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.
To analyze if methotrexate (MTX) resistance arises from gene amplification in a patient treated clinically with MTX, the cytogenetic and drug sensitivity profile of the tumor colony forming units (TCFUs) from a 58-year-old woman with stage III well-differentiated ovarian serous adenocarcinoma was studied. This patient had not received treatment directed against her tumor for nine months before this study, but had received oral-dose MTX (2.5 mg, twice weekly) for three years for the treatment of psoriasis. Analysis of TCFUs grown in nucleoside-free media demonstrated MTX resistance at concentrations of up to 100 micrograms/mL (2.2 X 10(-4)M). Cytologic evidence for dihydrofolate reductase (DHFR) gene amplification in TCFUs was determined by in situ hybridization, using radiolabeled cDNA to DHFR mRNA. Results localized the DHFR sequences to an abnormally staining region present on chromosome 4q. This study supports the notion that alterations in gene dosage (that is, gene amplification) play a role in the development of drug resistance in spontaneous human cancers.
A group of 158 patients with small cell carcinoma of the lung were followed for 174.5 person-years of observation to determine the risk of acute leukemia. Three cases of acute nonlymphocytic leukemia were observed at 2.3, 2.7, and 3.0 years. The relative risk of developing leukemia was 316 (95% confidence limit, 76-818) and the actuarial risk was 25% +/- 13% at 3.1 years. The relative risk for leukemia was significantly increased in these patients (p less than 0.0001).
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