High-dose leucovorin addition to fluorouracil (FUra) therapy has recently been shown to markedly increase response rates in patients with metastic colorectal adenocarcinoma. The rationale for leucovorin addition is increased percentage and duration of thymidylate synthase (TS) inhibition in tumors by 5- fluorodeoxyuridylate (FdUMP), by enhanced stabilities in TS FdUMP-folate ternary complexes. For greatest effect, leucovorin must be metabolized to 5,10-methylenetetrahydrofolate (CH2FH4) and its polyglumates, which are the only folates that form covalently-bonded ternary complexes. We have developed and shown the clinical applicability of assays of parameters related to TS inhibition performed on small biopsies from over 80 patients receiving FUra. These assays include free and total TS levels by (3H) FdUMP ligand-binding and (3H)dUMP tritium release; levels of FdUMP, total FdUMP-binding folates, and CH2FH4 levels by use of (3H) FdUMP binding to L. Casei TS; dUMP by (14C)dTMP synthesis; and levels of FUra incorporated into RNA by isolation and isotopic derivatization of fluorouridine. Recently, the clinical feasibility and usefulness of serial biopsies of tumor after FUra or leucovorin has been demonstrated. Two groups of patients receiving FUra plus high-dose leucovorin are proposed for study of correlations between clinical outcome and these biochemical parameters in biopsies of tumors. Patients with locally progressive, surgically incurable breast adenocarinoma will be treated with weekly FUra plus leucovorin give i.v. by a bolus schedule. Patients with metastatic colorectal adenocarcinoma will receive FUra and leucovorin by prolonged infusion. Biopsies of tumor will be obtained on days 1, 15 and 29 of therapy, and at relapse or progression. Superficial breast tumors will be sampled by serial biopsy over the period of drug administration. Intrahepatic colon tumor will be sampled by skinny needle aspirate (single time point). Results of CH2FH4 assays of tumor biopsies will be used as a basis for dose escalation of leucovorin in all cases, to achieve a proposed effective concentration of 5 muM. Additional observations in selected patients will include plasma pharmacokinetic studies of leucovorin, 5-methyltetrahydrofolate, FUra, and dihydroFUra; polyglutamate chain-length determination of folate pools in tumors; levels of thymidine in large tumor biopsy specimens; and TS and dihydrofolate reductase gene copy and mRNA levels. Statistical evaluations will include analysis of variance with repeated measures, to determine time trends in the biochemical data and differences between responders and non-responders, bolus vs infusion schedule, and between groups based on parameters of TS inhibition.
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