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. 2020 Sep 30;20(1):940.
doi: 10.1186/s12885-020-07422-y.

The evaluation of red blood cell folate and methotrexate levels during protocol M in childhood acute lymphoblastic leukemia

N Oosterom  1   2 , M Fiocco  3   4   5 , R Q H Kloos  6 , I M van der Sluis  3 , R Pieters  3 , B D van Zelst  7 , D E C Smith  8 , M M van den Heuvel-Eibrink  3 , R de Jonge  8 , S G Heil  7
Affiliations

The evaluation of red blood cell folate and methotrexate levels during protocol M in childhood acute lymphoblastic leukemia

N Oosterom et al. BMC Cancer. .

Abstract

Background: After High-Dose Methotrexate (HD-MTX), folinic acid rescue therapy (Leucovorin) is administered to reduce side effects in pediatric acute lymphoblastic leukemia (ALL) patients. Leucovorin and MTX are structural analogues, possibly competing for cellular transport and intracellular metabolism. We hypothesize that Leucovorin accumulates during consecutive courses, which might result in a lower MTX uptake.

Methods: We prospectively measured red blood cell (RBC) folate and MTX levels during four HD-MTX and Leucovorin courses in 43 patients treated according the DCOG ALL-11 protocol with 2-weekly HD-MTX (5 g/m2/dose) and Leucovorin (15 mg/m2/dose) using LC-MS/MS. We estimated a linear mixed model to assess the relationship between these variables over time.

Results: Both RBC MTX-PG and folate levels increased significantly during protocol M. MTX-PG2-5 levels increased most substantially after the first two HD-MTX courses (until median 113.0 nmol/L, IQR 76.8-165.2) after which levels plateaued during the 3d and 4th course (until median 141.3 nmol/L, IQR 100.2-190.2). In parallel, folate levels increased most substantially after the first two HD-MTX courses (until median 401.6 nmol/L, IQR 163.3-594.2) after which levels plateaued during the 3d and 4th course (until median 411.5 nmol/L, IQR 240.3-665.6). The ratio folate/MTX-PG decreased significantly over time, which was mostly due to the relatively higher increase (delta) of MTX-PG.

Conclusion: These results suggest that the increase in RBC folate levels does not seem to have a large effect on RBC MTX levels. Future studies, assessing competition of Leucovorin and MTX on other cellular mechanisms which might negatively affect treatment efficacy, are necessary.

Keywords: Acute lymphoblastic leukemia; Leucovorin; Methotrexate; Pediatric oncology.

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Conflict of interest statement

None of the authors have any competing interests to declare.

Figures

Fig. 1
Fig. 1
Mechanism of action MTX and LV. Overview of the folate pathway with separate folate isoforms and converting enzymes in relation to the mechanism of action of MTX. MTX enters the cell through RFC1, PCFT and MFR. MTX is then polyglutamated (−PG) by FPGS and depolyglutamated by GGH, after which MTX is exported out of the cell by ABC transporters. MTX(−PG) inhibits TS and DHFR. Leucovorin (5-formylTHF) is represented in bold / italic and bypasses the action of DHFR. Abbreviations: ABCB1 - ATP Binding Cassette Subfamily B Member 1; ABCC1–4 - ATP Binding Cassette Subfamily C Member 1–4; ABCG2 - ATP Binding Cassette Subfamily G Member 2; DHF – Dihydrofolate; DHFR – Dihydrofolate Reductase; FPGS – Folylpolyglutamate Synthetase; GGH – Gamma-Glutamyl Hydrolase; MFR – Membrane Folate Transporter; MTHFR - Methylene tetrahydrofolate reductase; MTHFD1 - Methylenetetrahydrofolate Dehydrogenase, Cyclohydrolase And Formyltetrahydrofolate Synthetase 1; PCFT – Proton-Coupled Folate Transporter; RFC1 – Reduced Folate Carrier; SHMT - Serine hydroxymethyltransferase; THF – tetrahydrofolate; TS – Thymidylate Synthase. This Figure was created using Microsoft Office Powerpoint
Fig. 2
Fig. 2
Median intracellular MTX-PG and folate levels during four consecutive courses. Measurements were performed in n = 8 patients at start of protocol M and in n = 43 patients every 2 weeks after a HD-MTX and LV course at time of qualification for the next course. Both intracellular MTX-PG (a) and folate (b) levels increased significantly during protocol M over time (p < 0.001). The ratio folate/MTX-PG (c) decreased significantly over time (p < 0.001). The bold line represents the median levels of patients over time. This Figure was created using Graphpad Prism version 8.3
Fig. 3
Fig. 3
Individual MTX-PG and folate levels over time. Measurements were performed in n = 43 patients every 2 weeks after a HD-MTX and LV course at time of qualification for the next course. Folate levels (blue line) and MTX-PG (red line) levels in 43 individual patients are depicted over time after four HD-MTX and LV courses (a). The ratio folate / MTX-PG (green line) is depicted over time (b) after four HD-MTX and LV courses. This Figure was created using RStudio version 1.1.442
See this image and copyright information in PMC

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