with a first relapse of acute lymphoblastic leukemia ...

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Prepublished online December 18, 2007; doi:10.1182/blood-2007-07-102525

High-dose as compared with intermediate-dose methotrexate in children with a first relapse of acute lymphoblastic leukemia Arend von Stackelberg, Reinhard Hartmann, Christoph Buhrer, Rudiger Fengler, Gritta Janka-Schaub, Alfred Reiter, Georg Mann, Kjeld Schmiegelow, Richard Ratei, Thomas Klingebiel, Jorg Ritter and Gunter Henze

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Blood First Edition Paper, prepublished online December 18, 2007; DOI 10.1182/blood-2007-07-102525 1

HIGH-DOSE AS COMPARED WITH INTERMEDIATE-DOSE METHOTREXATE IN CHILDREN WITH A FIRST RELAPSE OF ACUTE LYMPHOBLASTIC LEUKEMIA Arend von Stackelberg1, Reinhard Hartmann1, Christoph Bührer2, Rüdiger Fengler1, Gritta JankaSchaub3, Alfred Reiter4, Georg Mann5, Kjeld Schmiegelow6, Richard Ratei7, Thomas Klingebiel8, Jörg Ritter9, and Günter Henze1, for the ALL-REZ BFM Study Group

From the Departments of Pediatric Oncology/Hematology, Universities of 1Berlin (Charité Universitätsmedizin Berlin), 3Hamburg, 4Giessen, 8Frankfurt, 9Münster, 5St. Anna Kinderspital, Department of pediatric Hematology/Oncology Vienna, Austria, 6The Pediatric Clinic II, The University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark 7Department of Hematology, Oncology and Tumor Immunology, RobertRoessle-Clinic at the HELIOS Klinikum Berlin,Germany, and 2Basel University Children's Hospital, Basel, Switzerland S Correspondence: Dr. Arend von Stackelberg, Klinik für Pädiatrie m. S. Onkologie und Hämatologie Charité Campus Virchow-Klinikum, Universitätsmedizin Berlin Augustenburger Platz 1 D-13353 Berlin, Germany Tel. (+49) 30-450 666833, Fax (+49) 30-450 566906 e-mail [email protected]

Running Head: MTX dosage in relapsed childhood ALL Keywords: ALL-REZ BFM 90, acute lymphoblastic leukemia, relapse, methotrexate

Copyright © 2007 American Society of Hematology


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Abstract High dose Methotrexate (MTX) has been extensively used for treatment of acute lymphoblastic leukemia (ALL). To determine the optimal dose of MTX in childhood relapsed ALL, the ALL-REZ BFM Study Group performed this prospective randomized study. A total of 269 children with a first early/late isolated (n=156) or combined (n=68) bone marrow or any isolated extramedullary relapse (n=45) of precursor B-cell (PBC) ALL (excluding very early marrow relapse within 18 months after initial diagnosis) were registered at the ALL-REZ BFM90 trial and randomized to receive methotrexate infusions at either 1g/m2 over 36h (intermediate dose, ID) or 5g/m2 over 24h (high dose, HD) during 6 (or 4) intensive polychemotherapy courses. Intensive induction/consolidation therapy was followed by cranial irradiation, and by conventional dose maintenance therapy. Fifty-five children received stem-cell transplants. At a median follow-up of 14.1 years, the 10-year event-free survival probability was .36 ± .04 for the ID-group (n=141), and .38 ± .04 for the HD-group (n=128, p=.919). The two groups did not differ in terms of prognostic factors and other therapeutic parameters. Conclusion. Methotrexate infusions at 5 g/m2/24h, as compared with 1 g/m2/36h, are not associated with increased disease control in relapsed childhood PBC acute lymphoblastic leukemia.


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INTRODUCTION

Long-term event-free survival (EFS) can be achieved by aggressive polychemotherapy in a substantial proportion of children with acute lymphoblastic leukemia (ALL) who relapse after successful front-line induction and consolidation therapy.1-5 A second remission is achieved by more continuous elements using a 4-drug induction with corticosteroids, vinca alkaloids, anthracyclines, asparaginase and intrathecal therapy in some groups,2,6,7 or by more intensive short course elements containing high dose methotrexate and high dose cytarabine, as used by the ALLREZ BFM group.8,9 Various regimens for re-induction/consolidation have been used including more continuous elements, rapid alternating 2-drug elements, or again intensive short course multi-drug elements as used by the ALL-REZ BFM group.2,6-8 For treatment or prevention of CNS leukemia, intrathecal therapy is given during intensive treatment, and CNS irradiation is administered after the end of intensive chemotherapy.10-12 In case of testicular relapse, local irradiation or orchiectomy is performed.13-15 Less intensive maintenance therapy is given for up to 2 years.8,9 Despite heterogeneous approaches, rather comparable results are achieved. Remission- and long-term survival rates depend more on clinical and biological characteristics of the leukemia, than on the specific treatment regimen. The time-point and the site of relapse as well as the immunophenotype of the leukemia and a variety of translocation associated fusion genes have been identified as the most important prognostic determinants.8,16 Patients with poor prognostic features such as early time-point of relapse, bone marrow involvement, T-lineage disease, or BCR/ABL fusion transcript could not be salvaged even with intensive multi-drug chemotherapy and cranial radiation therapy and have been allocated to allogeneic stem-cell transplantation (SCT). Whereas superior results could be achieved with SCT from HLA identical siblings compared to chemo-/radiotherapy in patients with bone marrow relapse, the benefit of SCT from unrelated donors could be confirmed for high risk patients only. Furthermore, the role of allogeneic SCT in patients with extramedullary relapse remains controversial.17-20 Methotrexate (MTX) is one of the most active agents for the treatment of ALL, and as such is a component of most modern ALL front-line treatment worldwide. In contrast to other antineoplastic drugs, the cytotoxic effects of MTX can be antagonized by a specific antidote, activated folic acid


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(leucovorin). This allows for the use of escalating doses of MTX which are associated with greater antileukemic effects in randomized trials and up-front window studies, as assessed by peripheral blast cytoreduction.21,22 In addition, high dosages of MTX offer the advantage of targeting extramedullary leukemia by producing cytotoxic concentrations in sanctuary sites where low-dose MTX does not readily distribute (e.g., testes, cerebrospinal fluid).10,23 However, the optimal dose of MTX, the adequate duration of the drug-infusion, and the adequate folinic acid rescue remain controversial.1,24-28 While one randomized trial indicated that patients at increased risk of relapse treated initially with high-dose MTX (4 g/m2) had significantly better event-free survival (EFS) rates compared with patients treated with low-dose MTX (40 mg/m2),24 ultra-high dose MTX (12 g/m2) at a 4-hours infusion and an intensive folinic acid rescue was not found to be beneficial in relapsed ALL children, as compared with intermediate-dose MTX (1 g/m2) at a 36 hours infusion and a reduced folinic acid rescue.1,29 Since higher dosages of parenteral MTX are associated with significant toxicity, including long-term neurologic sequelae, and higher costs,30-32 its optimal use is an important question of clinical research. In 1990, the Berlin-Frankfurt-Münster (BFM) Relapse Study Group set out to address this issue in a prospective randomized fashion. The long-term results of this trial, ALL-REZ BFM 90, are the subject of the following report.

METHODS Patients Between July, 1990 and June, 1995, a total of 374 children and adolescents up to 18 years of age with a first relapse of precursor B-cell ALL including any isolated extramedullary relapse irrespectively of the timepoint of relapse and bone marrow (BM) relapses occurring at least 18 months after initial diagnosis were enrolled in the cooperative trial ALL-REZ BFM 90, conducted in 80 hospitals located in Germany, Austria, Switzerland, The Netherlands, Denmark and Russia. Patients with very early bone marrow relapse were excluded from the study and enrolled to experimental trials. All treatment protocols had been approved by the local ethical committee of each participating institution. Informed consent was obtained from the guardians and also patients if applicable.


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The diagnosis of a combined relapse was defined as the presence of at least 5% blast cells in the bone marrow with the presence of extramedullary disease, and isolated bone marrow relapse was defined by the presence of at least 25% blast cells in the bone marrow without evidence of extramedullary disease. Wright-Giemsa-stained BM and peripheral blood smears and cytocentrifuge preparations were subject to central review. Flow cytometric immunophenotyping according to EGIL classification,33 and polymerase chain reaction-based detection of bcr-abl fusion transcripts was done in central laboratories. Relapses were considered very early if they occurred within 18 month after initial diagnosis, early if they occurred after 18 and within 6 months after cessation of frontline therapy, or late if they occurred 6 months after elective cessation of frontline treatment. Patients with early isolated or combined BM relapse were stratified to the treatment group A, those with late isolated or combined BM relapse to group B, and those with any isolated extramedullary relapse to group C.

Treatment After confirmation of diagnosis, children were centrally randomized to receive 1 g/m2 methotrexate (MTX) over 36 h (ID-MTX), or 5 g/m2 over 24 h (HD-MTX), by continuous intravenous infusion during subsequent chemotherapy courses (R1 and R2). Therapy was started with prednisone (100 mg/m2) for five days, followed by alternating courses of polychemotherapy (R1, R2, R3) which are outlined in Table 1. Children with early (group A) or late (group B) isolated or combined BM relapse were scheduled to receive a total of 9 courses (i.e. 6 R1/R2 courses containing ID- or HD-MTX) whereas patients with isolated extramedullary relapse (group C) received 6 courses (i.e. 4 R1/R2 courses containing ID- or HD-MTX). Scheduled intervals between the start of the first 2 courses were two weeks, between all subsequent courses three weeks (figure 1). Ten percent of the MTX dose was administered intravenously over a period of 30 min, and the remaining 90 percent were given during the subsequent 23 1/2 or 35 1/2 hrs in children receiving 5 g/m2 or 1 g/m2, respectively. Fourty-two or 48 hours after initiation of MTX infusion, respectively, activated folic acid was given as rescue at a dosage of 15 mg/m2 every 6 h three or two times, respectively. The dosage was adjusted in cases of inappropriate excretion of MTX. No


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additional rescue was given when MTX serum concentrations had dropped to less than 0.25 µmol/L 54 hrs or later after the end of MTX infusion. To treat subclinical meningeal leukemia, triple intrathecal therapy was administered during each course, consisting of MTX (12 mg), cytarabine (30 mg), and prednisone (10 mg). Children with overt meningeal leukemia received initially 1 – 3 doses of intrathecal therapy until the CSF was cleared from leukemic blasts, and additional triple intrathecal injections at the end of each R2 course. The intensive polychemotherapy courses were followed by cranial irradiation (12 Gy) in children with BM relapse, in patients with overt meningeal leukemia craniospinal irradiation at a dose of 18 Gy was recommended (doses were reduced in children less than 2 years of age or with previous cranial irradiation). Any testis with overt disease was subjected to orchiectomy or local radiotherapy at a dose of 24 Gy, a contralateral clinically not involved testis was irradiated at a dose of 18 Gy, or 15 Gy if leukemic involvement was excluded by biopsy. Maintenance therapy consisted of daily 6-thioguanine (50 mg/m2) and every other week intravenous MTX (50 mg/m2) given for one year in isolated extramedullary disease and for two years in patients with BM relapse (figure 1). Stem cell transplantation (SCT) from an HLA-identical sibling was recommended for patients with isolated or combined BM relapse within 4 years after initial diagnosis after three to five courses of polychemotherapy. In increased-risk patients without a sibling donor, HLA-matched unrelated donors, HLA mismatched family donors or autologous transplants were considered as experimental alternative stem-cell sources during the course of the study. The preferred conditioning regimen was TBI 12 Gy and VP16 60mg/kg, GvHD prophylaxis after allogeneic transplants consisted in short course MTX, and cyclosporin A in most patients.

Statistical Analysis In a blinded fashion, enrolled patients were centrally randomized by the trial coordination center (Department of Pediatric Oncology / Hematology, Charité, Berlin) using a randomization list with equal probabilities for the two protocols. Randomization was stratified according to the treatment groups A/B/C.


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Differences in the distribution of variables among patient groups were assessed by the χ² test for categorical variables, and the Mann-Whitney-U or Kruskal-Wallis tests for continuous variables. Kaplan-Meier life table analysis was used to estimate the probability of event-free survival (EFS) from the start of salvage therapy until the date of death in remission, a second relapse, or a second malignant neoplasm, whichever first. Patients who were not in complete remission (CR) after 3 treatment courses (less than 5% blast cells in an otherwise normocellular marrow), were considered induction failures and censored at time zero. Children lost to follow-up were censored at the date of last contact. For the calculation of relapse-free survival times, the analysis was restricted to children achieving second CR, and all events were censored except for second relapses. Subgroups were compared by the two-sided log-rank test. In all tests, two-sided P values ≥ .05 were regarded as not significant (NS). A sample size calculation indicated a recruitment of 133 patients in each randomized arm to detect a superiority of the HD-MTX arm of 15% at an expected EFS rate of 35% of the ID-MTX arm with a power of 0.8 and a level of significance of 0.05. Multivariate Cox stepwise forward conditional regression analysis was performed to determine statistically significant independent indicators of outcome, including SCT as time-dependent covariate.

RESULTS Of the 374 children recruited during the 5-year trial, 269 children (72%) were randomized to IDMTX (n = 141) or HD-MTX (n = 128). Four patients randomized to the HD-MTX arm and included into the intention to treat analysis received nevertheless ID-MTX due to parents’ decision. Lack of the parents’ consent to subject their children to randomization resulted in 51 children to receive ID-MTX, and 54 children to receive HD-MTX out of the investigative protocol. The age of the randomized children did not differ between the ID-MTX and the HD-MTX group (8.5 / 1.8-17.3 vs. 8.8 / 3.3-18.0 years, median/range, p = .554), nor was there any significant difference with respect to the patients’ sex, time or site of relapse,1 peripheral blast count at relapse,4 immunophenotype33, detection of bcr-abl fusion transcripts34 or front-line treatment by BFM35 or CoALL36 protocols, as well as stem-cell transplantation and cranial irradiation at relapse (Table 2).


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Pro-B immunophenotype was more frequent in the ID-MTX group (10 versus 2 patients). Excluding patients with pro-B immunophenotype does not influence the results with respect to EFS. Events occurring after initiation of relapse treatment did not differ between the two groups (Table 3). This was also true for the rates of second CR (ID-MTX: 94%, HD-MTX: 89%, p = .197). Although the rate of subsequent isolated extramedullary relapses seems to be higher in patients treated with ID-MTX, this difference is not significant with respect to frequencies, or cumulative incidences of subsequent CNS-, testicular-, or any isolated and/or combined extramedullary relapses. Children of the two groups had virtually identical 10-year event-free survival (.36 ± .04 vs .38 ± .04, p = .919) and overall survival (OS) probabilities (.47 ± .04 vs .43 ± .04, p = .633). Kaplan-Meier plots for EFS probability revealed a highly similar temporal pattern of treatment failures in both groups (Figure 2). In contrast, OS probability is about 10 % higher in the ID-MTX group at 5 years, whereas at 15 years the OS probabilities approach nearly the identical level (Fig 3). Regarding SCT as censored event, EFS estimates between both groups where also not significantly different (ID-MTX: pEFS = .39 ± .05. HD-MTX: pEFS = .40 ± .05; p = .957). When data of children were analyzed for received treatment irrespective of the randomization, EFS rates (ID-MTX: n = 196; censored = 73; pEFS = .37 ± .04. HD-MTX: n = 178; censored = 62; pEFS = .35 ± .04; p = .564) did not differ significantly. Three secondary malignancies occurred in the population randomized to the ID-MTX arm: Two patients suffered an osteosarcoma 5.7 and 7.3 years after syngeneic and allogeneic SCT, respectively. A 17 years old girl suffered a myelodysplastic syndrome (MDS) 2.6 years after relapse diagnosis. Out of 71 patients of the IDMTX arm and 58 patients of the HD-MTX arm with subsequent relapse, 11 and 6 patients, respectively, are alive in 3rd CR (p = .455). The dose of MTX had no influence on results after SCT (p = .942) with comparable rates of treatment-related deaths (ID, n = 4; HD, n = 3) or subsequent relapses (ID, n = 15; HD, n = 12). Subgroup analyses did not reveal a prognostic impact of the MTX dose in the following categories: sex (male, p = .892; female, p = .744), time point of relapse (very early, p = .194; early, p = .957; late, p = .433), site of relapse (BM isolated, p = .90; BM combined, p = .593; isolated extramedullary, p = .765). Furthermore, the cumulative dose of intravenous (IV) MTX during frontline therapy had no impact of the efficacy of MTX at different dosages at relapse: EFS was not


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statistically different in 10 patients having received less than 20 g/m² IV MTX (p = .688), nor in 210 patients having received 20 g/m² as scheduled in the standard protocol M of the ALL-BFM studies for most patients (p = .665) or in 26 patients having received more than 20 g/m² IV MTX (p = .566). In 23 patients, the data on the cumulative IV MTX dosage at frontline therapy was not available. Multivariate Cox regression analysis including SCT as time-dependent covariate revealed time point (p < .001; results in univariate analysis: very early, n = 10, pEFS = .40 ± .16; early, n = 112, pEFS = .21 ± .04; late, n = 147, pEFS = .48 ± .04; p < .001) and site of relapse (p < .001; results in univariate analysis: isolated BM, n = 156, pEFS = .29 ± .04; combined BM n = 68, pEFS = .46 ± .06; isolated extramedullary, n = 45, pEFS = .50 ± .08; p = .010) as the only significant independent predictors for event-free survival, while sex (p = .666), age at relapse (p = .714), immunophenotype (missing values and 1 patient with biphenotypic ALL excluded; p = .761), peripheral blast count (p = .102), MTX dosage (p = .487) and SCT (p = .116) did not show an independent prognostic relevance.

DISCUSSION We report on a randomized trial ALL-REZ BFM 90 comparing the efficacy of MTX administered at a dose of 5 g/m² over 24 hrs followed by 3 scheduled doses of folinic acid versus MTX at a dose of 1 g/m² over 36 hrs followed by 2 doses of folinic acid in the treatment of childhood relapsed precursor B-cell ALL. Very high risk patients with very early bone marrow relapse and those with T-lineage ALL have been excluded and treated with individual protocols. MTX was part of alternating multi-drug chemotherapy courses. We did not find a difference in event-free and overall survival between the randomized groups, or in site or time patterns of subsequent relapses.

The antimetabolic effect of MTX and MTX-polyglutamates (MTX-PG) is mainly based on the inhibition of dihydrofolic acid reductase (DHFR), which reduces dihydrofolates to tetrahydrofolates as essential carriers of one-carbon groups in the synthesis of nucleotides and thymidilates. In addition, MTX-PG inhibit two enzymes involved in the de novo purine synthesis, 5’phosphoribosylgycinamid transformylase and aminoimidazole carboxamide transformylase.37,38


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Active transmembrane transport of MTX is regulated by the reduced folate carrier (RFC) and to a minor extent also by a folate receptor. At high serum concentrations, passive diffusion of the drug becomes an additional pathway for intracellular uptake of MTX.39 Hepatic and intracellular polyglutamination are important for the antileukemic activity since polyglutamates with more than 3 glutamyl residues have a higher affinity to the target enzymes and are retained intracellularly as active metabolites for prolonged periods of time.40 Polyglutamination is catalyzed by folylpolyglutamate sythetase (FPGS), polyglutamates are cleaved by folylpolyglutamate hydrolase (FPGH).41 Several mechanisms of resistance to MTX have been described: Impaired transmembrane transport due to decreased RFC activity42, elevated levels of DHFR and amplification of the DHFR gene43,44, impaired polyglutamination45 and increased activity of FGPH.46 For relapsed lymphoblastic leukemias, a 3-fold higher resistance to MTX measured in the in situ thymidilate synthetase inhibition assay as compared to initial ALL has been reported.47 In vitro studies on a series of antileukemic drugs revealed a 0.8 to 1.9 fold higher resistance of relapse ALL samples as compared to primary ALL samples to L-asparaginase, anthracyclines, and thiopurines and a comparable resistance to vinca-alkaloids, cytarabine, ifosfamide, and epipodophyllotoxins. Only resistance to glucocorticoids was more than 24-fold higher in relapse as compared to primary ALL-samples.48 Hence, at relapse sensitivity to MTX is substantially more impaired than to most other antileukemic drugs. Most likely, the resistance of relapse ALL samples towards MTX can be explained by an increased activity of DHFR44 as a consequence of gene amplification.43 In individual patients, lack of cytotoxicity was found despite complete inhibition of thymidylate synthetase. Therefore, an alternative pathway circumventing the thymidylate synthetase has been postulated as an additional mechanism of resistance.49

Several factors of the treatment design may influence the antileukemic efficacy of MTX: The dosage, the duration of administration, and the intensity of folinic acid rescue.50 Higher doses of MTX are administered using shorter infusion times, since the duration of exposure to toxic serum levels seems to be the major factor determining the toxicity.51,52 In 1993, the ALL-


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REZ BFM group had already reported on a randomized comparison between MTX at a dose of 1 g/m² administered over 24 hrs followed by 2 doses of folinic acid and 12 g/m² infused over 4 hrs with 12 doses of folinic acid.29 The median serum concentration during MTX infusion was more than 700µM/L in the high dose arm and 7.2 µM/L in the intermediate dose arm. Concentrations greater than 1 µmol/L were maintained for 36 hours with HD-MTX 12g/m² and 45 hours with IDMTX 1 g/m². EFS and survival were not significantly different but the toxicity was even higher in the ID arm due to the substantially longer infusion duration with longer lasting serum concentrations above 1 µM/L and probably also to the fact that the rescue regimens were different. Several studies have shown that ID-MTX at a dose of 1g/m² leads to higher intracellular concentrations of MTX and MTX-PG and subsequently to a better EFS than low-dose regimens.21 However, at higher doses, the intracellular uptake of MTX is not proportional to the serum concentration due to a limited capacity of the active mechanisms via RFC and FR and a limited passive infusion.53 In contrast, in the CNS the MTX level is correlated with the mean serum concentration of the drug.54 Hence, at steady state serum concentrations of even > 20µM/L the antileukemic effect of MTX is rather related to the higher levels in sanctuary sites like the CNS and the testes than to an increased intracellular accumulation of MTX and MTX-PG.53,55 This may be the explanation why the arm with the 5-fold higher MTX-dose did not lead to fewer subsequent BM relapses. Although the rate of subsequent isolated extramedullary relapses was higher in the ID-MTX group (16%) as compared to the HD-MTX group (5%) which would fit to the hypothesis of a better protection in extracompartimental sites with higher serum levels, this difference was not significant. Thus, our study design provided obviously sufficient protection to extracompartments even with ID-MTX.22 Infusion duration may be equally important for the antileukemic efficacy as the dosage. It is of note that the higher resistance towards MTX in relapsed ALL-samples could not be overcome by prolongation of in vitro exposure to the drug.48,56 Therefore, it would be an interesting question, whether 1 g/m² MTX administered over only 24 hrs would not be sufficient to exploit the


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antileukemic potential of MTX in this stadium of the disease. Until now, no prospective studies have been conducted investigating the importance of infusion duration of HD-MTX. The here reported trial does not allow to assess the isolated effect of the infusion duration because of the different doses of MTX. However, this question is being addressed in the ongoing trial Total XV performed at the St. Jude Children's Research Hospital, by giving 1 g/m² MTX upfront randomly at a 4 versus 24 hrs infusion.57 Toxicity of HD-MTX is usually limited to a moderate myelosuppression, reversible mucositis and moderate hepatic or renal dysfunction. It has been substantially reduced via hydration, urinary alkalization, and monitoring of renal function and methotrexate serum concentrations.58 In case of impaired elimination, an escalation of the leucovorin rescue, or enzymatic cleavage of the drug by carboxypetidase in selected cases, have succeeded in almost eliminating fatal toxicities.59,60 Transient neurotoxicity has been reported after diverse doses and application ways of MTX, although there are rare cases of severe encephalopathy occurring after HD-MTX.30,61-63 Thus, the toxicity profile of HD- or ID-MTX proves favorable in the context of intensive multidrug chemotherapy with myelosuppression as the prominent and dose-limiting side effect. Toxicity data on MTX therapy at several dosages had been collected in prior ALL-REZ BFM trials and have been published.29 Therefore, a detailed documentation of these data was not performed at the trial ALLREZ BFM 90.

The long-term results of the trial reported here demonstrate that more than 35 % of patients with relapsed precursor B-cell ALL (excluding very early bone marrow relapses) can be salvaged. This is in line with results reported by other collaborative trials.6,12,16,20,64 As some patients with a subsequent relapse can achieve even a long-lasting complete third remission, the overall survival rates actually exceed 45%. However, since very high risk patients (very early BM relapse, any Tlineage relapse) were excluded from the randomization, the results are not representative for an unselected population of children with ALL relapse. The dynamics of the Kaplan-Meier plot reveal


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a cascade of subsequent relapses with the latest relapse occurring 7.3 years after diagnosis. The rate of second malignancies is negligible in our patient cohort, 2 of them occurred after allogneic SCT. Nevertheless, still more than half of the patients suffer a subsequent relapse. It is the most important objective of ongoing trials to predict more precisely, which individual patient is at high risk for treatment failure and would therefore need an intensification of postremission therapy by allogeneic SCT, and which patients can be cured with chemo-/radiotherapy alone.65,66 Although SCT as timedependent covariate was not a significant prognostic factor for EFS in multivariate analysis, this trial does not allow to assess conclusively the role of allogeneic SCT in relapsed ALL due to its rather uncontrolled use. Although most successful protocols for treatment of ALL have incorporated ID- or HD-MTX, some studies show excellent results even for high-risk patients with intensive chemotherapy not including high dose MTX.67 The chosen study design does not permit to assess the overall impact of MTX in patients with relapsed ALL because it has been used in combination with intensive multidrug chemotherapy. However, since the results of this trial could not prove an advantage of HD over ID MTX but also not disprove the role of ID MTX in childhood relapsed ALL at all, we decided to use MTX at a dose of 1 g/m² over 36 hrs infusion as standard treatment in all subsequent ALL-REZ BFM trials.


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ACKNOWLEDGMENT

We are grateful to all nurses, physicians, technologists, and other support staff members involved in the BFM relapse trials. Special thanks to Marianne Löwke and Constanze Sukopp for laboratory assistance, and to Sabine Brühmüller, Andrea Kretschmann, and Steffanie Schober for preparing the data of this study.

All authors declare not to have any conflicting financial interests with the content of the article Explanation of authors contribution: Arend von Stackelberg wrote the paper and performed part of the data analysis; Reinhard Hartmann designed the statistical part of the trial and performed the data analysis; Christoph Bührer wrote part of the paper and reviewed it; Rüdiger Fengler designed the clinical part of the trial; Gritta Janka-Schaub warranted data exchange with the COALL frontline trial and reviewed the manuscript; Alfred Reiter warranted the data exchange with the ALL-BFM trial and reviewed the transplantation related data; Georg Mann reviewed the data of the Austrian patients; Kjeld Schmiegelow was national co-ordinator of the Danish patients and reviewed the paper; Richard Ratei was responsible for reference immunophenotyping and data transfer/analysis of corresponding results; Thomas Klingebiel was co-ordinator of stem-cell transplantation and reviewed the corresponding data; Jörg Ritter was contributing to the clinical design of the trial and was critically reviewing the paper; Günter Henze was responsible for the trial design and conduction of the trial as chair of the study group and has in part written/reviewed the manuscript. This work was kindly supported by Deutsche Krebshilfe, Bonn, Germany; the Deutsche Kinderkrebsstiftung and the BMBF (Federal Ministry of Education and Research, Germany)


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10. Bührer C, Henze G, Hofmann J, et al. Central nervous system relapse prevention in 1165 standard-risk children with acute lymphoblastic leukemia in five BFM trials. Haematol Blood Transfus. 1990;33:500-503. 11. Ritchey AK, Pollock BH, Lauer SJ, et al. Improved survival of children with isolated CNS relapse of acute lymphoblastic leukemia: A pediatric oncology group study. J Clin Oncol. 1999;17:3745-52. 12. Lawson SE, Harrison G, Richards S, et al. The UK experience in treating relapsed childhood acute lymphoblastic leukaemia: A report on the Medical Research Council UKALLR1 study. Br J Haematol. 2000;108:531-543. 13. Wofford MM, Smith SD, Shuster JJ, et al. Treatment of occult or late overt testicular relapse in children with acute lymphoblastic leukemia: A Pediatric Oncology Group study. J Clin Oncol. 1992;10:624-30. 14. Wolfrom C, Hartmann R, Brühmüller S, et al. Similar outcome on boys with isolated and combined testicular acute lymphoblastic leukemia relapse after stratified BFM salvage therapy. Haematol Blood Transfus. 1997;38:647-651. 15. Grundy RG, Leiper AD, Stanhope R, et al. Survival and endocrine outcome after testicular relapse in acute lymphoblastic leukaemia. Arch Dis Child. 1997;76:190-6. 16. Gaynon PS, Qu RP, Chappell RJ, et al. Survival after relapse in childhood acute lymphoblastic leukemia: Impact of site and time to first relapse - the Children's Cancer Group Experience. Cancer. 1998;82:1387-1395. 17. Barrett AJ, Horowitz MM, Pollock BH, et al. Bone marrow transplants from HLA-identical siblings as compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission. N Engl J Med. 1994;331:1253-8.


16 18. Dopfer R, Henze G, Bender-Götze C, et al. Allogeneic bone marrow transplantation for childhood acute lymphoblastic leukemia in second remission after intensive primary and relapse therapy according to the BFMand CoALL-protocols: Results of the German Cooperative Study. Blood. 1991;78:2780-4. 19. Borgmann A, von Stackelberg A, Hartmann R, et al. Unrelated donor stem cell transplantation compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission: a matched-pair analysis. Blood. 2003;101:3835. 20. Wheeler K, Richards S, Bailey C, et al. Comparison of bone marrow transplant and chemotherapy for relapsed childhood acute lymphoblastic leukaemia: The MRC UKALL X experience. Medical Research Council Working Party on Childhood Leukaemia. Br J Haematol. 1998;101:94-103. 21. Masson E, Relling MV, Synold TW, et al. Accumulation of methotrexate polyglutamates in lymphoblasts is a determinant of antileukemic effects in vivo. A rationale for high-dose methotrexate. J Clin Invest. 1996; 97:73-80. 22. Clarke M, Gaynon P, Hann I, et al. CNS-directed therapy for childhood acute lymphoblastic leukemia: Childhood ALL Collaborative Group overview of 43 randomized trials. J Clin Oncol. 2003;21:1798-809. 23. Brecher ML, Weinberg V, Boyett JM, et al. Intermediate dose methotrexate in childhood acute lymphoblastic leukemia resulting in decreased incidence of testicular relapse. Cancer. 1986;58:1024-1028. 24. Niemeyer CM, Gelber RD, Tarbell NJ, et al. Low-dose versus high-dose methotrexate during remission induction in childhood acute lymphoblastic leukemia (Protocol 81-01 update). Blood. 1991;78:2514-2519. 25. Chessells JM, Bailey C, Richards SM. Intensification of treatment and survival in all children with lymphoblastic leukaemia: results of UK Medical Research Council trial UKALL X. Medical Research Council Working Party on Childhood Leukaemia. Lancet. 1995;345:143-148. 26. Veerman AJ, Hahlen K, Kamps WA, et al. High cure rate with a moderately intensive treatment regimen in nonhigh-risk childhood acute lymphoblastic leukemia. Results of protocol ALL VI from the Dutch Childhood Leukemia Study Group. J Clin Oncol. 1996;14:911-918. 27. Pui CH, Simone JV, Hancock ML, et al. Impact of three methods of treatment intensification on acute lymphoblastic leukemia in children: long-term results of St Jude total therapy study X. Leukemia. 1992;6:150157. 28. Lange BJ, Blatt J, Sather HN, Meadows AT. Randomized comparison of moderate-dose methotrexate infusions to oral methotrexate in children with intermediate risk acute lymphoblastic leukemia: a Childrens Cancer Group study. Med Pediatr Oncol. 1996;27:15-20. 29. Wolfrom C, Hartmann R, Fengler R, et al. Randomized comparison of 36-hour intermediate-dose versus 4-hour high-dose methotrexate infusions for remission induction in relapsed childhood acute lymphoblastic leukemia. J Clin Oncol. 1993; 11:827-833. 30. Ochs J, Mulhern R, Fairclough D, et al. Comparison of neuropsychologic functioning and clinical indicators of neurotoxicity in long-term survivors of childhood leukemia given cranial radiation or parenteral methotrexate: a prospective study. J Clin Oncol. 1991;9:145-151. 31. Waber DP, Tarbell NJ, Kahn CM, et al. The relationship of sex and treatment modality to neuropsychologic outcome in childhood acute lymphoblastic leukemia. J Clin Oncol. 1992;10:810-817. 32. Waber DP, Tarbell NJ, Fairclough D, et al. Cognitive sequelae of treatment in childhood acute lymphoblastic leukemia: cranial radiation requires an accomplice. J Clin Oncol. 1995;13:2490-2496. 33. Bene MC, Castoldi G, Knapp W, et al. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia. 1995;9:1783-1786. 34. Beyermann B, Agthe AG, Adams HP, et al. Clinical features and outcome of children with first marrow relapse of acute lymphoblastic leukemia expressing BCR-ABL fusion transcripts. BFM Relapse Study Group. Blood. 1996;87:1532-1538.


17 35. Schrappe M, Reiter A, Zimmermann M, et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Munster. Leukemia. 2000;14:22052222. 36. Harms DO, Janka-Schaub GE. Co-operative study group for childhood acute lymphoblastic leukemia (COALL): long-term follow-up of trials 82, 85, 89 and 92. Leukemia. 2000;14:2234-2239. 37. Allegra CJ, Drake JC, Jolivet J, et al. Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates. Proc Natl Acad Sci U S A. 1985;82:4881-4885. 38. Allegra CJ, Chabner BA, Drake JC, et al. Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates. J Biol Chem. 1985;260:9720-9726. 39. Spinella MJ, Brigle KE, Sierra EE, et al. Distinguishing between folate receptor-alpha-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells. J Biol Chem. 1995;270:7842-7849. 40. Whitehead VM, Vuchich MJ, Lauer SJ, et al. Accumulation of high levels of methotrexate polyglutamates in lymphoblasts from children with hyperdiploid (greater than 50 chromosomes) B-lineage acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood. 1992;80:1316-1323. 41. Barrueco JR, O'Leary DF, Sirotnak FM. Metabolic turnover of methotrexate polyglutamates in lysosomes derived from S180 cells. Definition of a two-step process limited by mediated lysosomal permeation of polyglutamates and activating reduced sulfhydryl compounds. J Biol Chem. 1992;267:15356-15361. 42. Gorlick R, Goker E, Trippett T, et al. Defective transport is a common mechanism of acquired methotrexate resistance in acute lymphocytic leukemia and is associated with decreased reduced folate carrier expression. Blood. 1997;89:1013-1018. 43. Goker E, Waltham M, Kheradpour A, et al. Amplification of the dihydrofolate reductase gene is a mechanism of acquired resistance to methotrexate in patients with acute lymphoblastic leukemia and is correlated with p53 gene mutations. Blood. 1995;86:677-684. 44. Matherly LH, Taub JW, Wong SC, et al. Increased frequency of expression of elevated dihydrofolate reductase in T-cell versus B-precursor acute lymphoblastic leukemia in children. Blood. 1997;90:578-589. 45. Pizzorno G, Mini E, Coronnello M, et al. Impaired polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after short-term, high-dose treatment with this drug. Cancer Res. 1988;48:2149-2155. 46. Rots MG, Pieters R, Peters GJ, et al. Role of folylpolyglutamate synthetase and folylpolyglutamate hydrolase in methotrexate accumulation and polyglutamylation in childhood leukemia. Blood. 1999;93:1677-1683. 47. Rots MG, Pieters R, Peters GJ, et al. Methotrexate resistance in relapsed childhood acute lymphoblastic leukaemia. Br J Haematol. 2000;109:629-634. 48. Klumper E, Pieters R, Veerman AJ, et al. In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia. Blood. 1995;86:3861-3868. 49. Weigand M, Frei E, Graf N, et al. Mechanisms of resistance to methotrexate in childhood acute lymphoblastic leukemia: circumvention of thymidylate synthase inhibition. J Cancer Res Clin Oncol. 1999;125:513-519. 50. Skärby TV, Anderson H, Heldrup J, et al; Nordic Society of Paediatric Haematology and Oncology (NOPHO). High leucovorin doses during high-dose methotrexate treatment may reduce the cure rate in childhood acute lymphoblastic leukemia. Leukemia. 2006;20:1955-62. 51. Goldie JH, Price LA, Harrap KR. Methotrexate toxicity: correlation with duration of administration, plasma levels, dose and excretion pattern. Eur J Cancer. 1972;8:409-414.


18 52. Nathan PC, Whitcomb T, Wolters PL, et al. Very high-dose methotrexate (33.6 g/m(2)) as central nervous system preventive therapy for childhood acute lymphoblastic leukemia: results of National Cancer Institute/Children's Cancer Group trials CCG-191P, CCG-134P and CCG-144P. Leuk Lymphoma. 2006;47:2488-504. 53. Brenner T, Evans W. Rationale for High-Dose Methotrexate in Childhood Acute Lymphoblastic Leukemia. In: Pui CH, ed. Treatment of Acute Leukemias New Directions for Clinical Research. Totova, USA: Humana Press; 2002:339 - 356. 54. Shapiro WR, Young DF, Mehta BM. Methotrexate: distribution in cerebrospinal fluid after intravenous, ventricular and lumbar injections. N Engl J Med. 1975;293:161-166. 55. Evans WE, Crom WR, Abromowitch M, et al. Clinical pharmacodynamics of high-dose methotrexate in acute lymphocytic leukemia. Identification of a relation between concentration and effect. N Engl J Med. 1986;314:471477. 56. Rots MG, Pieters R, Peters GJ, et al. Circumvention of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates. Blood. 1999;94:3121-3128. 57. Pui CH, Relling MV, Sandlund JT, et al. Rationale and design of Total Therapy Study XV for newly diagnosed childhood acute lymphoblastic leukemia. Ann Hematol. 2004;83 Suppl 1:S124-126. 58. Relling MV, Fairclough D, Ayers D, et al. Patient characteristics associated with high-risk methotrexate concentrations and toxicity. J Clin Oncol. 1994;12:1667-1672. 59. Widemann BC, Balis FM, Murphy RF, et al. Carboxypeptidase-G2, thymidine, and leucovorin rescue in cancer patients with methotrexate-induced renal dysfunction. J Clin Oncol. 1997;15:2125-2134. 60. Buchen S, Ngampolo D, Melton RG, et al. Carboxypeptidase G2 rescue in patients with methotrexate intoxication and renal failure. Br J Cancer. 2005 14;92:480-7 61. Rubnitz JE, Relling MV, Harrison PL, et al. Transient encephalopathy following high-dose methotrexate treatment in childhood acute lymphoblastic leukemia. Leukemia. 1998;12:1176-1181. 62. Spiegler BJ, Kennedy K, Maze R, et al. Comparison of long-term neurocognitive outcomes in young children with acute lymphoblastic leukemia treated with cranial radiation or high-dose or very high-dose intravenous methotrexate. J Clin Oncol. 2006;24:3858-64 63. Reddick WE, Glass JO, Helton KJ, et al. Prevalence of leukoencephalopathy in children treated for acute lymphoblastic leukemia with high-dose methotrexate. AJNR Am J Neuroradiol. 2005;26:1263-9 64. Schroeder H, Garwicz S, Kristinsson J, et al. Outcome after first relapse in children with acute lymphoblastic leukemia: A population-based study of 315 patients from the Nordic Society of Pediatric Hematology and Oncology (NOPHO). Med Pediatr Oncol. 1995;25:372-378. 65. Eckert C, Biondi A, Seeger K, et al. Prognostic value of minimal residual disease in relapsed childhood acute lymphoblastic leukaemia. Lancet. 2001;358:1239-1241. 66. Henze G, von Stackelberg A. Treatment of Relapsed Acute Lymphoblastic Leukemia. In: Pui CH, ed. Treatment of Acute Leukemias New Directions for Clinical Research. Totova, USA: Humana Press; 2002:199-219. 67. Nachman JB, Sather HN, Sensel MG, et al. Augmented post-induction therapy for children with high-risk acute lymphoblastic leukemia and a slow response to initial therapy. N Engl J Med. 1998;338:1663-1671.


19

Table 1. Drugs and dosing of alternating polychemotherapy courses R1-3 Drug

Dose

Route

Given on Days

Dexamethasone

20 mg/m2

PO

1-5

6-Mercaptopurine

100 mg/m2

PO

1-5

IV

1, 6

IV

1

Course R1

Vincristine

2

1.5 mg/m

2

Methotrexate*

1 or 5 g/m

Methotrexate

12 mg

IT

1

Cytarabine

30 mg

IT

1

Prednisone

10 mg

IT

1

Cytarabine

2 g/m2 q 12 h

IV

5

IM/IV

6

PO

1-5

PO

1-5

L-Asparaginase

25 000 U/m

Dexamethasone

20 mg/m2

2

Course R2 6-Thioguanine

100 mg/m

2

2

IV

1

IV

1

12 mg

IT

1 (and 5)**

Cytarabine

30 mg

IT

1 (and 5)**

Prednisone

10 mg

IT

1 (and 5)**

IV

5

Vindesine

3 mg/m

Methotrexate*

1 or 5 g/m

Methotrexate

Daunorubicin

50 mg/m

2

2 2

Ifosfamide

400 mg/m

IV

1-5

L-Asparaginase

25 000 U/m2

IM/IV

6

Dexamethasone

20 mg/m2

PO

1-5

IV

1, 2

IV

3-5

Course R3 Cytarabine Etoposide

2

2 g/m q 12 h 150 mg/m

2

Methotrexate

12 mg

IT

5

Cytarabine

30 mg

IT

5

Prednisone

10 mg

IT

5

IM/IV

6

L-Asparaginase

25 000 U/m

2

PO, by mouth; IV, intravenously; IT, intrathecally; IM, intramuscularly. * MTX 1 g/m² and 5 g/m² at a 36 and 24 hours infusion, respectively; ** children with overt meningeal leukemia.


20

Table 2. Base-line patient characteristics and therapeutic parameters of the 269 randomized patients receiving intermediate-dose (ID-MTX) or high-dose methotrexate (HD-MTX). Total

ID-MTX

HD-MTX

N

%

N

%

N

%

269

(100)

141

(52)

128*

(48)

Male Female

174 95

65 35

89 52

63 37

85 43

66 34

Age at relapse < 5 years ≥ and < 10 years ≥ 10 years

26 143 100

10 53 37

13 79 49

9 56 35

13 64 51

10 50 40

Time point of relapse** Very early early Late

10 112 147

4 42 55

7 62 72

5 44 51

3 50 75

2 39 59

156 68

58 25

85 32

60 23 66 28

71 36 14 17 2 1 2 21 9 11 1

56 28

Total

P

Clinical parameters Sex

.611

.610

.313

Site

.587 Bone marrow isol. BM combined + CNS + Testis + CNS/Testis + CNS/Other + Other Isolated extramedullary CNS Testis CNS/Testis Other

52 38 3 2 6

35 26 2 1 4 17

45

21 9 2 24

40 56 2 2

18 25 1 1

9 14 1 -

6 17 38 58 4

.156 39 47 6 3 6

16

.538 43 52 5

Peripheral blast cell count < 1/µl 1 - < 10.000/µl ≥ 10.000/µl No data

93 142 32 2

35 53 12 (.7)

49 72 19 1

35 41 15 (.7)

44 70 13 1

35 55 10 (.8)

.673

BCR/ABL fusion transcript Positive Negative No data

11 115 143

9 91 (53)

6 73 62

8 92 (44)

5 42 81

11 89 (63)

Immunophenotype*** Pro B Common ALL Pre B Biphenotypic No data

12 191 58 1 7

5 73 22 .4 (3)

10 95 33 3

7 69 24 (2)

2 96 25 1 4

2 77 20 .8 (3)

179 31 59

67 12 22

98 16 27

70 12 19

81 15 32

63 12 25

88 94 37 46 4

33 36 14 17 (2)

47 45 21 24 4

34 33 15 18 (3)

41 49 16 22 -

32 38 13 17

214 32 9 4 10

80 12 3 2 4

110 17 7 1 6

78 12 5 1 4

104 15 2 3 4

81 12 2 2 3

.746

.082

Treatment Frontline protocol ALL-BFM COALL Other Irradiation at relapse None Cranial Craniospinal TBI No data Stem cell transplantation in CR2 None Matched family donor Unrelated donor Mismatched family donor Autologous

.887

.792

.426


21 Legend to table 2: * 4 Patients randomized to treat with HD-MTX received ID-MTX due to parental decision;** very early, within 18 month after primary diagnosis; early, after 18 months post primary diagnosis and within 6 months after elective cessation of front-line therapy; late, later than 6 months after elective cessation of 33 therapy; *** immunophenotype classified according to EGIL. BM, bone marrow; BFM, Berlin – Frankfurt – Münster Study Group; CNS, central nervous system; COALL, Cooperative ALL Study Group; CR2, 2nd complete remission; TBI, total body irradiation. Missing values are not included into the caluclation of percentages P-values.

Table 3. Treatment results in 269 randomized patients receiving intermediate-dose (ID-MTX) or highdose methotrexate (HD-MTX). Total Total Induction death Nonresponse Complete Remission achieved Lost to follow up Therapy related death Secondary malignancy Relapse

ID-MTX

HD-MTX

N

%

N

%

N

%

P

269

(100)

141

(52)

128

(48)

.275

8

3

4

3

4

3

15

6

5

4

10

8

246

91

132

94

114

89

2

1

-

2

2

15

6

8

7

6

6

3

1

3

2

-

129

48

71

50

58

.197

45

Site of relapse Bone marrow isolated BM combined + CNS + Testis + Other Isolated extramedullary CNS Testis Complete continuous Remission

.171 105 10 1 7 2 14 9 5 97

81 8 10 70 20 11 64 36 36

55 5 1 3 1 11 6 2 50

Legend to table 3: BM, bone marrow; CNS, central nervous system.

76 7 20 60 20 16 45 55

50 5

36

47

86 9 4 1 3 3 -

80 20 5 100 37

.565

.258


22

Figure 1

Group A/B

CP R1

R2

R3

R1

R2

R3

C

CP R1

R2

R3

R1

R2

R3 ↓ M 12

3

6

9

12

15

Week*

1

R1

18

R2

R3 ↓ M 24

21

24

27

Figure 1: Design of study ALL-REZ BFM 90. A, early isolated/combined bone marrow relapse; B, late isolated/combined bone marrow relapse; C, isolated extramedullary relapse (A/B/C, strategic groups); CP, cytoreductive prophase with prednisone; R1 / R2 /R3, multiagent chemotherapy courses (as described in Tab. 1); ↓ , cranial radiation therapy; M 12 / 24, maintenance therapy - duration in months. * Continuation of the next chemotherapy strictly according to the time schedule, as long as no remission was achieved. After achievement of a 2nd CR, the following criteria were mandatory to proceed with the next course: leukoytes > 2/nl; neutrophiles > .5/nl; platelets > 80/nl.


23

Figure 2

1.0

Probability

.8 .6 .4 .2 0.0 0

5

10

15

Years after relapse diagnosis

Figure 2. Kaplan-Meier event-free survival (EFS) estimates of randomized children receiving intermediate-dose (1 g/m2, solid line, n = 141; censored = 50; pEFS = 0.36 ± 0.04) or high-dose (5 g/m2, dashed line, n = 128; censored = 49; pEFS = 0.38 ± 0.04) methotrexate (p = 0.919).


24

Figure 3

1.0

Probability

.8 .6 .4 .2 0.0 0

5

10

15

Years after relapse diagnosis

Figure 3. Kaplan-Meier overall survival (OS) estimates at 10 years of randomized children receiving intermediate-dose (1 g/m2, solid line, n = 141; censored = 63; pOS = 0.47 ± 0.04) or high-dose (5 g/m2, dashed line, n = 128; censored = 55; pOS = 0.43 ± 0.04) methotrexate (p = 0.633).