Resistance to methotrexate due to gene amplification in a patient with acute leukemia

Abstract

A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR. Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562. Additional DHFR fragments were detected, not seen in the K562 blot, suggesting the presence of pseudogenes, or a result of gene rearrangements occurring as part of the amplification process. Resistance to MTX in this patient was therefore ascribed to gene amplification and overproduction of DHFR.