Through inhibition of purine de novo synthesis and enhancement of 6-mercaptopurine (6MP) bioavailability high-dose methotrexate (HDM) may increase the incorporation into DNA of 6-thioguanine nucleotides (6TGN), the cytoxic metabolites of 6MP. Thus, coadministration of 6MP could increase myelotoxicity following HDM. Twenty-one children with standard risk (SR) and 25 with intermediate risk (IR) acute lymphoblastic leukemia (ALL) were studied. During consolidation therapy they received either three courses of HDM at 2 week intervals without concurrent oral 6MP (SR-ALL) or four courses of HDM given at 2 week intervals with 25 mg/m2 of oral 6MP daily (IR-ALL). During the first year of maintenance with oral 6MP (75 mg/m2/day) and oral MTX (20 mg/m2/week) they all received five courses of HDM at 8 week intervals. In all cases, HDM consisted of 5000 mg of MTX/m2 given over 24 h with intraspinal MTX and leucovorin rescue. Erythrocyte levels of 6TGN (E-6TGN) and methotrexate (E-MTX) were, on average, measured every second week during maintenance therapy. When SR consolidation (6MP: 0 mg), IR consolidation (6MP: 25 mg/m2), and SR/IR maintenance therapy (6MP: 75 mg/m2) were compared, white cell and absolute neutrophil count (ANC) nadir, lymphocyte count nadir, thrombocyte count nadir, and hemoglobin nadir after HDM decreased significantly with increasing doses of oral 6MP. Three percent of the HDM courses given without oral 6MP (SR consolidation) were followed by an ANC nadir <0.5 × 109/l compared to 50% of the HDM courses given during SR/IR maintenance therapy. Similarly, only 13% of the HDM courses given as SR-ALL consolidation induced a thrombocyte count nadir <100 × 109/l compared to 58% of the HDM courses given during maintenance therapy. The best-fit model to predict the ANC nadir following HDM during maintenance therapy included the dose of 6MP prior to HDM (β = −0.017, P = 0.001), the average ANC level during maintenance therapy (β = 0.82, P = 0.004), and E-6TGN (β = −0.0029, P = 0.02). The best-fit model to predict the thrombocyte nadir following HDM during maintenance therapy included only mPLATE (β = 0.0057, P = 0.046). In conclusion, the study indicates that reductions of the dose of concurrently given oral 6MP could be one way of reducing the risk of significant myelotoxicity following HDM during maintenance therapy of childhood ALL.
Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood. During maintenance therapy of ALL with oral 6-mercaptopurine (6MP) and methotrexate (MTX), 6-thioguanine nucleotides (6TGN) and MTX polyglutamates accumulate in erythrocytes (E-6TGN and E-MTX), and E-6TGN and E-MTX have been related to the cure rate.12 6TGN are the primary mediators of the cytotoxic effect of 6MP through their incorporation into DNA.3 Another important metabolic pathway is the methylation by the enzyme thiopurine methyltransferase (TPMT, E.C. 184.108.40.206) of 6MP and some of its metabolites such as 6-thioinosine 5′-monophosphate.4 As a result of a common genetic polymorphism, large interindividual variations in TPMT activity exist, and this significantly influences the degree of methylation and intracellular 6TGN accumulation.56
High-dose methotrexate (HDM) is an important part of the therapy given to children with ALL during consolidation therapy with or without concurrent oral 6MP and as re-inductions during maintenance therapy with oral daily 6MP and weekly methotrexate (MTX) as the backbone.789 HDM often causes significant bone marrow toxicity which carries a risk of infections and a need for transfusions.10 This myelosuppression may lead to treatment interruptions and a reduction of the dose intensity, which can affect the cure rate.111213 MTX may increase the bioavailability of 6MP,14 and in addition, through inhibition of de novo purine synthesis, enhance the availability of 6TGN and its incorporation into DNA.15 We speculated that this drug interaction could in part be responsible for the bone marrow toxicity which follows HDM given during 6MP/MTX maintenance therapy. In the present study, we demonstrate that the dose of concurrently given oral 6MP and the individual variations in 6MP metabolism significantly influence the degree of myelotoxicity following HDM.
Patients and methods
The Nordic NOPHO ALL-92 protocol began on 1 January 1992.16 Patients were eligible for the present study and for statistical analyses, if they: (1) were diagnosed with standard risk (SR) or intermediate risk (IR) non-B cell ALL after 1 January 1992, at the University Hospital, Rigshospitalet, Copenhagen; (2) were treated according to the NOPHO ALL-92 program; (3) entered 6MP/MTX maintenance therapy before 1 January 1997, and (4) were in first remission when the study was closed, 1 January 1999. Fifty patients fulfilled these criteria. Four patients were excluded because they: (1) received only two courses of HDM due to severe neurotoxicity (n = 1); (2) were treated elsewhere (n = 2); or (3) data were lacking (n = 1). The remaining 30 boys and 16 girls included 21 cases of SR- and 25 cases of IR-ALL. The SR/IR risk classification depended on age and white blood cell count (WBC) (SR: age 2.0–9.9 years and WBC <10 × 109/l; IR: age 1.0–1.9 years or age 10.0–14.9 years and/or WBC 10–49 × 109/l) and the absence of high risk criteria (WBC 50 × 109/l, T cell disease, mediastinal or CNS or testicular or lymphomatous disease, t(4;11), t(9;22), a day 14 bone marrow with more than 25% lymphoblasts, or a day 29 bone marrow with more than 5% lymphoblasts).
Induction therapy consisted of prednisolone (60 mg/m2/day divided into three doses on days 1–36, then tapered), weekly vincristine (VCR, 2.0 mg/m2 ×6), doxorubicin (40 mg/m2 days 1, 22 and 36), Erwinia asparaginase (30 000 IU/m2 days 37 to 46), and i.s. MTX (days 1, 8, 15 and 29).16
From day 50, children with SR-ALL received three courses of HDM given at 2 week intervals without concurrent oral 6MP. Consolidation therapy for patients with IR-ALL consisted of: (1) from day 50: daily oral 6MP (75 mg/m2 for 2 × 2 weeks), cyclophosphamide (1 g/m2 ×2), four series of low-dose cytarabine (each with i.v. doses of 75 mg/m2 on 4 consecutive days), and i.s. MTX (×2); (2) from day 106: daily oral 6MP for 8 weeks (25 mg/m2 given once in the evening) with four courses of HDM given at 2 week intervals, (3) from day 169: 4 weeks of re-induction with dexamethasone (10 mg/m2/day divided into three doses for 3 weeks, then tapered), weekly VCR (2.0 mg/m2/day ×4), weekly daunorubicin (30 mg/m2/day ×4), and asparaginase four times (30 000 IU/m2 at 3–4 day intervals); and (4) from day 197: daily oral thioguanine (60 mg/m2 for 2 weeks), cyclophosphamide (1 g/m2 ×1), two series of low-dose cytarabine (each with i.v. doses of 75 mg/m2 on 4 consecutive days), and i.s. MTX (×1).
Maintenance therapy with starting 6MP doses of 75 mg/m2/day and MTX doses of 20 mg/m2/week given in the evening was initiated at week 14 (SR) or week 33 (IR). The doses of oral 6MP and oral MTX were to be targeted to a WBC of 1.5–3.5 × 109/l and reduced to 50% at a WBC <1.5 × 109/l and interrupted at a WBC <1.0 × 109/l and/or a thrombocyte count <100 × 109/l. During maintenance therapy blood counts were taken at least every 2 weeks. The median monthly number of blood counts for the 46 children was 3.6 (75% range: 2.8–5.3). As part of a randomized study, 10 patients with SR-ALL and 11 patients with IR-ALL were (in addition to dose adjustments by toxicity) been stratified to have their doses of oral 6MP and MTX adjusted by E-6TGN and E-MTX.217 However, even for these patients a WBC <1.5 × 109/l or a thrombocyte count <100 × 109/l indicated dose reductions as outlined above. During the first year of maintenance therapy, patients with SR- and IR-ALL received at 4 week intervals alternate pulses of either (1) VCR (2.0 mg/m2 ×1) and prednisolone (60 mg/m2/day for 1 week) or (2) HDM, the first being given at week 18 (SR) or week 33 (IR). These re-inductions were continued until five courses of HDM had been given.16 No oral MTX was given for a week from the start of HDM.
Five thousand milligrams of MTX/m2 were given over 24 h. Eighteen to 24 h after the beginning of the HDM, i.s. MTX was given in age-related doses (8–12 mg). Starting 36 h from the beginning of the HDM infusion, leucovorin rescue was given at a dose of 15 mg/m2 i.v. every 6 h until the serum MTX was below 200 nmol/l. If serum MTX was >1000 nmol/l at 42 h, the leucovorin doses were increased according to the serum MTX concentrations. Blood counts were taken from 10 days after the start of HDM and repeated at 1–3 day intervals until they were within the above given target range for maintenance therapy. For each HDM, we registered the hemoglobin, platelet counts, WBC, absolute neutrophil (ANC) and lymphocyte count nadirs, which were experienced 10 to 20 days from the start of HDM. Since changes in the dose of 6MP will change the intracellular 6TGN levels, the dose of 6MP prior to HDM was only included in the statistical analyses if they were unaltered for at least 14 days before HDM.
E-6TGN/MTX and TPMT
During maintenance therapy, blood samples were taken for 6MP/MTX metabolite determination together with every blood count. The median monthly number of blood samples for the 46 children was 2.0 (75% range: 0.9–2.8). E-MTX analyses were done at least 48 h after the latest oral dose of MTX. All analyses of E-6TGN and E-MTX were performed at The Laboratory for Pediatric Oncology, The University Hospital, Rigshospitalet. E-6TGN and E-MTX were measured by an HPLC method18 and a radioligand assay,19 respectively. One of the 46 patients was TPMT deficient.20 E-6TGN and E-MTX measurements were included in the multivariate regression analyses if available within 14 days prior to the start of HDM. If more than one measurement was available during this period, the one closest to the start of HDM was chosen.
For each patient, we calculated: (1) the mean neutrophil nadir and thrombocyte nadir following HDM during consolidation therapy and maintenance therapy (mANC-nadirMT-HDM, mPLATE-nadirMT-HDM); and (2) the mean WBC, neutrophil and thrombocyte counts during the last 38 weeks (IR) or 53 weeks (SR) of maintenance therapy which did not include HDM (mWBC, mANC, mPLATE). The mWBC, mANC and mPLATE were calculated as weighted means using as weight the interval between the sample in question and the next blood sample as previously described.21 The Mann–Whitney U test, Wilcoxon's test, and Spearman's rank order correlation analysis were applied to compare the distributions of parameters between subgroups, in related samples, and the correlations between parameters (rS = correlation coefficient).22 Since HDM was given right at the start of maintenance therapy for IR-ALL (at week 33) without preceding oral 6MP therapy, this HDM course was excluded from the multivariate regression analysis of the impact of oral 6MP on HDM toxicity. Stepwise, forward, multivariate linear regression analyses were done to identify the independent variables that best predicted the degree of bone marrow toxicity following HDM during maintenance therapy. Parameters were included in the models at significance limits of 0.05. Prior to the regression analyses natural logarithmic transformation of the ANC and the thrombocyte nadir were done to approximate normal distributions. Data analyses were done with SPSS statistical software.23 Two-sided P values <0.05 were regarded as being significant.
The median WBC nadir after HDM was significantly lower during maintenance therapy (median: 1.6 × 109/l; 75% range: 0.9–2.9) compared to SR consolidation (median: 5.1 × 109/l; 75% range: 3.3–6.9) and IR consolidation (median: 2.7 × 109/l; 75% range: 1.8–4.4) (Figure 1). Similarly, comparing SR consolidation, IR consolidation, and maintenance therapy, ANC nadir, lymphocyte count nadir, thrombocyte count nadir, and hemoglobin nadir after HDM decreased significantly with increasing doses of oral 6MP (Table 1). The mANC nadirMT-HDM and mPLATE nadirMT-HDM were significantly related (rS = 0.63, P < 0.001). Only 3% of the HDM courses given as SR-ALL consolidation was followed by an ANC nadir <0.5 × 109/l compared to 9% given as IR-ALL consolidation, and 50% of the HDM courses given during maintenance therapy. Similarly, only 13% of the HDM courses given as SR-ALL consolidation induced a thrombocyte count nadir <100 × 109/l compared to 15% of the HDM given as IR-ALL consolidation, and 58% of the HDM courses given during maintenance therapy. Due to neutropenia and/or thrombocytopenia, 6MP/MTX maintenance therapy was discontinued following 96 of the 230 HDM courses given during maintenance therapy. The median duration of these treatment interruptions was 10 days (75% range: 3–18 days, maximum: 30 days). The duration of treatment interruptions was significantly related to both the ANC nadir (rS = −0.55, P < 0.001) and thrombocyte nadir (rS = −0.72, P < 0.001).
The average ANC nadir experienced by the individual patient following HDM given as consolidation therapy was for all 21 patients with SR-ALL (mean of three courses) higher than the mANC nadirMT-HDM (mean of five courses) (P < 0.001, Wilcoxon) with a mean difference of 1.7 × 109/l (95% CI: 1.3–2.1). Similarly, the average ANC nadir after consolidation HDM (mean of four courses) was higher for 24 of the 25 patients with IR-ALL compared to the mANC nadirMT-HDM (P < 0.001) with a mean difference of 0.8 × 109/l (95% CI: 0.4–1.2) (Figure 2a). The average thrombocyte count nadir experienced by individual patients following HDM given as consolidation therapy was for 19 of 21 patients with SR-ALL higher than the mPLATE-nadirMT-HDM (P < 0.001) with a mean difference of 143 × 109/l (95% CI: 98–188), and similarly higher for 21 of 25 patients with IR-ALL (P < 0.001) with a mean difference of 83 × 109/l (95% CI: 55–111) (Figure 2b).
Predictive factors for myelotoxicity after HDM during maintenance therapy
In univariate analysis, both ANC nadir (rS = −0.20, P = 0.007) and thrombocyte nadir (rS = −0.13, P = 0.07), was negatively related to the dose of 6MP at the time of HDM. In contrast, the average dose of 6MP for individual patients during maintenance therapy was positively related to mWBC (rS = 0.54), to mANC (rS = 0.48), and to average thrombocyte counts (rS = 0.48). This reflected that during the last part of maintenance therapy the patients with the least myelotoxicity would be prescribed the highest dose of 6MP. The dose of 6MP prior to HDM was not correlated to the time spend on maintenance therapy (rS = 0.03, P = 0.22).
The degree of neutropenia following HDM did not correlate significantly with the time spent on maintenance therapy either for children with SR-ALL (rS = −0.03, P = 0.79) or for those with IR-ALL (rS = −0.16, P = 0.10).
We found no significant difference in the degree of neutropenia following HDM during maintenance therapy for SR- vs IR-ALL (median (75% range) mANC nadirMT-HDM; SR: 0.56 × 109/l (0.22–1.3), IR: 0.74 × 109/l (0.32–1.5); P = 0.49). Similarly, there was no significant difference in the degree of thrombocytopenia following HDM during maintenance therapy for SR- vs IR-ALL (median (75% range) mPLATE nadirMT-HDM; SR: 95 × 109/l (33–165), IR: 108 × 109/l (39–191); P = 0.52). The mANC nadirMT-HDM and the mPLATE nadirMT-HDM were related to mANC (rS = 0.49, P = 0.001) and to mPLATE (rS = 0.47, P = 0.01), respectively. Thus, the degree of neutropenia and thrombocytopenia-induced HDM reflected the relative bone marrow suppression in relation to the patient's average blood counts.
With multivariate linear regression analysis, we tested the impact on ANC nadir (or thrombocyte nadir) following HDM during maintenance therapy of gender (0 = girls vs 1 = boys), age at diagnosis, risk group (0 = SR, 1 = IR), number of days on maintenance therapy, mANC (or mPLATE), dose of 6MP, E-6TGN and E-MTX levels prior to HDM, serum MTX concentration at the end of the MTX infusion, and 42 h serum MTX concentration. The regression analyses were performed in two ways: (1) only a single data set was used for each child using the mean neutrophil and thrombocyte nadirs following HDM, and the mean of the individual parameters to be tested in the regression analyses; or (2) each of the HDM courses were regarded as independent. In the first alternative, only mANC and the mPLATE, respectively, were found to be related to the average degree of neutropenia and thrombocytopenia following HDM. In the second alternative, the best-fit model to predict ANC nadir following HDM during maintenance therapy included in the following order: dose of 6MP prior to HDM (β = −0.017, P = 0.001), average ANC level during maintenance therapy (β = 0.82, P = 0.004), and E-6TGN (β = −0.0029, P = 0.02). The age of the patient (β = 0.19, P = 0.09) and the risk group (β = −0.19, P = 0.08) were of borderline significance. Thus, those who received the highest doses of 6MP, those with the lowest average ANC during maintenance therapy, and those with the highest E-6TGN levels prior to HDM had the most severe degree of neutropenia following HDM. Similarly, we tested the impact on thrombocyte count nadir following HDM. The best-fit model to predict thrombocyte nadir following HDM during maintenance therapy included only mPLATE (β = 0.0057, P = 0.046). Thus, those with the lowest average thrombocyte level during maintenance therapy also had the most severe thrombocytopenia following HDM.
HDM has been given to children with ALL since the 1960s to reduce the risk of systemic as well as extramedullary relapse. A number of clinical studies during the last 10 to 15 years has increased our pharmacokinetic and pharmacodynamic understanding of low- and high-dose MTX.2425 However, most of these studies have focused on MTX pharmacokinetics only, whereas the possible pharmacodynamic interaction between MTX and other drugs has received far less attention.
Patients with delayed clearance have long been known to be at increased risk for myelotoxicity, and for this reason routine monitoring of serum MTX concentrations is an integrated part of HDM therapy. However, apart from the myelotoxicity in patients who have delayed MTX clearance, most cases of clinically significant bone marrow toxicity leading to treatment withdrawal have been unpredictable.26
HDM and low-dose oral 6MP can interact in several ways. MTX is an inhibitor of the enzyme dihydrofolate reductase,27 which converts folates to their active reduced (tetra- hydrofolate) form. The subsequent accumulation of dihydrofolate can inhibit de novo purine synthesis.1528 In addition, MTX can inhibit purine de novo synthesis by virtue of the polyglutamate metabolites which directly inhibit aminoimidazole carboxamide ribonucleoside monophosphate.28 Finally, low-dose MTX as well as HDM can inhibit xanthine oxidase and enhance the bioavailability of 6MP.1429 These interactions between 6MP and MTX were indicated by clinical studies more than 30 years ago when 6MP/MTX combination maintenance therapy for childhood ALL was shown to be superior to monotherapy with either MTX or 6MP.3031 In recent years both in vitro and in vivo studies have supported that the interaction between 6MP and MTX is of clinical significance.215203233 However, this is the first study which, in detail, explores the influence of 6MP dose and pharmacokinetics on the myelotoxicity of HDM.
Theoretically, treatment intensity prior to administration of HDM could, to some extent, have influenced the degree of myelotoxicity. However, several findings make it unlikely that this explains our results: (1) there was no significant difference in the degree of neutropenia and thrombocytopenia experienced by the SR and IR group during maintenance therapy in spite of the difference in their consolidation therapy (in fact myelosuppression was slightly more pronounced for SR patients); (2) the degree of myelosuppresision was more pronounced for SR patients on maintenance therapy compared to IR patients on consolidation who have previously received combinations of cyclophosphamide, low-dose ARA-C and 6MP; (3) the degree of neutropenia after HDM did not progress during maintenance therapy, which indicates that repetitive HDM courses in themselves do not progressively reduce the bone marrow reserve; and (4) the significant influence of the dose of 6MP during maintenance therapy on the degree of myelotoxicity following HDM supports that the coadministration of 6MP during IR consolidation and during SR/IR maintenance therapy is a major determinant of the risk of HDM-induced myelotoxicity.
The correlation between the dose of 6MP and ANC nadir even with the E-6TGN levels included in the linear regression model indicates (1) that other 6MP metabolites such as the 6-methyl-thioinosine 5′-monophosphate could have influenced the degree of myelotoxicity, since these are strong inhibitors of purine de novo synthesis, and/or (2) that E-6TGN levels do not sufficiently reflect DNA-6TGN levels. In support hereof, we recently demonstrated that the risk of secondary myelodysplasia or myeloid leukemia following 6MP/MTX maintenance therapy of childhood ALL was related not only to E-6TGN levels but also to the intracellular levels of methylated 6MP metabolites, which could reflect an increased incorporation of DNA-6TGN due to inhibition of de novo purine synthesis mediated by these methylated metabolites.17
Seemingly contradictory findings are: (1) the negative correlation between the individual dose of 6MP prior to HDM during maintenance therapy and the ANC and thrombocyte nadirs, and (2) the positive correlation between the average dose of 6MP during maintenance therapy and mANC and mPLATE. However, whereas the latter reflect the tolerance to standard doses of 6MP, which for a large part are based on inter- and intraindividual variations in 6MP pharmacokinetics, the former reflects the impact of the dose of 6MP on HDM-induced bone marrow toxicity when the differences in the average ANC and thrombocyte count levels are also taken into account (as demonstrated by the multivariate regression analyses). Thus, the impact of a certain dose of 6MP prior to HDM will be stronger for a patient with a poor tolerance to 6MP than for a patient with a high average neutrophil level at standard doses of 6MP.
Treatment interruptions following HDM should, if possible, be avoided since at least theoretically these periods of treatment withdrawals could increase the risk of treatment failure.1213 But how clinically significant myelosuppression following HDM can be avoided is not clear. The data of the present study indicate that reductions of the dose of oral 6MP prior to the administration of HDM rather than reductions of the dose of HDM could be one approach, although this needs to be confirmed in clinical trials.
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The commitment and skillful technical assistance of Jannie Gregers, Kristine Nielsen, and Michael Timm are greatly appreciated. The study has received financial support from The Carl and Ellen Hertz Foundation, The Danish Childrens Cancer Foundation, The Danish Cancer Society (grant Nos 91-048, 92-017, 93-017, 95-100-28), The JPC Foundation, The Lundbeck Foundation (38/99), and The Minister Erna Hamilton Foundation.
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Schmiegelow, K., Bretton-Meyer, U. 6-Mercaptopurine dosage and pharmacokinetics influence the degree of bone marrow toxicity following high-dose methotrexate in children with acute lymphoblastic leukemia. Leukemia 15, 74–79 (2001). https://doi.org/10.1038/sj.leu.2401986
- Bone marrow toxicity
- acute lymphocytic leukemia
- thioguanine nucleotides
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