Mechanisms of innate resistance to thymidylate synthase inhibition after 5-fluorouracil

Fifty-four patients with metastatic adenocarcinoma received i.v. bolus 5-fluorouracil, 500 mg/m2, prior to surgical biopsy of tumor at 20-400 min, for analysis of biochemical parameters of resistance to thymidylate synthase (TS) inhibition. The majority of patients, 37, had colon or rectal adenocarcinoma, five had breast cancer, five had gastric primary disease, four had pancreatic adenocarcinoma, and three had hepatocellular adenocarcinoma. Fluorodeoxyuridylate (FdUMP) was assayed by isotope dilution of [3H]FdUMP binding to bacterial TS; free and total TS was determined by [3H]FdUMP binding; and deoxyuridylate (dUMP) was assayed by conversion to [14C]thymidylate. Free levels of TS were lower in breast cancers, 0.08 +/- 0.06 pmol/g, than in other histologies (overall average, 1.41 +/- 2.25), associated with significantly greater percentages of TS inhibition (88.6% versus 62.0% overall). Colorectal tumors showed significantly greater FdUMP levels than other gastrointestinal malignancies, associated with somewhat lower free TS values. Plots of FdUMP levels, or (FdUMP/dUMP) x 100 values versus percentages of TS inhibition suggested minima of 75 pmol/g and 0.10, respectively, for achieving maximal enzyme inhibition. Analyses of normal tissues showed: poor TS inhibition in liver and normal colonic mucosa, related to low FdUMP levels; and very high dUMP levels in bone marrow leukocytes suggestive of reactive increases in dUMP as an important mechanism of recovery in this tissue. Among the 30 of the 37 colorectal tumors that showed suboptimal (less than 85%) inhibition of TS, 16 (53%) showed FdUMP levels less than 75 pmol/g, 8 (27%) showed relatively high dUMP levels (over 35 nmol/g), and 16 (53%) showed poor efficiency of inhibition of TS, with the major overlap between these mechanisms of resistance being high dUMP and poor binding in 6 (20%). These data provide a strong rationale for administration of leucovorin to the majority of patients receiving 5-fluorouracil, since increased intratumoral reduced folates potentially can overcome multiple mechanisms of resistance including low FdUMP, high dUMP, and high total TS levels, in addition to that caused by isolated folate deficiency.