Introduction

Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood. High-dose methotrexate (HDM) is an important part of the therapy given to these patients to reduce the risk of systemic and extramedullary relapse.¹ HDM is commonly given during consolidation therapy with or without concurrent oral 6-mercaptopurine (6MP) and as reinductions during maintenance therapy with daily oral 6MP and weekly oral methotrexate (MTX) as the backbone.

We have previously demonstrated that the risk of significant bone-marrow suppression is increased if oral 6MP is coadministered with HDM.² MTX may increase the bioavailability of 6MP through inhibition of xanthine oxidase, which catabolizes 6MP.³ In addition, several studies of cytotoxicity have indicated that MTX and 6MP act synergistically.^4,5,6,7,8 In hemopoietic cells, 6MP is anabolized to 6-thioguanine nucleotides (6TGNs), which are the most important mediators of the cytotoxic effect of 6MP through incorporation into DNA.⁹ Erythrocytic accumulation of 6TGN correlate with the degree of myelotoxicity and remission duration in childhood ALL.^3,10,11 Another important metabolic pathway of 6MP is the methylation of 6MP and some of its metabolites by thiopurine methyltransferase (TPMT). Methylated products of 6MP as well as MTX polyglutamates inhibit purine de novo synthesis and, thus, may enhance the incorporation of 6TGN into DNA.^12,13

Myelotoxicity following HDM may lead to treatment interruptions of maintenance therapy and a reduction of dose intensity, which could affect survival rate.^2,14,15 We here report that reduction of the dose of concurrently given oral 6MP significantly reduces the degree of myelosuppression and risk of treatment interruptions following HDM.

Patients and methods

Patients

Between March 1, 1996 and April 1, 2001, 50 patients were diagnosed with standard risk (SR) or intermediate risk (IR) non-B-cell ALL at the University Hospital, Rigshospitalet, Copenhagen, and completed therapy according to the NOPHO ALL-92 program.¹ The SR/IR risk classification has been described elsewhere.² A total of 26 patients were included in the present analyses, because they had their dose of oral 6MP reduced to approximately 50% from 2 weeks before until 2 weeks after at least one HDM treatment during maintenance therapy because of a thrombocyte nadir 60 10⁹/l and/or an absolute neutrophil count (ANC) nadir 0.7 10⁹/l following their previous HDM. The cohort consisted of seven girls and 19 boys including 12 cases of SR- and 14 cases of IR-ALL. Their median age was 4.9 years. One patient heterozygous for TPMT (mutations G460A and A719G) has developed acute myeloid leukemia, and one patient has experienced a bone-marrow relapse.¹⁶ All of the remaining 24 patients are alive in first remission.

Therapy

Induction therapy, consolidation therapy, and maintenance therapy have been described in detail previously.² Patients received five courses of HDM at 8-week intervals during their first year of maintenance therapy with oral MTX (20 mg/m²/week) and 6MP (75 mg/m²/day). HDM was given at a dose of 5000 mg/m² over 24 h with intrathecal MTX. 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 level 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.² A total of 95 HDM treatments given during maintenance therapy were included in the study including 45 courses with standard 6MP dosage and 50 courses with reduced 6MP dosage. Treatments of HDM with standard 6MP dosage were given three times to six patients (HDM treatment no. -3, -2 and -1), twice to seven patients (HDM treatment no. -2 and -1) and once (HDM treatment no. -1) to 13 patients. Treatments of HDM with reduced 6MP dosage were given three times to 8 patients (HDM treatment no. +1, +2 and +3), twice to eight patients (HDM treatment no. +1 and +2), and once to 10 patients (HDM treatment no. +1) (Table 1).

Table 1 - Myelotoxicity following HDM with coadministered 6MP.

Full table

6-MP dose reduction

Based on our previous observations showing that the degree of myelotoxicity following HDM was related to the dose of 6MP,² patients who developed myelotoxicity following a HDM treatment (ie a thrombocyte nadir 60 10⁹/l and/or an ANC nadir 0.7 10⁹/l) had their dose of 6MP reduced at all subsequent HDM treatments from 2 weeks before HDM and until the white blood cell count (WBC) was 1.0 10⁹/l and the thrombocyte count 100 10⁹/l, however, at least until 2 weeks after the HDM infusion. This duration of 6MP dose reduction was chosen as the steady state of 6TGN on daily oral 6MP is obtained after approximately 2 weeks and the myelotoxicity of HDM occurs between 1 and 3 weeks after HDM. Both the patients and the physicians were aware of the 6MP dose reductions. The 6MP dose reductions were not done as part of a clinical trial but on compassionate basis approved by the Ethical Committee of Copenhagen, Denmark. The NOPHO ALL-92 protocol included no recommendations for patients who experienced significant myelosuppression or treatment interruptions following HDM.

The total doses of 6MP that the patients received during the period with reduced 6MP dosage were compared to standard 6MP dosage using two separate control materials:

1. A historic comparison: Patients elected from our previous study,² who experienced the same degree of myelotoxicity as the patients included in the present study and/or treatment interruption following HDM because of myelotoxicity. Of 46 patients, 37 fulfilled these criteria. Their average dose of 6MP/m²/day was calculated by dividing the total cumulated prescribed dose/m² by the number of days from start of maintenance therapy until 4 weeks after the last HDM treatment.
2. A matched comparison: The average dose of 6MP/m²/day was calculated as above from 2 weeks before and until 4 weeks after the first HDM treatment with reduced 6MP treatment (treatment no. +1) and compared to the average dose of 6MP/m²/day from 2 weeks before and until 4 weeks after the HDM leading to treatment interruption (treatment no. -1).

Thiopurine methyltransferase

The TPMT genotype was determined using polymerase chain reaction-based methods specific for the mutations G460A and A719G detecting the TPMT^*3A, ^*3B, ^*3C, and ^*3D mutations.¹⁷ Six patients were TPMT heterozygotes, all with both mutations present.

Statistics

For each patient, we calculated the mean hemoglobin nadir, thrombocyte nadir, WBC nadir, ANC nadir, and the mean length of treatment interruption following HDM blocks with standard doses of 6MP (HDM treatment no. -3 to -1) as well as following HDM blocks with reduced 6MP dosage (HDM treatment no. +1 to +3). Since HDM was given right at the start of maintenance therapy for IR-ALL without preceding oral 6MP, this first HDM was excluded from the analyses. Spearman's rank-order correlation analysis was used for correlations between parameters (r_s=correlation coefficient). The Mann–Whitney U-test and Wilcoxon's test were applied to compare the distributions of parameters between subgroups and in matched samples. Data analyses were performed using the SPSS statistical software.¹⁸ Two-sided P values <0.05 were regarded as being significant.

Results

The doses of 6MP given during blocks of HDM with standard 6MP dosage (HDM no. -3, -2, and -1) are shown in Table 1. The doses were not significantly related to risk group (median: SR=79 mg/m²/day; IR=70 mg/m²/day; P=0.07), age (r_s=0.008; P=0.97), gender (median: female=72 mg/m²/day; male=71 mg/m²/day; P=0.61), or to TPMT genotype (median: wild type=72 mg/m²/day; heterozygote=66 mg/m²/day; P=0.46). The degree of myelotoxicity and the length of treatment interruption following blocks of HDM with standard 6MP dosage are given in Table 1. The degree of neutropenia following blocks of HDM with standard 6MP dosage was not significantly related to TPMT genotype (median ANC: wild type=0.3 10⁹/l; heterozygote=0.5 10⁹/l; P=0.33). There were no significant differences between HDM no. -3, -2, and -1 regarding the degree of myelotoxicity or length of treatment interruption.

Owing to myelotoxicity and treatment interruption, the doses of 6MP during subsequent HDM treatments were reduced with a median of 51% (75% range: 39–62%, maximum 74%) of the doses in previous HDM blocks. This degree of 6MP dose reduction was related to risk group (median: SR=55%; IR=45%, P=0.02), but not significantly to age (r_s=-0.03; P=0.89), gender (median: female=50%; male=52%; P=0.53), or to TPMT genotype (median: wild type=52%; heterozygote=45%; P=0.66).

When the dose of 6MP was reduced from 2 weeks before and until at least 2 weeks after HDM, the degree of myelotoxicity was significantly reduced (Table 1). This was the case both in a paired comparison of the mean degree of myelotoxicity following HDM blocks with standard 6MP dosage vs reduced 6MP dosage (Table 1, Figure 1), and if only HDM no. -1 was compared to HDM no. +1 (Table 1). The mean hemoglobin nadir and thrombocyte nadir were raised for all but one patient each (Figure 1a), and the mean WBC nadir was increased for all patients except three. Differential counts with ANCs were available for 25 patients of whom 21 patients experienced less neutropenia while one patient had an unchanged value and three patients experienced more neutropenia following the HDM treatments given with 6MP dose reduction (Figure 1b). Of the latter three patients, two fell 0.2 10⁹/l in ANC nadir while one patient fell 0.4 10⁹/l compared to the mean ANC nadir following HDM blocks with standard 6MP dosage. Two of these three patients were TPMT heterozygotes (Figure 1b). The effect of dose reductions was not significantly related to risk group (thrombocytes: P=0.56; ANC: P=0.81), gender (thrombocytes: P=0.53; ANC: P=0.69), age (thrombocytes: P=0.24; ANC: P=0.97), or TPMT genotype (thrombocytes: P=0.93; ANC: P=0.64).

Figure 1.

(a) Average thrombocyte nadirs following HDM in relation to coadministered 6MP doses (median dose reduction 51% (75% range: 39–62%, maximum 74%)). Each line represents one patient with the start of the line reflecting the reduced 6MP doses and the end of the line the standard 6MP doses. The dashed lines indicate patients with TPMT G460A and A719G mutations. (b) Average neutrophil count nadirs following HDM in relation to coadministered 6MP doses (median dose reduction 51% (75% range: 39–62%, maximum 74%)). Each line represents one patient with the start of the line reflecting the reduced 6MP doses and the end of the line the standard 6MP doses. The dashed lines indicate patients with TPMT G460A and A719G mutations.

Full figure and legend (60K)

There were no significant differences between the 23 h serum MTX concentrations during HDM blocks with standard 6MP dosage (median: 64 590 nM (range: 11 320–11 4070)) compared to the 23 h serum MTX concentrations during HDM blocks with reduced doses of 6MP (median: 67 011 nM (range: 22 100–53 4725); P=0.99). The 42 h serum MTX concentrations were higher during HDM blocks with standard 6MP dosage (median 42 h MTX with standard 6MP dosage: 757 nM (range: 290–12 470); median with reduced 6MP dosage: 394 nM (range: 130–743); P=0.001). Similarly, the total dose of leucovorin rescue was higher in HDM blocks with standard 6MP dosage (median: 120 mg/m² (range: 60–4559)) compared to HDM blocks with reduced 6MP dosage (median: 75 mg/m² (range: 53–165); P<0.001). The 42 h serum MTX concentrations were not related to bone marrow suppression following HDM (thrombocytes: r_S=-0.11, P=0.28; ANC: r_S=-0.14, P=0.16).

The duration of treatment interruption following HDM was significantly shorter when 6MP was reduced prior to HDM. Thus, with 6MP dose reductions, the median treatment interruption was reduced from 8 to 0 days (P<0.001) (Table 1), and only nine of 26 patients had treatment interruptions following 6MP dose reduction compared to 24 of 26 patients when treated with standard doses of 6MP (P<0.001).

According to the historical as well as the matched comparisons, the overall 6MP doses were significantly reduced during HDM with 6MP dose reductions. Thus, the median dose of 6MP given from the start of maintenance therapy until 4 weeks after the last HDM treatment was 51 mg/m²/day (range: 22–78) compared to 61 mg/m²/day (range: 3–84) in the historic comparison (P=0.04). The median dose of 6MP given from 2 weeks before and until 4 weeks after HDM no. -1 was 55 mg/m²/day (range: 24–88) compared to 40 mg/m²/day (range: 16–80) during HDM treatment no. +1 (P=0.01).

Discussion

Although HDM has been used since the 1960s, the optimal way to administer HDM is still debated. HDM is given in combination with oral 6MP in order to increase the antileukemic efficacy of the treatment; however, this may in part be counteracted by an increased risk of toxicity with subsequent treatment interruption. We have previously shown that HDM given during maintenance therapy with coadministered oral daily 6MP causes significant pancytopenia.² Owing to myelosuppression, 42% of the treatments with HDM (5 g/m²/24 h) and oral 6MP were found to be followed by treatment interruptions of 6MP and low-dose MTX with a median duration of 10 days(2). In addition, fatal infections because of immune suppression have been reported to occur in 1–6% of patients in remission.^1,19 To decrease or even prevent myelotoxicity and treatment interruptions following HDM in maintenance therapy several approaches could be chosen:

(1) The total dose of MTX could be reduced. However, this approach carries several drawbacks. Higher doses of MTX increase the accumulation in blasts of MTX polyglutamates and specifically long-chain MTX polyglutamates. Hence, decreased MTX doses could reduce the antileukemic effect of MTX.^20,21 In addition, MTX dose reduction would limit penetration of MTX into the CNS,²² which could increase the risk of CNS relapse. Supporting the clinical significance of high systemic MTX exposure, Evans et al²³ found that pharmacokinetically guided MTX dosage to achieve higher MTX concentration in patients with rapid clearance of the drug lead to a better outcome. Further, it is uncertain whether a reduction in MTX dose would lead to reduced myelotoxicity. Thus, HDM followed by leucovorin rescue within 36 h carries only little myelotoxicity if given without concurrent oral 6MP.^2,22 Thus, with the leucovorin regimen used in the NOPHO ALL-92 protocol, the median neutrophil and thrombocyte nadirs were only 1.9 10⁹/l and 240 10⁹/l, respectively, when HDM was given without concurrent 6MP.² Finally, the duration of MTX exposure above a certain threshold is probably more important than the peak MTX concentration.²⁴

(2) The leucovorin rescue could be given earlier. The toxicity of HDM is primarily related to the duration of exposure to the drug prior to leucovorin administration rather than to the peak MTX concentration.^22,24,25 MTX and its polyglutamated metabolites inhibit dihydrofolate reductase, thereby lowering cellular pools of reduced folates.¹² The lack of reduced folate cofactors is responsible for the inhibition of the synthesis of purines and pyrimidines. Thus, leucovorin rescue administered earlier after the start of MTX infusion could be another approach to reduce myelotoxicity and the duration of treatment interruption. However, even though this has not been tested in randomized studies, this approach could to lead to a decreased antileukemic efficacy.²⁶

(3) The approach chosen in the present study was to reduce 6MP doses during HDM. This approach was based on our previous observations that significant myelotoxicity following HDM was seen when the treatment was given with coadministered 6MP.² It was also based on the assumption that MTX, through inhibition of the de novo purine synthesis, increases the levels of phosphoribosyl pyrophosphate, and thereby increases both the build up of 6TGN and their incorporation into DNA, although the latter has not yet been proven in clinical studies.²⁷ However, it is not known whether uninterrupted maintenance therapy with reduced doses of 6MP is superior to full-dose therapy with intermittent treatment interruptions. A few studies have indicated that reductions of the overall 6MP dose intensity because of treatment interruptions may affect the cure rate.^14,15 Since the half-life of the cytosol 6TGN levels is only 3–5 days at least in erythrocytes (unpublished observations), it is possible that a period of dose reductions of 1 week before HDM and until the nadirs have been passed could be sufficient to prevent clinically significant myelotoxicity. If this is the case, such a 6MP dose reduction approach may not reduce the overall 6MP dose intensity during MT. This approach is now an option in the NOPHO ALL-2000 protocol for patients who have experienced an ANC nadir <0.5 10⁹/l and/or a thrombocyte nadir <60 10⁹/l following a HDM treatment with standard 6MP dosage during maintenance therapy.

It is unlikely that the reduced myelotoxicity subsequent to 6MP dose reduction found in the present analyses is a random effect or explained by an increasing tolerance of the bone marrow to HDM during maintenance therapy. Thus, the degree of myelotoxicity following HDM at 8-week intervals does not decrease during maintenance therapy.² In fact, we found an increase in the degree of myelotoxicity during HDM treatment -3 to -1 (Table 1). Furthermore, we found no significant difference in post-HDM myelotoxicity in SR and IR patients in spite of a more intensive treatment in IR patients during their preceding consolidation therapy.^1,2

We found a delayed excretion of MTX following HDM blocks with standard 6MP dosage compared to reduced 6MP dosage. The majority of MTX is bound to albumin in the blood and, when HDM is administered, approximately one-third of MTX is metabolized in the liver. It is well known that MTX/6MP maintenance therapy causes hepatotoxicity²⁸ and this may in part be because of methylated metabolites of 6MP.²⁹ It is possible that a reduced degree of hepatotoxicity second to the reduced 6MP doses could have caused an improvement in the hepatic clearance of MTX and explain the moderate decrease in 42 h MTX concentrations observed in the present study. However, the MTX concentrations were not related to the degree of bone-marrow toxicity.

The degree of myelotoxicity following HDM treatments with standard doses of 6MP in this cohort of patients were very similar to what we have reported previously with respect to the median nadirs of hemoglobin, thrombocytes, white blood cells, and neutrophil counts.² Even though reduced 6MP dosage during HDM was able to decrease myelotoxicity and treatment interruption, the situation is still less than optimal since the efficacy of dose reductions could not be predicted for the individual patient and the overall 6MP dose intensity was reduced. Thus, further pharmacokinetic and biological studies are needed to improve the prediction of the patients who are at risk to develop severe myelo-toxicity following HDM and to improve individual preventive measures.

The frequency of TPMT heterozygotes (including TPMT G460A and A719G mutations) in this study was 23% (95% range: 9–44%) compared to the expected 8–9%.^30,31 Although, this indicates that TPMT heterozygosity may be a risk factor for myelotoxicity following HDM with concurrent oral 6MP, larger studies are needed to explore if TPMT heterozygotes as a rule should have their dose of 6MP reduced to HDM.

On the basis on the present findings, we conclude that reducing the dose of 6MP from 2 weeks before to 2 weeks after HDM is one way to decrease bone-marrow toxicity and treatment interruptions in patients who have previously developed significant myelotoxicity after HDM/6MP. However, further studies are needed to be able to predict the effect of 6MP dose reduction in the single patient.

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Acknowledgements

The commitment and skillful technical assistance of Jannie Gregers, Kristine Nielsen, and Michael Timm are greatly appreciated. This study has received financial support from the Lundbeck Foundation (Grant No. 38/99); the Danish Childrens Cancer Foundation; the Childrens Cancer Foundation, Sweden (Grant No. 1999/080).