Mechanisms of resistance to methotrexate in childhood acute lymphoblastic leukemia: circumvention of thymidylate synthase inhibition

Purpose: In about 25% of patients suffering from acute lymphoblastic leukem ia (ALL) treatment failures occur that are most likely due to development o f resistance to methotrexate (MTX). Blasts from patients with ALL were eval uated for MTX uptake, formation of long-chain MTX polyglutamates (MTX-Glu(5 +6)), cytotoxicity and thymidylate synthase inhibition by MTX and compared to blasts from patients with acute myelogenous leukemia (AML). Methods: Rad ioactively labeled MTX-Glu(n) were analyzed by means of HPLC. Thymidylate s ynthase activity was measured by a tritium-release assay. Cytotoxicity was determined by trypan blue exclusion. Results: In most ALL blasts (n = 9) la rge amounts of MTX-Glu(5+6) (1.06-7.03 pmol/10(7) cells) and high cytotoxic ity (43.5%-92.7%) were found, while in others small amounts of MTX-Glu(5+6) (0.0-0.39 pmol/10(7) cells) caused only weak cytotoxicity (6.0%-27.9%) (n = 5, 2 relapsed patients). Resistance to MTX in blasts from AML patients (n = 5) was also caused by reduced synthesis of MTX-Glu(5+6) (0.00.42 pmol/10 (7) cells). In contrast, some ALL blasts (n = 7, 4 relapsed patients) were able to survive MTX treatment despite large amounts of MTX-Glu(5+6) (1.5-5. 05 pmol/10(7) cells) and extensive thymidylate synthase inhibition. Conclus ions: Since the majority of ALL patients were examined at first diagnosis, an inherent mechanism of resistance seems most likely. We propose a mechani sm based on the switch of thymidylate synthesis to the salvage pathway.