Cancer Chemotherapy and Pharmacology

, Volume 83, Issue 1, pp 53–60 | Cite as

Methotrexate polyglutamate levels and co-distributions in childhood acute lymphoblastic leukemia maintenance therapy

  • Jacob NerstingEmail author
  • Stine Nygaard Nielsen
  • Kathrine Grell
  • Maria Paerregaard
  • Jonas Abrahamsson
  • Bendik Lund
  • Olafur Gisli Jonsson
  • Kaie Pruunsild
  • Goda Vaitkeviciene
  • Jukka Kanerva
  • Kjeld Schmiegelow
  • the Nordic Society of Paediatric Haematology and Oncology (NOPHO)
Original Article



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.


Acute lymphoblastic leukemia maintenance therapy Methotrexate polyglutamates Thiopurine Therapeutic drug monitoring Personalized/individualized therapy 



Dihydrofolate reductase


DNA (-incorporated) thioguanine nucleotides






High dose






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




Thioguanine nucleotides


White blood cell count



The authors thank the dedicated staff at the laboratory of Pediatric Oncology, Copenhagen for their valuable work.

Author contributions

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.


  1. 1.
    Schmiegelow K, Nielsen SN, Frandsen TL, Nersting J (2014) Mercaptopurine/methotrexate maintenance therapy of childhood acute lymphoblastic leukemia: clinical facts and fiction. J Pediatr Hematol Oncol 36(7):503–517. CrossRefGoogle Scholar
  2. 2.
    Karran P, Attard N (2008) Thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer. Nat Rev Cancer 8(1):24–36. CrossRefGoogle Scholar
  3. 3.
    Nielsen SN, Frandsen TL, Nersting J, Hjalgrim LL, Schmiegelow K (2015) Pharmacokinetics of 6-thioguanine and 6-mercaptopurine combination maintenance therapy of childhood ALL: hypothesis and case report. J Pediatr Hematol Oncol 37(3):e206–e209. CrossRefGoogle Scholar
  4. 4.
    Schmiegelow K (2009) Advances in individual prediction of methotrexate toxicity: a review. Br J Haematol 146(5):489–503. CrossRefGoogle Scholar
  5. 5.
    Nielsen SN, Grell K, Nersting J, Frandsen TL, Hjalgrim LL, Schmiegelow K (2016) Measures of 6-mercaptopurine and methotrexate maintenance therapy intensity in childhood acute lymphoblastic leukemia. Cancer Chemother Pharmacol 78(5):983–994. CrossRefGoogle Scholar
  6. 6.
    Chabner BA, Allegra CJ, Curt GA, Clendeninn NJ, Baram J, Koizumi S, Drake JC, Jolivet J (1985) Polyglutamation of methotrexate. Is methotrexate a prodrug? J Clin Investig 76(3):907–912. CrossRefGoogle Scholar
  7. 7.
    Schroder H, Fogh K (1988) Methotrexate and its polyglutamate derivatives in erythrocytes during and after weekly low-dose oral methotrexate therapy of children with acute lymphoblastic leukemia. Cancer Chemother Pharmacol 21(2):145–149Google Scholar
  8. 8.
    Nielsen SN, Grell K, Nersting J, Abrahamsson J, Lund B, Kanerva J, Jonsson OG, Vaitkeviciene G, Pruunsild K, Hjalgrim LL, Schmiegelow K (2017) DNA-thioguanine nucleotide concentration and relapse-free survival during maintenance therapy of childhood acute lymphoblastic leukaemia (NOPHO ALL2008): a prospective substudy of a phase 3 trial. Lancet Oncol 18(4):515–524. CrossRefGoogle Scholar
  9. 9.
    Kamen BA, Takach PL, Vatev R, Caston JD (1976) A rapid, radiochemical-ligand binding assay for methotrexate. Anal Biochem 70(1):54–63CrossRefGoogle Scholar
  10. 10.
    Toft N, Birgens H, Abrahamsson J, Griskevicius L, Hallbook H, Heyman M, Klausen TW, Jonsson OG, Palk K, Pruunsild K, Quist-Paulsen P, Vaitkeviciene G, Vettenranta K, Asberg A, Frandsen TL, Marquart HV, Madsen HO, Noren-Nystrom U, Schmiegelow K (2018) Results of NOPHO ALL2008 treatment for patients aged 1–45 years with acute lymphoblastic leukemia. Leukemia 32(3):606–615. CrossRefGoogle Scholar
  11. 11.
    Toft N, Birgens H, Abrahamsson J, Bernell P, Griskevicius L, Hallbook H, Heyman M, Holm MS, Hulegardh E, Klausen TW, Marquart HV, Jonsson OG, Nielsen OJ, Quist-Paulsen P, Taskinen M, Vaitkeviciene G, Vettenranta K, Asberg A, Schmiegelow K (2013) Risk group assignment differs for children and adults 1–45 years with acute lymphoblastic leukemia treated by the NOPHO ALL-2008 protocol. Eur J Haematol 90(5):404–412. CrossRefGoogle Scholar
  12. 12.
    den Boer E, Meesters RJ, van Zelst BD, Luider TM, Hazes JM, Heil SG, de Jonge R (2013) Measuring methotrexate polyglutamates in red blood cells: a new LC–MS/MS-based method. Anal Bioanal Chem 405(5):1673–1681. CrossRefGoogle Scholar
  13. 13.
    van Haandel L, Becker ML, Leeder JS, Williams TD, Stobaugh JF (2009) A novel high-performance liquid chromatography/mass spectrometry method for improved selective and sensitive measurement of methotrexate polyglutamation status in human red blood cells. Rapid Commun Mass Spectrom 23(23):3693–3702. CrossRefGoogle Scholar
  14. 14.
    Jacobsen JH, Schmiegelow K, Nersting J (2012) Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard. J Chromatogr B Anal Technol Biomed Life Sci 881–882:115–118. CrossRefGoogle Scholar
  15. 15.
    Shipkova M, Armstrong VW, Wieland E, Oellerich M (2003) Differences in nucleotide hydrolysis contribute to the differences between erythrocyte 6-thioguanine nucleotide concentrations determined by two widely used methods. Clin Chem 49(2):260–268CrossRefGoogle Scholar
  16. 16.
    Becker ML, van Haandel L, Gaedigk R, Lasky A, Hoeltzel M, Stobaugh J, Leeder JS (2010) Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations. Arthritis Rheum 62(6):1803–1812. CrossRefGoogle Scholar
  17. 17.
    Cook JD, Cichowicz DJ, George S, Lawler A, Shane B (1987) Mammalian folylpoly-gamma-glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis. Biochemistry 26(2):530–539CrossRefGoogle Scholar
  18. 18.
    de Rotte MC, den Boer E, de Jong PH, Pluijm SM, Calasan MB, Weel AE, Huisman AM, Gerards AH, van Schaeybroeck B, Wulffraat NM, Lindemans J, Hazes JM, de Jonge R (2015) Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in patients with rheumatoid arthritis. Ann Rheum Dis 74(2):408–414. CrossRefGoogle Scholar
  19. 19.
    Calasan MB, den Boer E, de Rotte MC, Vastert SJ, Kamphuis S, de Jonge R, Wulffraat NM (2015) Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in juvenile idiopathic arthritis patients. Ann Rheum Dis 74(2):402–407. CrossRefGoogle Scholar
  20. 20.
    Stamp LK, Barclay ML, O’Donnell JL, Zhang M, Drake J, Frampton C, Chapman PT (2011) Effects of changing from oral to subcutaneous methotrexate on red blood cell methotrexate polyglutamate concentrations and disease activity in patients with rheumatoid arthritis. J Rheumatol 38(12):2540–2547. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pediatrics and Adolescent Medicine, RigshospitaletUniversity of CopenhagenCopenhagenDenmark
  2. 2.Section of Biostatistics, Department of Public HealthUniversity of CopenhagenCopenhagenDenmark
  3. 3.Department of Pediatrics, Institution for Clinical SciencesSahlgrenska Academy, University of GothenburgGothenburgSweden
  4. 4.Department of Pediatrics, St. Olavs HospitalTrondheim University HospitalTrondheimNorway
  5. 5.Department of PediatricsLandspitali University HospitalReykjavíkIceland
  6. 6.Tallinn Children’s HospitalTallinnEstonia
  7. 7.University Hospital Santariskiu KlinikosVilniusLithuania
  8. 8.Children’s Hospital, Helsinki University Central HospitalUniversity of HelsinkiHelsinkiFinland
  9. 9.Institute of Clinical MedicineUniversity of CopenhagenCopenhagenDenmark

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