Methotrexate polyglutamate levels and co-distributions in childhood acute lymphoblastic leukemia maintenance therapy
Methotrexate polyglutamates (MTXpg) facilitate incorporation of thioguanine nucleotides into DNA (DNA-TG, the primary cytotoxic thiopurine metabolite and outcome determinant in MTX/6-mercaptopurine treatment of childhood ALL). We hypothesized that mapping erythrocyte levels of MTXpg with 1–6 glutamates and their associations with DNA-TG formation would facilitate future guidelines for maintenance therapy dosing.
Methods and results
Summed MTX with 1–6 glutamates resolved by LCMS [median (interquartile): 5.47 (3.58–7.69) nmol/mmol hemoglobin] was in agreement with total MTX by radio ligand assay. In 16,389 blood samples from 1426 ALL maintenance therapy patients, MTXpg3 21.0 (15.2–27.4)% was the predominant metabolite, and MTXpg1 (the maternal drug) constituted 38.6 (27.2–50.2)% of MTXpg1–6. All subsets correlated; the strongest associations were between metabolites with similar polyglutamate lengths. Correlations of MTXpg1 with MTXpg2 and MTXpg3,4,5,6 were rs = 0.68 and rs = 0.25–0.42, respectively. Intercorrelations of MTXpg3,4,5,6 were all rs ≥ 0.51. MTXpg4 accounted for 29.8 (24.7–33.3)% of MTXpg3–6, yet explained 96% of the summed MTXpg3–6 variation. MTXpg1–4, MTXpg1–6, MTXpg2–6 and MTXpg3 were all associated with DNA-TG levels (p < 0.00001), but collinearity precluded identification of the most informative subset.
Measuring erythrocyte MTXpg4 simplifies and can replace longer chain MTXpg monitoring. Resolving individual MTXpg identifies samples that are unsuitable for dose guidance due to high levels of MTXpg1 remaining in the plasma fraction because of recent MTX intake. All tested MTXpg subsets correlated with DNA-TG and may be used for ALL maintenance therapy dose adjustments, but the most informative subset remains to be identified.
KeywordsAcute lymphoblastic leukemia maintenance therapy Methotrexate polyglutamates Thiopurine Therapeutic drug monitoring Personalized/individualized therapy
DNA (-incorporated) thioguanine nucleotides
Liquid chromatography tandem mass spectrometry
Early and late maintenance therapy phases
Polyglytamated MTX with X γ-linked glutamates
Total MTX determined by radio ligand assay
Radio ligand assay
White blood cell count
The authors thank the dedicated staff at the laboratory of Pediatric Oncology, Copenhagen for their valuable work.
JN drafted the manuscript, developed, implemented, supervised all pharmacological analyses and performed together with MP the statistical analysis in the method comparison and co-distribution study. SNN and KG compiled data and did the statistical analysis of associations of DNA-TG with MTXpg subsets and cytoplasmic metabolites. JA, BL, JK, OGJ, GV, and KP developed the study protocol and coordinated the national blood sample and data collection for each country. KS initiated, supervised, and was the principal investigator for this study and of the NOPHO ALL2008 protocol. All authors approved the final manuscript.
The Danish Childhood Cancer Foundation (Grant nos. 2012/13, PROJ12/059), The Danish Cancer Society, The Nordic Cancer Union, The Swedish Childhood Cancer Foundation, Otto Christens Foundation, University Hospital Rigshospitalet, and Novo Nordic Foundation.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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