Comparison of a thymine challenge test and endogenous uracil-dihydrouracil levels for assessment of fluoropyrimidine toxicity risk.

PURPOSE: Standard dosages of fluoropyrimidine chemotherapy result in severe toxicity in a substantial proportion of patients, however, routine pre-therapeutic toxicity prediction remains uncommon. A thymine (THY) challenge test can discriminate risk of severe gastrointestinal toxicity in patients receiving fluoropyrimidine monotherapy. We aimed to measure endogenous plasma uracil (U) and its ratio to dihydrouracil (DHU), and assess the performance of these parameters compared with the THY challenge test to evaluate risk of severe toxicity. METHODS: Plasma samples, previously collected from 37 patients receiving 5-fluorouracil (5-FU) or capecitabine monotherapy for a THY challenge test (ACTRN12615000586516; retrospectively registered), were assessed for endogenous plasma concentrations of U and DHU using a validated LC-MS/MS method. Renal function was estimated from blood creatinine, and patients with ≥ grade 3 toxicity (CTCAE v4.0) were classified as cases. RESULTS: There were no differences in median endogenous U plasma concentrations or U/DHU ratios between severe toxicity cases and non-cases. Significant differences between cases and non-cases were noted when these measures were normalised to the estimated renal function (CrCL), Unorm p = 0.0004; U/DHUnorm p = 0.0083. These two parameters had a sensitivity of 29%, compared with 57% for the THY challenge test in the same patients. Genotyping for clinically relevant DPYD variants was inferior to either of these pyrimidine phenotyping tests (sensitivity of 14%). CONCLUSIONS: The endogenous uracil-based parameters, adjusted to CrCL, were more predictive of increased risk of severe fluoropyrimidine toxicity than DPYD genotyping. However, endogenous U measurement detected fewer cases of severe toxicity than the THY challenge test.

5-Fluorouracil Response Prediction and Blood Level-Guided Therapy in Oncology: Existing Evidence Fundamentally Supports Instigation.

5-Fluorouracil (5-FU) response prediction and therapeutic drug monitoring (TDM) are required to minimize toxicity while preserving efficacy. Conventional 5-FU dose normalization uses body surface area. It is characterized by up to 100-fold interindividual variability of pharmacokinetic (PK) parameters, and typically >50% of patients have plasma 5-FU concentrations outside the optimal range. This underscores the need for a different dose rationalization paradigm, hence there is a case for 5-FU TDM. An association between 5-FU PK parameters and efficacy/toxicity has been established. It is believed that 5-FU response is enhanced and toxicity is reduced by PK management of its dosing. The area under the concentration-time curve is the most relevant PK parameter associated with 5-FU efficacy/toxicity, and optimal therapeutic windows have been proposed. Currently, there is no universally applied a priori test for predicting 5-FU response and identifying individuals with an elevated risk of toxicity. The following two-step strategy: prediction of response/toxicity and TDM for subsequent doses seems plausible. Approximately 80% of 5-FU is degraded in a three-step sequential metabolic pathway. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme. Its deficiency can cause toxicity with standard 5-FU doses. DPD also metabolizes uracil (U) into 5,6-dihydrouracil (UH2). The UH2/U ratio is an index of DPD activity and a credible biomarker of response and toxicity. This article outlines the UH2/U ratio as a parameter for 5-FU response/toxicity prediction and highlights key studies emphasizing the value of 5-FU TDM. Broad application of 5-FU response/toxicity prediction and blood level-guided therapy remains unmet, despite ever-increasing clinical interest. Considered collectively, existing evidence is compelling and fundamentally supports universal instigation of response/toxicity prediction and TDM.

Development, validation and application of a novel liquid chromatography tandem mass spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-fluorouracil, α-fluoro-ß-ureidopropionic acid and α-fluoro-ß-alanine in human plasma.

The plasma 5,6-dihydrouracil/uracil (UH2/U) ratio is a possible phenotypic marker of dihydropyrimidine dehydrogenase (DPD) activity, hence an index of 5-fluorouracil (5-FU) response and toxicity. Studies have re-affirmed the value of 5-FU and 5,6-dihydro-5-fluorouracil (FUH2) for therapeutic drug monitoring (TDM). However, FUH2 has limited stability in plasma, necessitating expedited plasma separation and freezing, where routine compliance may not be easy. The metabolites α-fluoro-ß-ureidopropionic acid (FUPA) and α-fluoro-ß-alanine (FßAL) are stable in plasma and are probable candidates for TDM. This paper describes development, validation and application of an LC-MS/MS assay quantifying U, UH2, 5-FU, FUH2, FUPA and FßAL in human plasma. Extraction was by salt-assisted liquid-liquid extraction (LLE) in two-stages with pH adjustment. The supernatants were mixed, dried and reconstituted. Analytes were resolved on the Luna PFP (2) (150×2.00mm, 3µ) column by gradient elution and analyzed by tandem mass spectrometry via electrospray ionisation in positive polarity. The analytical response was linear (r2≥0.99) in the concentration (ng/mL) ranges: 50-10 000 for FßAL and FUH2, 50-5 000 for FUPA, 50-100 000 for 5-FU, 5-200 for U and 10-400 for UH2. Within- and between-run accuracy and precision were ≤ 10.2% and ≤ 9.8% respectively across the QC range and inclusive of LLOQ. The internal standard (IS) normalised matrix effects were within 93-112% with CV of ≤ 9.7% and normalised recoveries were within 91-107% with CV of ≤ 9.8%. This robust assay was successfully applied to samples from rectal and colorectal cancer patients (n=10) on 5-FU. Deviations ≤ 2.0% from the mean values were observed when incurred samples were reanalysed.