PURPOSE: 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.

CONCLUSIONS: 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.