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Mechanisms of Natural Resistance to Antifolates in Human Soft Tissue Sarcomas¹

Laboratory of Molecular Pharmacology [W-W. L., J. T. L., W. P. T., T. M. T., J. R. B.] and Department of Surgery [M. F. B.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Efforts to use fresh human sarcoma cells for evaluating antifolate resistance with an in situ thymidylate synthesis assay using 5-[³H] deoxyuridine were unsuccessful because of low thymidylate synthesis activity in enzymatically disaggregated tumors. By incubating tumor cell suspensions in supplemented RPMI-1640 medium with 10% fetal bovine serum for 3 days, activity of the in situ thymidylate synthesis assay markedly increased (1.42 versus 0.03 pmol/h/10⁷ cells), thus allowing 75% of samples to be evaluated for antifolate sensitivity. By criteria developed with a methotrexate-resistant and -sensitive cell line, this assay indicated that most sarcomas are naturally resistant to methotrexate (12 of 15). Natural resistance to 10-ethyl-10-deazaaminopterin and trimetrexate was also observed in 60% of the samples (nine of 15, respectively). The results from the 3-day in situ assay were confirmed by specific tests for resistance mechanisms in most sarcoma samples. The resistance mechanisms detected were impaired polyglutamylation, an increased level of dihydrofolate reductase, and amplification of this gene. These results encourage further exploration of this assay to predict response to antifolates in individual patients and to evaluate efficacy of new antifolates as candidates for clinical trial.

¹ Supported by Grant CA-47179. J. R. B. is American Cancer Society Professor of Medicine and Pharmacology.

² To whom requests for reprints should be addressed.

Received 8/ 8/91. Accepted 1/ 6/92.

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