Gene amplification in a leukemic patient treated with methotrexate.

Abstract

Resistance to methotrexate (MTX) has been shown in mouse, hamster, and human cell lines grown in sequentially increased MTX concentrations to be due to increased dihydrofolate reductase (DHFR) synthesis and a proportional increase in DHFR gene copy number. Leukemia cells of a patient were studied to assess change in DHFR gene copy number after MTX treatment. The patient presented with chronic myeloid leukemia which rapidly evolved into blast crisis; 90% of peripheral white cells were lymphoblasts. Treatment included intrathecal and intravenous MTX; the lymphoblasts were reduced to undetectable levels. Three months later a second blast crisis occurred; 90% of peripheral white cells were lymphoblasts. Cells from each blast crisis were obtained by leukapheresis and stored for study. Quantification of DHFR gene copy number in DNA from lymphoblasts before and after MTX treatment was done: a radiolabeled cloned cDNA constituting the mouse DHFR coding sequence was used to probe high molecular weight pretreatment and posttreatment DNA bound to nitrocellulose filters. Posttreatment DNA contained two- to three-fold more DHFR gene sequences than pretreatment DNA. Quantitative Southern blotting of EcoRI-digested pretreatment and posttreatment DNA confirmed amplification of the DHFR gene. Karyotype analysis showed no double minute chromosomes or homogeneously staining regions. This is the first study demonstrating DHFR gene amplification in leukemia cells sampled in vivo from a patient treated with MTX.