazole i.v. Dosage 2 mg/kg/d, i.v. in 2 SD and/or 4 mg/kg/d, p.o. in 2 SD; dose-reduction in case of restricted GFR 0.25 mg/kg/d, i.v. in 1 SD and/or mg/kg/d, p.o. in 2 SD

72 72 14 Supportive Therapy EURO-LB Extravasation of administered drugs General measures stop of the injection, leave venous catheter, renew the system aspirate and discard 3-5 ml blood in order to eliminate rests of cytostatic agent try to aspirate the content of any blisters with a fine needle and remove the venous catheter (Vincristine: leave venous catheter and use it for the application of parts of Hyaluronidase) local application of the recommended antidote see table below watch and document the process in case of necrosis, despite local measures (absence of physiological redness of the skin is often an early sign): early consultation of a plastic surgeon and consideration of an early surgical revision of the necrosis and the inflamed surroundings 61. Paravenous injection Anthracycline Vincristine Measure Cooling Dimethylsulfoxide (DMSO 99%) No cooling! Hyaluronidase (diluted in NaCl 0.9%) Ice-application 15 minutes each time, 4-6 hours a day for few days (Caution: frostbite) Optional: careful application of 4 drops on a 10 cm² area (drying on air, no bandage) U s.c./intracutaneous into the localisation of paravenous injection/infusion 14.8 Sagittal Sinus Thrombosis For children with or without prothrombotic risk factors there is an increased risk of sagittal sinus thrombosis during NHL-therapy. During comparable ALL-type therapy the risk for children with an inherited thrombophilic tendency is approximately 20%, whereas children without a detectable prothrombotic risk factor show a risk of 2.2% 63. Sagittal sinus thrombosis occurred especially during Induction Protocol I (14 of 17 thrombosis), in contrast to Reinduction Protocol II (3 of 17 dural sinus thrombosis). But in this study the impact of a central venous catheter as an important prothrombotic risk factor was disregarded. A sagittal sinus thrombosis can be diagnosed by MRI or CT after injection of intravenous contrast 64. Concerning the therapy of sagittal sinus thrombosis there are no randomised studies with paediatric patients and there are no evidence based therapeutic recommendations. In a randomised study within 20 adult patients with sagittal sinus thrombosis, the application of heparin versus a placebo was examined. It showed a significant benefit concerning survival and complete clinical reconstitution for the heparin-group 65. From a Canadian not-randomised pilot study within 30 children with sagittal sinus thrombosis it appeared that low-molecular-weight heparin was as efficacious as standard heparin. The start of a multi-centre-study in order to evaluate the necessity and mode of heparin-application was demanded in this study 66. Regarding these aspects it results, that a standard proceeding for paediatric patients with sagittal sinus thrombosis cannot be recommended yet. Anticoagulation seems to be justified based on data from studies in adult patients.

73 EURO-LB Evaluation of the Kinetics of Treatment Response Evaluation of the Kinetics of Treatment Response The evaluation of the prognostic impact of the kinetics of treatment response is a goal of the study. The kinetics of tumour response to treatment is to be determined by CT-scan evaluation of tumour volume regression from day 1 to day 8 and day 15 tumour volume determined by CT-scan diagnosis day 8 (after beginning of cytoreductive prephase) day 15 (after beginning of cytoreductive prephase) day 33 (after beginning of cytoreductive prephase) X X X * *) At day 33 CT-scan only, if there was no tumour regression of at least 35% at day 15. CT-scan for determination of tumour volume at days 0, 8, 15 is chosen due to the following reasons: The Method delivers reproducible results. Automated computation of tumour volume is possible. It is available in all participating institutions and scans can be done immediately after admittance of a patient. This may not be the case with MRI. Due to short processing time and possibility of continued patient monitoring CT-scan is feasible, even in a severe ill patient. This contrasts with MRI. A detailed Protocol for chest scan acquisition and tumour volume measurement is given in Appendix Imaging Guidelines page 100. There is a currently ongoing pilot-study concerning Minimal Residual Disease Monitoring of children with lymphoblastic lymphoma. One aim of this pilot study is to find out about the feasibility of MRD Monitoring for these patients. If the study will show, that MRD Monitoring is feasible, an accompanying research project of the EURO-LB 02 Protocol with MRD Monitoring will be started.

74 74 16 Progressive Disease EURO-LB Progressive Disease Disease progression is suspected in case of: bone marrow progression is diagnosed in case of > 25 % lymphoblasts in the bone marrow. If disease progression has been otherwise diagnosed by biopsy, then the BM is considered involved, if it contains 5% lymphoblasts. CNS progression is diagnosed, if lymphoma cells are present in the CNS and cell count 5 µl and/or in case of (re)appearance of an intra-cerebral tumour. appearance of new manifestations local manifestations: reappearance or increase in size of residual remnants testes: increase in volume If there is an increase in the diameter of a residual mass which is not obviously a progress it is recommended to repeat the examination within a short interval. The diagnosis of progressive disease must be proven by biopsy and histology, except where it can be established by a simpler procedure, e. g. by examination of the bone marrow. In case of progressive disease the initial diagnostic and staging procedures should be repeated including Histology and Cytology Immunophenotyping Cytogenetics Gene expression profiling Matrix-CGH See chapter 8, page 26 and chapter 10, page 31. As soon as progression is diagnosed, the study centre should be informed to evaluate the individual second line treatment strategy. The event-form for progressive disease should be completed and sent to the study centre (see Appendix, page 124).

75 EURO-LB Late Effects and Follow-Up Studies Follow-Up Studies of Disease Status Routine follow-up studies are to be performed to follow and document the status of the disease. Imaging methods for follow-up studies depend on the localisation. Localisation mediastinal tumour, lung head, brain, spinal peripheral nodes abdominal sites bone soft tissue skin testes Method for follow-up studies x-ray clinical symptoms: MRI physical examination, ultrasound ultrasound, if doubtful: CT or MRI clinics, if symptoms: x-ray, MRI physical examination, ultrasound, MRI physical examination physical examination Time points of follow-up studies of disease status Time point Local manifestations appropriate imaging as described above day 33 of Induction x if positive initially* BM CSF with each i.th. therapy before Protocol M x only if progression with i.th. therapy*** before Reinduction x suspected with i.th. therapy*** (stage III and IV only) before Maintenance x with i.th. therapy*** during Maintenance first year during Maintenance second year 4-weekly 8-weekly 3 rd year 6-monthly or if progression suspected 4 th year once a year or if progression suspected later if progression suspected x** only if progression suspected*** * ) In case of bone marrow infiltration at diagnosis, please send slides to the national study centre. ** ) In case of blasts in CSF at diagnosis, please send CSF cytospin preparations at day 33 to the national study centre. **** ) In case of blasts in CSF, please send cytospin preparations to the national study centre. In case of non-response at day 33, pleas fill in an event-form and send it to the responsible data centre. Persistent tumour remnants may not have therapeutic consequences except where there is virtually no treatment response until day 33 of Induction. In such a case, contact national study coordinator, see chapter 11.5 and 11.6, page 35.

76 76 18 Late Effects EURO-LB Monitoring of late Effects Echocardiogram Echocardiograms must be performed 3 months after beginning of maintenance treatment and then every year. Renal evaluation After completion of therapy renal function (creatinine-clearance, Na-reabsorption fraction) should be performed every six months during the first two years and later on once per year. FSH and LH levels for boys: FSH and LH levels must be measured at the completion of puberty and any abnormality must be investigated. Osteonecrosis In case of bone pain, osteonecrosis should be considered and ruled out. Diagnostic method: MRI. Futher examinations may be perforemed according to local practice.

77 EURO-LB Research Projects Research Projects The vast majority of tumour failures, especially in T-LBL patients, occur within the first year of treatment, and most relapsed T-LBL are highly resistant to salvage therapy. This observation suggests a major biological difference of the tumour in these patients. The identification of the patients at risk for this kind of early failure is essential to be able to alter their treatment in future trials. The program to identify prognostic factors in study EURO-LB 02 includes: Kinetics of early tumour response to treatment Cytogenetics Gene expression profile of tumour cells Matrix comparative genomic hybridisation SELDI (Surface Enhanced Laser Desorption/Ionization) In study EURO-LB 02 any effort has to be undertaken to ascertain these parameters for every patient. Collection and preservation of appropriate material for the biological studies is described in chapters 7, 8 and 9. Kinetics of Tumour Response to Therapy The elimination of leukaemia blasts in the peripheral blood during a seven-day Prednisone therapy (plus one intrathecal Methotrexate dose) is the most important prognostic factor in childhood T-ALL 67. It can be assumed that the kinetics of tumour response is also an important prognostic factor in T-LBL patients. Unfortunately, such an easy reproducible and always available parameter as counting blasts in the blood is not available for the evaluation of the response to therapy in lymphoma patients. In study EURO-LB 02 the prognostic impact of the kinetics of tumour response to therapy will be evaluated by means of evaluation of the regression of tumour volume from day 1 to day 8 and day 15 (for details see Appendix Imaging Guidelines, page 100). Minimal Residual Disease Monitoring Monitoring the decrease of leukemia blasts in the bone marrow by means of clone specific probes (minimal residual disease monitoring) technology resulted in an even higher predictive power for the subsequent relapse in childhood ALL 30,68. Because clone specific probes are not always available, due to the shortness of diagnostic tumour material, this method is difficult to apply in lymphoma patients. More recently, immunological methods have become available, which allow also the detection of rare residual lymphoblasts by phenotype in blood or bone marrow at a detection level of 10 3 to 10 4 even during the course of treatment 69,70. In an international cooperation, a consensus panel of antibodies useful for MRD monitoring, in particular in T-cell acute lymphoblastic leukemia, has been recently accepted. According to pilot observations, this panel can also be adopted for T-LBL. The technique of MRD monitoring could therefore also be used to estimate the presence of minimally disseminated disease (MDD) in blood and bone marrow of patients with clinically localised T-LBL. There is a currently ongoing pilot-study concerning Minimal Residual Disease Monitoring of children with lymphoblastic lymphoma. One aim of this pilot study is to find out about the feasibility of MRD Monitoring for these patients. If the study will show, that MRD Monitoring is feasible, an accompanying research project of the EURO-LB 02 Protocol with MRD Monitoring will be started. Cytogenetics Cytogenetic aberrations are known to be powerful prognostic parameters in many malignant diseases. Chromosomal aberrations are also known in lymphoblastic lymphomas. However, data on cytogenetics are lacking in large series of uniformly treated patients, mainly due to the lack of appropriate tumour material. Therefore, gaining appropriate material for cytogenetics from all study patients has a very high priority.

78 78 19 Research Projects EURO-LB 02 Microarrays Microarrays are a new tool to analyse the gene expression profile of defined cell populations. In tumour research, microarray analyses have been performed to classify tumours, to define new prognostic subgroups and to dissect the molecular networks deregulated in transformation and progression. Matrix-Comparative Genomic Hybidization (Matrix-CGH) Matrix-CGH is a microarray-based technique to detect genomic imbalances like deletions or trisomies. In contrast to microarrays designed to evaluate gene expression profiles, which usually uses oligonucleotids or cdna-clones, for matrix-cgh clones with large human inserts are spotted on glass surfaces and hybridised with differentially labelled tumour and reference DNA. Amplifications and deletions of small genomic regions not detectable by chromosome analyses or CGH can then be recognised. SELDI Surface Enhanced Laser Desorption/Ionization (SELDI) is a new mass spectrometry to enable protein profiling. It is based on the binding of matrices and cell lysates or other protein mixtures on a surface, which can be changed in order to identify different protein modifications like acetylation or phosphorylation. Moreover, proteins binding to an antibody coupled to the surface can be found. We plan to use these advanced technologies to perform large-scale genomics and proteomics. The aim is to identify genomic changes, genes and proteins deregulated during the malignant transformation and progression of lymphoblastic lymphomas of childhood. The parallel use of these methods enables us to compare genomic and proteomic changes. It is agreed that for these biological studies, appropriate material will be made available from the study patients from all participating groups. Data generated in these studies will be recorded in the database of the study. The statistician of the study will carry out statistical analysis aimed at the evaluation of the association of treatment outcome with biological parameters generated in these studies. The national study groups organise archiving and processing of the appropriate material. The best way to assure that appropriate tumour material will be available for biological studies according to the priority list may be that the participating clinics have to limit the number of laboratories requiring diagnostic material. Ideally a central laboratory will be responsible for the primary processing of the tumour material for these research studies. Because cytogenetics depends on fresh material, the national cytogenetics laboratory may be the most logical centre for this primary processing of the tumour for research purposes. From there, the laboratories carrying out the different biological studies shall receive the appropriate material according to the agreed priority list. It is further agreed that the researchers responsible for the biological studies will have access to anonymised clinical data of the study for their own analysis. The statistician of the study will support those analyses. Moreover, analyses of the combined data of all biological studies and of the data on clinical features and treatment outcome are one the aims of the study. The rules for publication of data are described in chapter 23, page 89. Tumour cell/dna/rna-banking In order to ascertain appropriate material for additional projects, which may become available during the study, tumour cell/dna/rna-banking is another project of the study. The biological committee will be responsible for approval of research projects, which should have access to that material. It is agreed, that research projects, approved by the biological committee, will have access to the material of the patients from all participating groups as well as to the clinical data of the study in anonymous way. Vice versa, data generated in research projects will be made

79 EURO-LB Research Projects 79 available to the database of the study, in order to connect as much information on the disease as possible. The rules for publication of data are described in chapter 23, page 89.

80 80 20 Organisational Aspects and Documentation EURO-LB Organisational Aspects and Documentation 20.1 Status of Study This is a collaborative study between several equal participating national groups. The core group includes AIEOP (Italy), BFM (Austria, Czechia, Germany, Switzerland), CoALL (Germany), DCOG (Netherlands), EORTC (Belgium, France), NOPHO (Denmark, Finland, Iceland, Norway, Sweden), PPLLSG (Poland), SFCE (France), Spain and UKCCSG (United Kingdom). Other national groups may join the study after discussion with the study committee. Each of these groups has established own infrastructures in their countries regarding data management, quality control of diagnostics and treatment application and consulting support for the participating hospitals. It is agreed that this Protocol should exploit these established infrastructures. Moreover, they necessarily form the basis for the quality of this inter-group study as well as for the provision of optimal patient care. Therefore, each participating group remains responsible for its national patients regarding the execution of the Protocol and data management. The common constitutional organs of the inter-group study will be the common Protocol, the Steering Committee, a committee consisting of the national reference pathologists, the study commission and an external Data Safety and Monitoring Committee. A common biological committee for issues of biological investigations and research may be set up in the future The Protocol One common Protocol will be used for the international study by all national groups. The finalised master Protocol in English can be obtained from the NHL-BFM data centre at Giessen, Germany. Translations of the English master Protocol into the local language will be prepared, if necessary, by each national group. The data centre of each national group will be responsible for distribution of Protocols to hospitals within that national group. Addenda may be added or removed independently by any of the national groups to address local needs, provided they have no bearing on the essential aims of the international Protocol. Subsequent to finalisation, any amendments to the Protocol must be agreed by the International Study Committee and by all the national groups. The originals of all documents of the study have to be stored in the responsible data centres for at least 15 years after preparation of the final report International Study Committee One or two paediatric oncologists of each national group and at least two statisticians will participate in the International Study Committee of the study. The International Study Committee shall meet as appropriate to consider patients treatment, eligibility and outcome to ensure the smooth running of the study. The information given twice yearly to the International Study Committee are: accrual rate, description of causes of ineligibility group allocation toxicity data description of the events

81 EURO-LB Organisational Aspects and Documentation 81 The members of the International Study Committee will receive the survival curves (overall and event-free survivals) of the whole population of the interim analysis. The interim analyses of the randomised trials will be given to the International Study Committee in blinded form (curves without the identification of the groups and with tests). All scientific decisions concerning stopping, continuation or any amendment of the study will be made by the International Study Committee after discussion with the Data Safety and Monitoring Committee Data Safety and Monitoring Committee (DMC) An independent Data Safety and Monitoring Committee composed of 4 international experts will monitor the progress of the study on ethical and scientific grounds. The role of the DMC will be: 1. to review accrual rate 2. to examine interim analyses Two sequential interim analyses and a final analysis are planned. O Brien-Fleming boundaries require very convincing evidence that a treatment effect is real before early termination of the study. These interim analyses will remain confidential. On the basis of these analyses, the DMC may recommend whether the study should continue or whether it should be changed or terminated prematurely. 3. to monitor toxicity Every 6 months the statistician for the trial will circulate a report to the members of the DMC about toxicity. The DMC will review these interim toxicity data although this is primarily the responsibility of the International Study Committee. This biannual procedure prevents against problems of major toxicity. 4. to examine other trials The DMC will review reports of related studies performed by other groups or organisations to determine whether such information materially affects the aims or preliminary findings of the trial. 5. other The DMC will be asked to review any major modification to the study proposed by the study committee prior to its implementation The Master Database The Master Database for the entire study will be held at the NHL-BFM Data Centre (Children s University Hospital, Feulgenstr. 12, D Giessen/Germany) Registration Each national group will carry out registration of patients in their own Data Centre and each group is responsible for data quality, data plausibility and data completeness of their registered patients. After assurance of diagnosis of a lymphoblastic lymphoma, a registration fax has to be sent to the responsible data centre within 7 days after the beginning of the prephase (see Appendix, page 119). In return the treating hospital will receive both a confirmation of registration and the registration number, which clearly identifies the patient in the common database. The registration number may be used for data transfer and for the master database. The use of names or initials as patient identifiers on paper forms and on national databases will be handled according to national practice. All eligible patients will be registered in the study, regardless of whether they are evaluable for treatment response and/or for the randomised trial. For each study patient there will be an individual file at the responsible data centre, which includes all information concerning this patient. These files will be stored with an open-end. Nevertheless, the participating hospitals are bound to store the patient-files for at least 15 years.

82 82 20 Organisational Aspects and Documentation EURO-LB Randomisation Each national group will decide whether the two randomisations will be carried out in its own data centre or sent by fax to the International Data Centre First Randomisation The first randomisation should be obtained for eligible patients within the first 7 days of prephase. The fax randomisation form should be completed and sent to the responsible data centre (see Appendix, page 122). This could be done together with the registration. The treating hospital will promptly receive the result of randomisation. If a randomisation on weekend is unavoidable, please inform the responsible data centre in time, at least on Friday by noon Second Randomisation The second randomisation should be obtained within the first 21 days after the beginning of the maintenance treatment (so there is enough time to check the disease status after the end of intensive chemotherapy and before randomisation). The fax randomisation form should be completed and sent to the responsible data centre (see Appendix, page 123). The treating hospital will promptly receive the result of randomisation Mode of Randomisation The random assignment will be produced by an automatic procedure based on random permuted blocks. Due to the small number of patients entered by each group, it is reasonable to define blocks of small size. The minimisation technique, when available, could be used instead of the permuted blocks. Randomisation will be stratified according to: participating study group stage of disease The second randomisation will additionally be stratified according to the therapy given in Protocol I Refusal of Randomisation Patients refusing randomisation should be treated according to the reference arm. However, patients/guardians may choose the treatment arm. In case of refusal of one of the randomisations, a patient remains eligible for the other randomisation Study Forms and Data Collection General Principles The International Coordination Centre provides a common set of forms for data collection. Each national group will be responsible for distribution of forms to centres within this national group. Subsequent to finalisation, amendments to the forms must be agreed by the International Study Committee. The International Coordination Centre will be responsible for the issue of amended forms. Additional forms may be produced independently by any national group for the collection of data additional to that required for the international study. Each national group shall collect forms for its own patients and shall be responsible for data quality according to local practice. The master database for the entire study will be held at the BFM Data Centre (Children s University Hospital, Feulgenstr. 12, D Giessen/Germany). Each national group may choose either to forward the forms directly to the master data centre at Giessen or data may be collected at the national centre and sent electronically to the master data base, using a common coding system, a common format and identical cross-checks. The data are requested to be sent to the international coordination centre at least once every 6 months.

83 EURO-LB Organisational Aspects and Documentation Data Collection Forms and Data Flow for each Patient The common set of data collection forms consists of the forms listed in the table below. Furthermore, this table gives an overview about the data flow for each patient. A flow sheet for this data flow is enclosed in Appendix, page 118. Form Time Point for Sending to the Responsible Data Centre After Assurance of Diagnosis Informed consents asking for the informed consents (for data processing, for participation in the study, for randomisation etc.); Registration initially (within the first 7 days of prephase); (for German patients an additional registration form has to be filled in and to be sent to the "Deutsches Kinderkrebsregister") First Randomisation initially (within the first 7 days of prephase); only for T-LBL Initial Observation initially (within the first month from beginning of therapy); should be sent together with local cytomorphological report and local histological report pathological report immunological report genetics cyto-/moleculargenetical report have to be filled in and sent by the corresponding reference institutes After Induction Phase I/a Kinetics of Treatment Response have to be filled in and sent by the corresponding reference institutes After End of Intensive Treatment Therapy documentation of intensive treatment (Prephase, I/a- Prednisone, I/a-Dexamethasone, I/b, M, II/a, II/b) together with toxicity forms each approximately 2 weeks after end of the treatment element, all at least at the end of intensive treatment Second Randomisation within the first 21 days after the beginning of the maintenance treatment; only for T-LBL After End of Maintenance Treatment Documentation of Maintenance treatment at least at the end of maintenance treatment; forms could be used as - handout for parents/patients - internal hospital documentation End of treatment at least one month after end of treatment In General SAE In case of a SAE (see chapter 20.10, page 83): immediately after occurrence of the event. Event In case of any event (death from any cause, disease progression, secondary malignancy, late event): at least two weeks after occurrence of the event Late Effects In case of a late effect the responsible data centre will send this form to the treating hospital the form should be returened to the data centre soon Follow-Up-Forms will be sent to the treating hospitals at regular intervals; have to be returned to the responsible data centre as soon as possible

84 84 20 Organisational Aspects and Documentation EURO-LB 02 Please fill in the forms with permanent ink or ball-pen only. Entries with pencil are not acceptable. Corrections are to be made as described in the following: The false entry is crossed out with a simple line. The correct information is inscribed aside and signed by the attending physician; with specification of the date and if necessary the reason for correction. Data fields, which cannot be completed because of missing information, are to be commented. The forms are to be filled out promptly. Subsequently, they are to be controlled from the attending physician and to be signed with date. Please send the forms to the responsible data centre promptly (see also the table above). A copy shall remain in the treating hospital. Please keep in mind not to send to the International Coordination Centre any copies of written results, nor any medical letter, except in case of an appropriate request of the International Coordination Centre. All necessary information inquired is on the documentation forms; supplementary documents are required only in special cases Follow-Up Follow-up-requests will be performed six-monthly for patients within the first three years from diagnosis once a year for patients in the fourth and fifth year from diagnosis every two years for patients after the fifth year from diagnosis The international data centre offers to start requests centrally. In this way the international data centre will send the request-lists to the national study groups for further distribution to the national centres. The national centres should send the completed forms back to the national study group, which will then forward them to the international data centre. If there will be relevant changes to the data in the database (for example new events), there will be a subsequent request. Or requests can be conducted according to local practice Reporting of Severe Adverse Events (SAE) The treatment of the reference arm of this study has been published and is considered to be high standard and the toxicity is well known (see chapter 1, page 12). The modifications in the experimental arms concern only the steroid in the Induction and the duration of the maintenance therapy. In case of severe adverse events, these are to be reported on a special event form (SAE-form). This form has to be filled in in each of the following cases: SAE if death results SAE with permanent handicap/damage in consequence SAE, which impair further therapy according to the Protocol or make it impossible SAE, which is life-threatening Unexpected, severe side effects, which can not be documented on the toxicity form The SAE-form has to be sent by fax to the responsible data centre within 48 hours after the beginning resp. the detection of the SAE. The responsible data centre will forward the form directly to the International Coordination Centre.

85 EURO-LB Statistical Considerations Statistical Considerations 21.1 Study questions The aim of the study is to prove in a randomised way for patients with T-LBL: 1. the efficacy of Dexamethasone in Induction therapy (Protocol I), as compared to standard treatment (Prednisone) 2. the equivalence of reduced duration of maintenance (18 months) vs. standard (24 months) total therapy duration, calculated from the first day of cytoreductive therapy In order to collect data for biological studies, all patients with other lymphoblastic lymphoma will be registered but not randomised. These patients will be treated in the reference arm (Prednisone and 24 months maintenance therapy) Criteria of Assessment Main End Point The main end point is the conditional event free survival (EFS c ), defined as minimum time from the date of randomisation to: - death by any cause - progressive disease - non-response at day 33: > 5% blasts in BM and/or blasts in CSF or/and <35% regression of primary tumour (see chapter 11.6, page 35). In case of non-response, the date of event will be considered the date of the beginning of treatment. - second malignancy - late event (malignancy more than 3 years after diagnosis of T-LBL; no differentiation between progression and second malignancy possible) EFS c will be estimated using the Kaplan-Meier method Secondary End Points - overall survival: defined as time of death by any cause, measured from the date of randomisation - acute and long term toxicity - non-lymphoma related deaths and early deaths (excluding deaths occurring after second line treatment for failure or relapse) 21.3 Number of Subjects required First Randomisation (Efficacy of Dexamethasone in Induction Therapy) Assuming that the 3-year EFS is about 80% with the reference treatment, about 270 patients per group have to be randomised (overall 82 events) 71 to be able to show a 3-year EFS c absolute difference of 10% between the two arms, with α of 5%, β of 10% and bilateral formulation log-rank test. With this number of patients, the power will be 82%, 71% and 59% for a difference of 9%, 8% and 7% respectively. Under the assumption that about 10% of the patients will not be randomised because of very early events or refusal, 600 patients have to be recruited. The expected number of patients newly diagnosed with lymphoblastic T-cell lymphoma in the core group of participating countries/study groups is about 170 per year.

86 86 21 Statistical Considerations EURO-LB 02 BFM-Austria 4 BFM-Czechia/Germany/Switzerland 38 AIEOP-Italy 16 DCOG-Netherlands 8 EORTC 12 SFCE-France 24 UKCCSG-U.K. 20 PPLLSG-Poland 24 NOPHO-Scandinavia 12 Spain 12 Israel 3 Total 173 With 170 patients per year, the duration of the recruitment should be 3.5 years. The Data Safety and Monitoring Committee will decide if more or less patients are required, according to the recruitment rate and the proportion of randomised patients Second Randomisation (Reduced Duration of Maintenance vs. Standard) The probability of 3-years EFS c after having survived event-free for 15 months is estimated to be 95%. With an equivalence range of 4% and a first type error of 5%, 450 patients have to be randomised in each randomisation arm to prove equivalence with a power of 80% 72. The power will be 85%, 74% and 69% with a baseline of 96%, 94% and 93% respectively. Under the assumption that about 25% of the patients will not be randomised because of early events or refusal, T-LBL-patients have to be enrolled. Therefore, the second randomisation will be continued in the subsequent inter-group study on lymphoblastic lymphoma Analysis According to the factorial design of the study each randomisation will be analysed separately. If there is evidence of interaction, this has to be taken into account in the final analysis First Randomisation The test of the null hypothesis (no difference) for first randomisation will be carried out, according to the intention to treat principle for all randomised patients in order to ensure an unbiased estimation of treatment effect. The EFS c in the two treatment arms will be compared with the log-rank test stratified by participating group. A combined estimate of treatment effect and the confidence interval will be given, adjusting by participating group, if no significant heterogeneity of the effects will be detected Second Randomisation The test for second randomisation will be based on a one-sided confidence interval of the estimated probability of 3-years EFS c from randomisation (pefs c ). Reduced maintenance will be considered equivalent if the lower limit of a one sided confidence interval for the difference of the pefs c is below 4% (equivalence range 4%). Primary analysis will be a per-protocolanalysis. All randomised patients, except those who fail before month 18 of treatment, will be included in this analysis, but patients who switch the arm will be included in the treatment arm actually given. Patients, who fail from month 18 to 24 of therapy, remain in their randomised arm if no switch was intended. If there are conflicting results for the per-protocol and the intent-to treat analysis, the International Study Committee and the Data Safety and Monitoring Committee will have to

87 EURO-LB Statistical Considerations 87 decide about the implication on conclusions from the main analysis for first and second randomisation Interim Analysis of Event Free Survival Two interim analyses are planned for each of the randomisations. The O Brien and Fleming rules will be followed to conclude at each sequential analysis 73. The boundary proposed in such rules requires very strong evidence of an effect to terminate at the first interim test, whereas the criteria at the final test are rather close to those for a single sample design (i.e. a design with no interim testing). On the discretion of the DMC other time-points and frequencies of interim analysis may be chosen. The p-values will then be based on a Lan-DeMets spending function approach with O Brien-Fleming type spending function. If any of the boundaries are reached, patient recruitment will be stopped by the DMC, the international study coordinator will be informed and a meeting of the International Study Committee will be held to discuss further continuation or modification of the trial. Final analysis will be performed three years after the inclusion of the last patient First Randomisation Two interim analyses are planned after observing 33% and 66% of the expected events. p value* events** Approximate time of the analysis first analysis years after the beginning of patient enrolment second analysis years after beginning of patient enrolment final analysis years after the end of patient enrolment p value *) events **) nominal p values for overall type I error of 0.05 O Brien-Fleming boundaries number of events required for the interim analysis Second Randomisation For practical reasons (planning of subsequent studies) the first interim analysis will be conducted at the end of patient enrolment of this study, the second after the end of patient enrolment of the subsequent study. For safety reasons, the interim analysis will be a log-rank test of difference instead of the equivalence test planned for the final analysis (p-values see chapter , page 86) Stopping Rule (Monitoring Toxicity) Interim analysis on severe toxicity (grade 3 and 4) and toxic deaths will take place twice a year under supervision of the Data Safety and Monitoring Committee. The absolute death rate observed in each treatment arm (and globally in the whole population) will be compared to a reference rate in order to detect an absolute excess of toxic deaths with a Wald sequential plan. In the studies NHL-BFM 90 and 95 (04/ /2001) four toxic deaths were observed among 387 patients of therapy group I (Non-B-NHL) = 1% [95% confidence interval %]. Based on this previous experience, we choose the following parameters: p 0 = 1% and p 1 = 4%, with α = 5% and β = 1%. This means that the risk to wrongly conclude that there is an excess of toxic deaths (whereas the real rate is equal to p 0 1%) is equal to α = 5%. On the other hand, the power to detect an excess of toxic deaths (if the real rate is equal to p 1 = 4%) is equal to 1-β = 99%. Using the Wald s test with these parameters, the boundary is:

88 88 21 Statistical Considerations EURO-LB 02 Number of patients with at least 30 weeks follow up or toxic death Number of toxic deaths If the number of toxic deaths observed reaches the boundary defined in this sequential plan (for example a third death occurs amongst the first 30 patients), then the International Study Committee has to decide in cooperation with the DMC, whether the trial has to be stopped.

89 EURO-LB Information of Patient and Consent Information of Patient and Consent Prior to the therapy, parents and guardians must be informed about the possibilities of treatment and therapeutical side effects in an understandable way. The explanation should be based on the purpose and directives of the clinical study and also on the most current knowledge and understanding of scientific research. Parents should be informed that the participation in the clinical study, especially the participation in the randomised trials is voluntary and that they may choose to receive treatment as part of the clinical study. During the course of treatment, they may also choose the randomised group. An alternative to this treatment Protocol is for instance the treatment strategy of study NHL-BFM 90. Compared to international literature, the treatment based on this Protocol has shown a high rate of long remission in patients. If irradiation is intended, the radiotherapists must explain the treatment and the risk of radiation therapy. In case of surgery, surgeons as well as anaesthesiologists are obliged to inform about the risks and complications. In the Declaration of Helsinki (current version of October 2000), and in the actual GCP- Principles, it is stated that, aside from parents, underage patients are to be included in being informed about the possibilities of treatment and can participate in giving their consent or assent to treatment, if they are physically and psychologically in the state of mind to do so. This declaration is also valid for this study, especially for the adolescent between 14 and 18, who may be well informed and who are familiar with their sickness. This may be also possible with younger children. It is the duty of the attending physician to decide, whether or not to allow the minor to participate in the discussions. If so, the conversation and explanations should be made carefully in an understandable and appropriate manner for the child. If the minor patient turns 18 years old and becomes liable, another statement of consent must be given by the patient for data processing and statistical purposes. We recommend, in case treatment is chosen, to ask the parents and guardians to give their consent to the treatment according to the directives of the Protocol. Aside from the formal statement, the points of explanations should be recorded individually below. After signing the consent, we also recommend to give a copy to the parents or patients. It is also necessary, to ask the parents and guardians or patients for their consent to allow to have their personal and therapy records and data to be forwarded and processed by the Principal Investigators to the National Coordinators and the master database of the study in Giessen/Germany, and to the Central Reference Laboratories for special diagnostics. Each of the patients personal data and records have serial numbers and are handled anonymously in evaluations or in the publications of results. The use of names or initials as patient identifiers on paper forms and on national databases will be according to national practice. For forms for patients informed consents, see Appendix, page 107 ff.

90 90 23 Publication Rules/Presentation of Results EURO-LB Publication Rules/Presentation of Results Final results of the study will be published irrespective of whether the aims of the study have been reached or not. Publication will follow the CONSORT-Statement 74 and include a thorough safety analysis. Data relating to the study must not be reported or published without prior consultation with the International Study Committee of the study. Any publication arising from the trial will have as its authors those who have produced the paper and acknowledgement to the inter-group members.

91 EURO-LB Declaration of Helsinki 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 th WMA General Assembly, Venice, Italy, October st WMA General Assembly, Hong Kong, September th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 and the 52nd WMA General Assembly, Edinburgh, Scotland, October 2000 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 fulfilment 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.

92 92 24 Declaration of Helsinki EURO-LB 02 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

93 EURO-LB Declaration of Helsinki 93 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. 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. 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 patientphysician 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.

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96 96 25 Reference List EURO-LB 02 (39) Sutor AH, Mall V, Thomas KB. Bleeding and thrombosis in children with acute lymphoblastic leukaemia, treated according to the ALL-BFM-90 protocol. Klin Padiatr. 1999;211: (40) Nowak-Gottl U, Wermes C, Junker R et al. Prospective evaluation of the thrombotic risk in children with acute lymphoblastic leukemia carrying the MTHFR TT 677 genotype, the prothrombin G20210A variant, and further prothrombotic risk factors. Blood. 1999;93: (41) Nowak-Gottl U, Wermes C, Junker R et al. Prospective evaluation of the thrombotic risk in children with acute lymphoblastic leukemia carrying the MTHFR TT 677 genotype, the prothrombin G20210A variant, and further prothrombotic risk factors. Blood. 1999;93: (42) Nowak-Gottl U, Wermes C, Junker R et al. Prospective evaluation of the thrombotic risk in children with acute lymphoblastic leukemia carrying the MTHFR TT 677 genotype, the prothrombin G20210A variant, and further prothrombotic risk factors. Blood. 1999;93: (43) Sutor AH, Mall V, Thomas KB. Bleeding and thrombosis in children with acute lymphoblastic leukaemia, treated according to the ALL-BFM-90 protocol. Klin Padiatr. 1999;211: (44) Halton JM, Mitchell LG, Vegh P, Eves M, Andrew ME. Fresh frozen plasma has no beneficial effect on the hemostatic system in children receiving L-asparaginase. Am J Hematol. 1994;47: (45) Nowak-Gottl U, Wermes C, Junker R et al. Prospective evaluation of the thrombotic risk in children with acute lymphoblastic leukemia carrying the MTHFR TT 677 genotype, the prothrombin G20210A variant, and further prothrombotic risk factors. Blood. 1999;93: (46) Pizzo PA. Management of fever in patients with cancer and treatment-induced neutropenia. N Engl J Med. 1993;328: (47) Feusner JH, Hastings CA. Infections in children with acute myelogenous leukemia. Concepts of management and prevention. J Pediatr Hematol Oncol. 1995;17: (48) Winston DJ, Chandrasekar PH, Lazarus HM et al. Fluconazole prophylaxis of fungal infections in patients with acute leukemia. Results of a randomized placebo-controlled, double-blind, multicenter trial. Ann Intern Med. 1993;118: (49) Weinthal J, Frost JD, Briones G, Cairo MS. Successful Pneumocystis carinii pneumonia prophylaxis using aerosolized pentamidine in children with acute leukemia. J Clin Oncol. 1994;12: (50) Gershon AA, Steinberg SP. Persistence of immunity to varicella in children with leukemia immunized with live attenuated varicella vaccine. N Engl J Med. 1989;320: (51) Wagstaff AJ, Faulds D, Goa KL. Aciclovir. A reappraisal of its antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs. 1994;47: (52) Asano Y, Yoshikawa T, Suga S et al. Postexposure prophylaxis of varicella in family contact by oral acyclovir. Pediatrics. 1993;92: (53) Feldman S, Lott L. Varicella in children with cancer: impact of antiviral therapy and prophylaxis. Pediatrics. 1987;80: (54) Nyerges G, Meszner Z. Treatment of chickenpox in immunocompromised children. Am J Med. 1988;85: (55) Arikoski P, Komulainen J, Voutilainen R et al. Reduced bone mineral density in long-term survivors of childhood acute lymphoblastic leukemia. J Pediatr Hematol Oncol. 1998;20: (56) Rauch F, Travers R, Norman ME et al. Deficient bone formation in idiopathic juvenile osteoporosis: a histomorphometric study of cancellous iliac bone. J Bone Miner Res. 2000;15: (57) Srivastava T, Alon US. Bisphosphonates: from grandparents to grandchildren. Clin Pediatr (Phila). 1999;38: (58) Shoemaker LR. Expanding role of bisphosphonate therapy in children. J Pediatr. 1999;134:

97 EURO-LB Reference List 97 (59) Brumsen C, Hamdy NA, Papapoulos SE. Long-term effects of bisphosphonates on the growing skeleton. Studies of young patients with severe osteoporosis. Medicine (Baltimore). 1997;76: (60) van Persijn van Meerten EL, Kroon HM, Papapoulos SE. Epi- and metaphyseal changes in children caused by administration of bisphosphonates. Radiology. 1992;184: (61) von Heimburg D, Pallua N. [Early and late treatment of iatrogenic injection damage]. Chirurg. 1998;69: (62) Bertelli G, Gozza A, Forno GB et al. Topical dimethylsulfoxide for the prevention of soft tissue injury after extravasation of vesicant cytotoxic drugs: a prospective clinical study. J Clin Oncol. 1995;13: (63) Wermes C, Fleischhack G, Junker R et al. Cerebral venous sinus thrombosis in children with acute lymphoblastic leukemia carrying the MTHFR TT677 genotype and further prothrombotic risk factors. Klin Padiatr. 1999;211: (64) Einhaupl KM, Masuhr F. [Cerebral sinus and venous thrombosis]. Ther Umsch. 1996;53: (65) Einhaupl KM, Villringer A, Meister W et al. Heparin treatment in sinus venous thrombosis. Lancet. 1991;338: (66) deveber G, Chan A, Monagle P et al. Anticoagulation therapy in pediatric patients with sinovenous thrombosis: a cohort study. Arch Neurol. 1998;55: (67) Reiter A, Schrappe M, Parwaresch R et al. Non-Hodgkin's lymphomas of childhood and adolescence: results of a treatment stratified for biologic subtypes and stage--a report of the Berlin-Frankfurt-Munster Group. J Clin Oncol. 1995;13: (68) van Dongen JJ, Macintyre EA, Gabert JA et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia. 1999;13: (69) Coustan-Smith E, Sancho J, Hancock ML et al. Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood. 2000;96: (70) Dworzak MN, Froschl G, Printz D et al. Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia. Blood. 2002;99: (71) Freedman LS. Tables of the number of patients required in clinical trials using the logrank test. Stat Med. 1982;1: (72) Rodary C, Com-Nougue C, Tournade MF. How to establish equivalence between treatments: a one-sided clinical trial in paediatric oncology. Stat Med. 1989;8: (73) O'Brien PC, Fleming TR. A multiple testing procedure for clinical trials. Biometrics. 1979;35: (74) Begg C, Cho M, Eastwood S et al. Improving the quality of reporting of randomized controlled trials. The CONSORT statement. JAMA. 1996;276:

98 98 26 Index of Abbreviations EURO-LB Index of Abbreviations AIEOP Associazione Italiana di Ematologia ed Oncologia Pediatrica ALL acute lymphatic leukaemia APC activated protein C APCR activated protein C resistance, Factor V Leiden mutation AT-III antithrombin III AUT Austria B-ALL B-cell acute lymphatic leukemia BFM Berlin-Frankfurt-Münster BM bone marrow BMP bone marrow puncture B-NHL B-cell Non-Hodgkin Lymphoma BSA body surface area Ca calcium CG Cardiogram CGH Comparative Genomic Hybridization CHF congestive heart failure CI confidence interval CMV cytomegalovirus CNS central nervous system CoALL Co-operative study group for childhood acute lymphoblastic leukemia CRT cranial radiation therapy CSF cerebrospinal fluid CT computerized tomography d day DCLSG Dutch Childhood Leukemia Study Group DIC disseminated intravascular coagulation DMC Data Safety and Monitoring Committee DNA desoxyribunucleic acid EBV Epstein-Barr virus ECG electrocardiogram ECOG Eastern Cooperative Oncology Group EDTA ethylenediaminetetracetic acid EF ejection fraction EFI event-free interval EFS event-free survival EFS c conditional event free survival EGIL European Group for Immunophenotyping of Leukemias EORTC European Organisation for research and treatment of cancer FAB French-American-British (cytomorphological classification) FACS flourescence-activeted cell sorter FFP fresh frozen plasma FSH follicle stimulating hormone GCP good clinical practice GER Germany GFR glomerular filtration rate Glc glucose GOT glutamic-oxaloacetic-transaminase GPOH Gesellschaft für Pädiatrische Hämatologie und Onkologie GPT glutamic-pyruvic-transaminase GVHD graft versus host disease Gy Gray h hour HD high-dose HIV human immunodeficiency virus

99 EURO-LB Index of Abbreviations 99 i.m. i.th. i.v. kg LBL LDH LH LLN LP LRT MDD mg ml MRD MRI MTHFR N Na NB-NHL NCI-CTC NHL NOPHO OS P p.a. p.o. pb-lbl pefs pos PPLLSG PRED pts PTT RNA s.c. SAE SD SE SELDI SF SFOP SGOT SGPT SIADH STIKO tbl. T-LBL TPM-SMZ TPMT TPN U UKCCSG UNL WBC WNL intramuscular intrathecal intravenous kilogram lymphoblastic lymphoma lacate dehydrogenase luteinising hormone lower limit of normal lumbar puncture local radiotherapy minimally disseminated disease milligram millilitre minimal residual disease magnetic resonance imaging methylentetrahydrofolatreductase number sodium Non-B Non-Hodgin lymphoma National Cancer Institute-Common Toxicity Criteria Non-Hodgkin lymphoma Nordic Society of Pediatric Haematology and Oncology overall survival phosphate posterior-anterior per os precursor-b-cell-lymphoblastic lymphoma probability of event-free survival probability of overall survival Polish Paediatric Leukaemia/Lymphoma Study Group Prednisone, Prednisolone patients partial thromboplastine time ribonucleic acid subcutaneous severe adverse events single dose standard deviation surface enhanced laser desorption/ionization shortening fraction Société Française d Oncologie Pédiatrique serum glutamic-oxaloacetic-transaminase serum glutamic-pyruvic-transaminase syndrome of inappropriate antidiuretic hormone Ständige Impfkomission tablet T-cell-lymphoblastic lymphoma Trimethoprim-Sulfamethoxazole (Cotrim) Thiopurinmethyltransferase total parenteral nutrition units United Kingdom Children Cancer Study Group upper normal limit white blood cell/count within normal limits

100 Appendix EURO-LB Appendix Appendix. 99 Imaging Guidelines for mediastinal Disease. 100 Therapy schema of High-Dose Methotrexate 104 Schema Processing of Tumour Material Reference Laboratories: Address and Dispatch Note Consent Forms Patient Informed Consent Form for Data Exchange Patient Informed Consent Form for Participation in the EURO-LB 02 Study Patients Information Protocol Patient Informed Consent Form for 1 st Randomisation 113 Patient Informed Consent Form for 2 nd Randomisation Patient Informed Consent Form for Participation in Research Projects and Tumour Banking. 115 Forms Flow Sheet for Data Flow and Collection 118 Registration Initial Observation st Randomisation nd Randomisation Events Severe Adverse Events (SAE). 125 Late Effects Maintenance Therapy 127 End of Treatment Toxicity Form Kinetics of Treatment Response Histopathological and Immunohistochemical Review Genetics 133 Immunological Report 134 Participating Groups and Centres Contract of Participation of a Clinical Institution 136 Principal Investigators of the Study Groups/National Reference Centres. 137 Participating Centres of Paediatric Haematology and Oncology 140 Index. 141

101 EURO-LB Appendix Appendix Imaging Guidelines for mediastinal Disease Therapy Schema of High-Dose Methotrexate Schema Processing of Tumour Material Reference Laboratories: Addresses and dispatch notes

102 102 Imaging Guidelines EURO-LB 02 IMAGING GUIDELINES FOR MEDIASTINAL DISEASE Last up-date: 13/10/02 INTRODUCTION Objectives of the radiological evaluation: To define the precise volume and extension of the disease at diagnosis. To define the precise response to initial chemotherapy, since the absence of response at the end of I/a phase of Induction leads to a more intensive treatment regimen (high risk ALL Protocol and possibly local irradiation). To search for new prognostic factors such as the absolute volume at day 8 (after prephase) and kinetics of tumour response during Induction therapy. Tumour volume can be estimated: Either on «classical» elliptical extrapolation from the three one-dimensional measurements, and this represents the current reference. Either on direct volume measurement with 3D-softwares and manual tumour delineation on each slice (Breiman, Nawaratne, Hopper, Eggli). This method is currently considered as more precise but also more time-consuming for radiologists and not yet available in each institution. OBJECTIVES OF THE RADIOLOGICAL STUDY Main objectives: To study the prognostic signification of the tumour response during Induction therapy: shrinking of tumour volume from Day 1 to Day 8 and Day 15. To study the prognostic signification of the absolute tumour volume at Day 8. Secondary objectives: To homogenise the imaging acquisition Protocols and the radiological response criteria. To study the feasibility of a centralised imaging review in each national group. To compare the calculated and the computed-direct 3D evaluation of tumour volumes. CHEST IMAGING MODALITIES AND TIMING Time x-ray CT-scan Day 1 Pre-treatment + + Day 8 End of Prephase - + Day 15 During I/a-phase Induction - + Day 33 End of I/a-phase Induction + +* Before Protocol M + - Before Reinduction (stage III and IV only) + - Before Maintenance + - During Maintenance 4-weekly 1 st year 8-weekly 2 nd year if progression suspected only or if progression suspected After Completion 6-monthly in 3 rd and once a year in 4 th year if progression suspected only or if progression suspected *) CT-scan only, if there was no tumour regression of at least 35% at day 15. Important note: The evaluation of the prognostic impact of the kinetics of tumour response to treatment is a major goal of this study. Therefore, the primary mediastinal tumour should be

103 EURO-LB 02 Imaging Guidelines 103 documented by CT-scan for accurate calculation of tumour volume and to gain reproducible and comparable data for all patients in all countries. CHEST CT SCAN ACQUISITION PROTOCOL General case: Spiral mode acquisition, with breath-holding when possible Contiguous 3 to 7 mm slice thickness Tube potential: kv Rotation time of the tube, ma and pitch are adjusted in order to optimise the radiation dose while keeping sufficient diagnostic-quality signal-to-noise ratio. An acquisition pitch of 1 to 2 is recommended in order to reduce the acquisition time. Field of view, between 250 and 500 mm, is adapted to the patient size. Axillary areas must always be included in the FOV. Two reconstructions are mandatory: one for the mediastinum and one for the lung with adapted reconstruction filters and windowing. Intravenous iodine contrast agent is mandatory and may be directly used (vascular contrast agent between 240 and 300 mg I/l, volume: 2 ml/kg, injection rate ml/sec). Automatic injection is recommended. Important note: In order to achieve comparable data, identical scan acquisition conditions at different time points of investigation are mandatory, at least in the same patient. In case of respiratory distress (orthopnea) at diagnosis: In case of respiratory distress at diagnosis the advisability of pre-treatment CT-scan should be discussed with caution. In critically ill children the initial CT-scan may be postponed after stabilisation of the patient. When finally accepted, CT scan should be obtained: without any sedation with continuous visual and transcutaneous SaO2 control with a dorsal block if necessary (appropriate axial slices may be obtained with multiplanar post-reconstruction) with reduced time acquisition: pitch 1:2 or more, lowest time of tube rotation and adjusted ma In case of patients with personal history of severe allergic adverse effect (asthma, Quincke edema, anaphylactic shock) after a previous iodine injection: initial imaging will be limited to the chest-x-ray, and non-injected CT-scan if MRI is not available or feasible (respiratory distress) following imaging may be done with MRI CHEST MR: Only when history of severe allergic severe adverse effect after iodine injection limits the use of CT-scan. Cardiac and respiratory gating are recommended Axial 5-7 mm thickness slices FOV adjusted (see above) Acquisition matrix: 256 x 192 or more Non enhanced and enhanced-t1-weighted and T2-weighted sequences Spin echo (or Fast spin echo) sequences are recommended Intravenous gadolinium injection (0.2ml/kg) is mandatory but not directly (after a pre-contrast acquisition) Specific T1w or T2w 3D-acquisition (gap 0) for volume calculation is needed

104 104 Imaging Guidelines EURO-LB 02 TARGETS AND MEASUREMENTS Targets: Measurable targets: Thymic mass Enlarged lymph nodes: short axis > or = 10 mm Other targets: Pleural or pericardial effusion Pulmonary involvement Measurements: Measurements are made at the highest tumour size level. The largest diameter and the largest perpendicular diameter are chosen (Axial Dim1 and Axial Dim2, see figure below). Height of the tumour may be obtained either from vertical reconstruction or from subtraction of the highest and the lowest reference levels. Volume calculation: Tumour volume [ml] = Axial Dim1 [cm] x Axial Dim2 [cm] x Height [cm] x 0,52 Response criteria: Response 3D response criteria* Corresponding CT Pattern Stable disease < 35% Abnormal enlarged thymus Objective effect 35-65% or mediastinal mass Partial response 65 85% Very good partial response > 85% Complete Remission Normal thymus and no mediastinal residual mass and no residual pleuro-pericardial effusion and no residual pulmonary involvement *) 3D response criteria are adapted and calculated from classical 2D response criteria of the WHO classification: SD < 25%; OE 25-50%; PR > 50% (Therasse et al.). 2D-VGPR is defined as > 70%. Important Note: Definition of the non-responders during Induction: Volume response < 35%. Non-response at Day 33 (after I/a phase of Induction) leads to a more intensive treatment regimen (see chapter 11.6, page 35). Definition of progressive disease: reappearance or increase in size of residual remnants (see chapter 16, page 74).

105 EURO-LB 02 Imaging Guidelines 105 DATA STORAGE AND NATIONAL REVIEW CT data should be printed on films (including scales). As far as possible, CT data should also be burned on a CD-ROM using DICOM format with a Dicom.directory file. This CD-ROM will be transmitted to the national reference radiologist: A centralised review process will confirm the volume evaluations at Day 1, 8 and 15 (reference measurement, using the three dimensions and the formula). If available, a direct volume will be measured with 3D-software and manual tumour delineation on each slice (values also transmitted to the database, for further comparison to the reference measurements). CONTRIBUTORS: C Baunin 1, C Bergeron 2, B Bourlière 3, H Brisse 4, D Couanet 5, S Neuenschwander 4, L Ollivier 4, H Pacquement 4, C Patte 5, P Thiesse 2 1 Hôpital Mère-Enfant, Toulouse 2 Centre Régional Léon Berard, Lyon 3 CHU La Timone, Marseille 4 Insitut Curie, Paris 5 Institut Gustave Roussy, Villejuif REFERENCES Nawaratne S, Fabiny R, Brien JE, Zalcberg J, Cosolo W, Whan A, Morgan DJ. Accuracy of volume measurement using helical CT. J Comput Assist Tomogr 1997 May-Jun;21(3):481-6 Breiman RS, Beck JW, Korobkin M, Glenny R, Akwari OE, Heaston DK, Moore AV, Ram PC. Volume determinations using computed tomography. AJR Am J Roentgenol 1982 Feb;138(2): Brisse H, Pacquement H, Burdairon E, Plancher C, Neuenschwander S. Outcome of residual mediastinal masses of thoracic lymphomas in children: impact on management and radiological follow-up strategy. Pediatr Radiol 1998 Jun;28(6): Thiesse P, Ollivier L, Di Stefano-louineau D, Negrier S, Savary J, Pignard K, Lasset C, Escudier B for the Groupe Français d Immunothérapie of the Fédération Nationale des Centres de Lutte Contre le Cancer. Response rate accuracy in oncology trials: reasons for interobserver variability. J Clin Oncol 1997 Dec; 15(12): Therasse P et col. New Guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst. 2000; 92: Eggli KD, Close P, Dillon PW, Umlauf M, Hopper KD. Three-dimensional quantitation of pediatric tumor bulk. Pediatr Radiol 1995;25(1):1-6 Hopper KD, Kasales CJ, Eggli KD, TenHave TR, Belman NM, Potok PS, Van Slyke MA, Olt GJ, Close P, Lipton A, Harvey HA, Hartzel JS. The impact of 2D versus 3D quantitation of tumor bulk determination on current methods of assessing response to treatment. J Comput Assist Tomogr 1996 Nov-Dec;20(6):930-7 St Amour TE, Siegel MJ, Glazer HS, Nadel SN. CT appearances of the normal and abnormal thymus in childhood. J Comput Assist Tomogr 1987 Jul-Aug;11(4): WHO handbook for reporting results of cancer treatment. Geneva (Switzerland): World Health Organization Offset Publication N 48; 1979.

106 106 Therapy Schema of High Dose MTX EURO-LB 02 Therapy schema of High-Dose Methotrexate Protocol M starts 2 weeks after completion of Protocol I and is composed of 6-MP over 56 days and 4 courses of high-dose MTX (5g/m 2 as 24 h infusion day 8, 22, 36 and 50). Name: Date of Birth: Weight: Height: BSA: Requirements: Good general condition without serious infections, renal function within normal age-adapted limits, GOT/GPT 5xUNL, bilirubin normal, WBC 1.5 x 10 9 /l, platelets 50 x 10 9 /l Therapy management: Due to MTX-metabolism interactions Co-trimoxazole should not be given for at least 6 days prior to the beginning of the MTX-therapy and it should be resumed only after completion of the MTX-therapy. Days 1 56 mg 6-Mercaptopurine p.o. (25 mg/m 2 /d), in the evening ( - ) (1 tbl. Puri-Nethol contains 50 mg) mg/d x 56 d = mg cumulative dose mg cumulative dose :50 mg = tbl. Puri-Nethol distributed over 56 days Therapy schema of high-dose Methotrexate: Day 1 ( ) Pre-Hydration ml NaHCO 3 8.4% (2 ml/kg) in ml aqua dest. (2 ml/kg) infusion over 1 h, then 500 ml NaCl 0.45%:Gluc 5% + 40 ml NaHCO 3 8.4% + 10 ml KCl 7.45% over at least 2 hrs, in order to achieve urine ph 7 and urine output 100 ml/m²/h Day 1 (14 00 h) if urine ph > 7.0 ( ) mg Methotrexate 500 mg/m 2 initially as infusion over 30 min mg Methotrexate 4.5 g/m 2 as infusion over 23 ½ hours + ml NaCl 0.45%:Gluc 5% (3 000 ml/m 2 /d) + ml NaHCO 3 8.4% (180 ml/m 2 /d) + ml KCl 7.45% (90 ml/m 2 /d) ml/h Day 1 (16 00 h) Methotrexate i.th. in age-adapted dose: LP! 2 hours after start of MTX years: <1 <2 <3 3 dose (mg i.th.): (lowered head, prone position for 2 hrs) Days 1-3 Fluid balance every 12 hours, urine ph (test strips) at every miction ( - ) if urine ph < 7.0 NaHCO 3 8.4% (2 ml/kg) in aqua dest. (2 ml/kg) as infusion over 30 min. if input > output ml/m 2 /12 hrs: Furosemide i.v. (0.5 mg/kg, maximum 20 mg SD) Days 2+3 Hydration ( - ) ml NaCl 0.45%:Gluc 5% (3 000 ml/m 2 /d) ml NaHCO 3 8.4% (180 ml/m 2 /d) ml KCl 7.45% (90 ml/m 2 /d) Days 2+3 Folinic Acid Rescue (Leucovorin) ( - ) mg LCV i.v. (15 mg/m 2 /SD) at hour 42, 48 and 54 MTX-level measurement: 24, 42 and 48 hrs after start of MTX-infusion if MTX 24 > 150 µmol/l, MTX 42 > 1 µmol/l or MTX 48 > 0.4 µmol/l then forced diuresis, MTX-levels every 6 hrs and LCV as described below Supportive Therapy: Amphomoronal: Date: TMP-SMZ: Colistin: Signature 1: Signature 2:

107 EURO-LB 02 Schema Processing of Tumour Material 107 Schema Processing of Tumour Material (Lymphoma) Biopsy sterile Tumour touch imprints Sterile specimen in NaCl further proceeding see below NHL NHL questionable no NHL Proceeding according to diagnosis sterile Devide sterile specimen into two parts Local pathologist sterile Reference-pathologist Keep sterile! Send specimen (in NaCl) to the NHL-laboratory within 24 hours Local pathologist NHL-laboratory