Dihydropyrimidine Dehydrogenase Phenotyping Using Pretreatment Uracil: A Note of Caution Based on a Large Prospective Clinical Study.

In clinical practice, 25-30% of the patients treated with fluoropyrimidines experience severe fluoropyrimidine-related toxicity. Extensively clinically validated DPYD genotyping tests are available to identify patients at risk of severe toxicity due to decreased activity of dihydropyrimidine dehydrogenase (DPD), the rate limiting enzyme in fluoropyrimidine metabolism. In April 2020, the European Medicines Agency recommended that, as an alternative for DPYD genotype-based testing for DPD deficiency, also phenotype testing based on pretreatment plasma uracil levels is a suitable method to identify patients with DPD deficiency. Although the evidence for genotype-directed dosing of fluoropyrimidines is substantial, the level of evidence supporting plasma uracil levels to predict DPD activity in clinical practice is limited. Notwithstanding this, uracil-based phenotyping is now used in clinical practice in various countries in Europe. We aimed to determine the value of pretreatment uracil levels in predicting DPD deficiency and severe treatment-related toxicity. To this end, we determined pretreatment uracil levels in 955 patients with cancer, and assessed the correlation with DPD activity in peripheral blood mononuclear cells (PBMCs) and fluoropyrimidine-related severe toxicity. We identified substantial issues concerning the use of pretreatment uracil in clinical practice, including large between-center study differences in measured pretreatment uracil levels, most likely as a result of pre-analytical factors. Importantly, we were not able to correlate pretreatment uracil levels with DPD activity nor were uracil levels predictive of severe treatment-related toxicity. We urge that robust clinical validation should first be performed before pretreatment plasma uracil levels are used in clinical practice as part of a dosing strategy for fluoropyrimidines.

Evaluation of clinical implementation of prospective DPYD genotyping in 5-fluorouracil- or capecitabine-treated patients.

AIM: Fluoropyrimidines are commonly used anti-cancer drugs, but lead to severe toxicity in 10-30% of patients. Prospective DPYD screening identifies patients at risk for toxicity and leads to a safer treatment with fluoropyrimidines. This study evaluated the routinely application of prospective DPYD screening at the Leiden University Medical Center. METHODS: Prospective DPYD screening as part of routine patient care was evaluated by retrospectively screening databases and patient files to determine genotype, treatment, dose recommendations and dose adjustments. RESULTS: 86.9% of all patients with a first fluoropyrimidine prescription were screened. Fourteen out of 275 patients (5.1%) carried a DPYD variant and received a 25-50% dose reduction recommendation. None of the patients with a DPYD variant treated with a reduced dose developed toxicities. CONCLUSION: Prospective DPYD screening can be implemented successfully in a real world clinical setting, is well accepted by physicians and results in low toxicity.

Evaluation of an oral uracil loading test to identify DPD-deficient patients using a limited sampling strategy.

AIM: Dihydropyrimidine dehydrogenase (DPD) deficiency can lead to severe toxicity following 5-fluorouracil (5FU) or capecitabine (CAP) treatment. Uracil (U) can be used as a probe to determine systemic DPD activity. The present study was performed to assess the sensitivity and specificity of a U loading dose for detecting DPD deficiency. METHODS: Cancer patients with Common Toxicity Score (CTC) grade III or IV toxicity after the first or second cycle of 5-FU or CAP treatment were asked to participate. Based on DPD activity in PBMCs, patients were divided into two groups: DPD activity in peripheral blood mononuclear cells (PBMCs) <5 nmol mg(-1) *h(-1) (deficient group) and ≥ 5 nmol mg(-1) *h(-1) . U 500 mg m(-2) was administered orally and plasma concentrations of U and dihydrouracil (DHU) were determined. In the deficient group, polymerase chain reaction amplification of all 23 coding exons and flanking intronic regions of DPYD was performed. A U pharmacokinetic model was developed and used to determine the maximum enzymatic conversion capacity (Vmax ) of the DPD enzyme for each patient. The sensitivity and specificity of Vmax, U concentration and the U/DHU concentration ratio were determined. RESULTS: A total of 47 patients were included (19 DPD deficient, 28 DPD normal). Of the pharmacokinetic parameters investigated, a sensitivity and specificity of 80% and 98%, respectively, was obtained for the U/DHU ratio at t = 120 min. CONCLUSIONS: The high sensitivity of the U/DHU ratio at t = 120 min for detecting DPD deficiency, as defined by DPD activity in PBMCs, showed that the oral U loading dose can effectively identify patients with reduced DPD activity.

Influence of metastatic disease on the usefulness of uracil pharmacokinetics as a screening tool for DPD activity in colorectal cancer patients.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD) deficiency can lead to severe toxicity in patients treated with a standard dose of a fluoropyrimidine such as 5-fluorouracil or capecitabine (CAP). Administration of oral uracil and subsequent measurement of uracil and dihydrouracil (DHU) plasma concentrations has been used to identify patients with DPD deficiency. Liver metastasis might influence systemic DPD activity. The aim of the study was to investigate the effect of metastatic disease on the pharmacokinetics of uracil and DHU after oral administration of uracil. METHODS: 500 mg/m(2) uracil was administered orally to 12 subjects with stages II-III colorectal cancer (CRC) who were treated in the adjuvant setting and to 12 subjects with stage IV metastasized CRC, all treated with CAP containing therapy. All subjects had a normal DPD activity defined as >6 nmol/mg/h determined in peripheral blood mononuclear cells. RESULTS: The mean uracil clearance [CL 51.7 (SD 6.4) vs. 46.7 (SD 13.0) l/h], area under the curve [AUC0-220min 20.6 (SD 6.4) vs. 21.0 (SD 5.7) h mg/l], elimination half-life [t 1/2 21 (SD 7) vs. 21 (SD 8) min], maximum concentration time [T max 27 (SD 9) vs. 25 (SD 9) min], volume of distribution [V 26.58 (SD 10.11) vs. 21.10 (SD 8.48) l] and the elimination constant [k el 2.01 (SD 0.56) vs. 2.41 (SD 0.72) h(-1)] did not differ significantly (p > 0.05) non-metastatic CRD versus metastatic CRC. CONCLUSIONS: Metastasis does not alter uracil pharmacokinetics and is similar in CRC patients with and without metastasis. Therefore, the uracil test dose could be used as a DPD phenotype test in both adjuvantly treated and metastatic CRC patients using similar cutoff criteria to identify patients with DPD deficiency.

Pharmacokinetics of orally administered uracil in healthy volunteers and in DPD-deficient patients, a possible tool for screening of DPD deficiency.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD) deficiency can lead to severe toxicity in patients treated with standard doses of 5-fluorouracil (5-FU). Oral uracil administration and subsequent measurement of uracil and dihydrouracil (DHU) plasma concentrations might detect patients with DPD deficiency. This study compares the pharmacokinetics of uracil and DHU after oral uracil administration in subjects with normal and deficient DPD status. METHODS: Five hundred milligrams of uracil per metre square was administered orally to 11 subjects with normal DPD status and to 10 subjects with reduced DPD activity. Repeated administration (n = 3) of this dose was performed in 4 subjects, and 1,000 mg uracil/m(2) was administered to 4 subjects to assess intra-individual variation and linearity of pharmacokinetics. RESULTS: In subjects with normal DPD status, 500 mg/m(2) uracil resulted in uracil C (max) levels of 14.4 ± 4.7 mg/L at T (max) = 30.0 ± 11.6 min, and in DPD-deficient subjects, 20.0 ± 4.5 mg/L at 31.5 ± 1.1 min. The uracil AUC(0>180) was 31.2 ± 5.1 mg L/h in DPD-deficient subjects, which was significantly higher (P < 0.05) than in the subjects with normal DPD status (13.8 ± 3.9 mg L/h). Repeated uracil dosing showed reproducible uracil PK in subjects with normal DPD status, and dose elevation of uracil suggested linear pharmacokinetics. CONCLUSION: The pharmacokinetics of uracil differs significantly between subjects with a normal DPD activity and those with a deficient DPD status. The AUC and C (max) of uracil can be useful as a diagnostic tool to differentiate patients with regard to DPD status.