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

Abstract

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 r s = 0.68 and r s = 0.25-0.42, respectively. Intercorrelations of MTXpg3,4,5,6 were all r s ≥ 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.

Glutamic Acid Increased Methotrexate Polyglutamation and Cytotoxicity in a CCRF-SB Acute Lymphoblastic Leukemia Cell Line

“…ATRA is used in the treatment of acute promyelocytic leukemia and myelodysplastic syndrome. Two derivatives of ATRA (containing glutamic acid or its sodium salt), RAE and RAENa, have been synthesized, and they showed improved aqueous solubility and were more effective in mice bearing S-180 tumors and in childhood acute leukemia [14,15]. In the present study, we proposed glutamic acid as an adjuvant to increase MTX efficacy by promoting polyglutamate synthesis.…”

Effects of germline DHFR and FPGS variants on methotrexate metabolism and relapse of leukemia

“…To avoid the interference caused by recent drug intake, we excluded samples with excessive levels of MTX pg1 , defined as those in which the sum of MTX pg1-6 exceeded 2.7 3 MTX pg2-6 1 2.5, since we have shown that MTX short-chain measurements in samples above this threshold are excessively affected by recently administered MTX pg1 . 28 For each metabolite, we calculated time-weighted means (prefix wm ), weighting each measurement by the time between the previous and the adjacent measurements and allowing for a maximum weight of 7 days (ie, MTX pg measurements were regarded as representing the MTX pg level in the period from 3 days before until 3 days after blood sampling, unless other MTX pg measurements were available within that interval 24,28 ). Because each of the wm MTX pg fractions were distributed differently (supplemental Figure 1, available on the Blood Web site), we applied rank normalization to allow for uniform statistical modeling, despite different distributions.…”

“…wm MTX pg2 and wm MTX pg4 among the 6 types of MTX polyglutamates were prioritized for further GWASs, based on our previous study on erythrocyte MTX polyglutamate profiling. 28 GWASs were then performed to evaluate the correlation between the 2 MTX polyglutamates (ie, wm MTX pg2 and wm MTX pg4 ) and genotypes at 1 495 495 SNPs in the discovery cohort (n 5 447) after quality control. A linear regression was applied with adjustment for sex, risk group, and genetic ancestry, as determined by principal components analyses.…”

“…Given that the 6 types of MTX polyglutamates are classified as either short chain (ie, MTX pg1 and MTX pg2 ) or long chain (ie, MTX pg4 -MTX pg6 ), we selected MTX pg2 and MTX pg4 as representatives of each class for GWAS for the following reasons. 28 First, quantification of MTX pg1 can be disturbed by residual plasma MTX, whereas MTX pg2 measurements more accurately reflect its pure metabolite levels. Second, levels of the long-chain metabolites correlate strongly with each other, with MTX pg4 being the most strongly associated with the sum of MTX pg3-6 .…”

“…Second, levels of the long-chain metabolites correlate strongly with each other, with MTX pg4 being the most strongly associated with the sum of MTX pg3-6 . 28 Accordingly, we performed GWASs with MTX pg2 and MTX pg4 as phenotypes of interest.…”