A Simple and Rapid UPLC-UV Method for Detecting DPD Deficiency in Patients With Cancer.

Detecting patients with dihydropyrimidine dehydrogenase (DPD) deficiency is becoming a major concern in clinical oncology. Monitoring physiologic plasma uracil and/or plasma uracil-to-dihydrouracil metabolic ratio is a common surrogate frequently used to determine DPD phenotype without direct measurement of the enzymatic activity. With respect to the increasing number of patients rquiring analysis, it is critical to develop simple, rapid, and affordable methods suitable for routine screening. We have developed and validated a simple and robust ultraperformance liquid chromatography-ultraviolet (UPLC-UV) method with shortened (i.e., 12 minutes) analytical run-times, compatible with the requirements of large-scale upfront screening. The method enables detection of uracil (U) over a range of 5-500 ng/ml (265 nm) and of dihydrouracil (UH2) over a range of 40-500 ng/ml (210 nm) in plasma with no chromatographic interference. When used as part of routine screening for DPD deficiency, this method was fully able to discriminate nondeficient patients (i.e., with U levels < 16 ng/ml) from deficient patients at risk of severe toxicity (i.e., U > 16 ng/ml). Results from 1 month of routine testing are presented and, although no complete deficits were detected, 10.7% of the screened patients presented DPD deficiency and would thus require s decresed dose. Overall, this new method, using a simple preanalytical solid-phase extraction procedure, and based on use of a standard UPLC apparatus, is both cost- and time-effective and can be easily implemented in any laboratory aiming to begin routine DPD testing.

DPD functional tests in plasma, fresh saliva and dried saliva samples as predictors of 5-fluorouracil exposure and occurrence of drug-related severe toxicity.

OBJECTIVE: to evaluate plasma and salivary uracil (U) to dihydrouracil (UH2) ratios as tools for predicting 5-fluorouracil systemic exposure and drug-related severe toxicity, and clinically validate the use of dried saliva spots (DSS) as an alternative sampling strategy for dihydropyrimidine dehydrogenase (DPD) deficiency assessment. METHODS: Pre-chemotherapy plasma, fresh saliva and DSS samples were obtained from gastrointestinal patients (N = 40) for measurement of endogenous U and UH2 concentrations by LC-MS/MS. A second plasma sample collected during 5FU infusion was used for 5FU area under the curve (AUC) determination by HPLC-DAD. Data on toxicity was reported according to CTCAE. RESULTS: 15% of the patients developed severe 5FU-related toxicity, with neutropenia accounting for 67% of the cases. U, UH2 and [UH2,]/[U] were highly correlated between fresh and dried saliva samples (rs = 0.960; rs = 0.828; rs = 0.910, respectively). 5FU AUC ranged from 11.3 to 37.31 mg h L-1, with 46.2% of under-dosed and 10.3% over-dosed patients. The [UH2]/[U] ratios in plasma, fresh saliva and dried saliva samples were moderately correlated with 5FU AUC and adverse events grade, indicating a partial contribution of the variables to drug exposure (r = -0.412, rs = -0.373, rs = 0.377) and toxicity (r = -0.363, rs = -0.523, rs = 0.542). Metabolic ratios were lower in patients with severe toxicity (P < .01 salivary ratios, and P < .5 plasma ratios), and 5FU AUC were in average 47% higher in this group than in moderate toxicity. The diagnostic performance of [UH2]/[U] ratios in fresh saliva and DSS for the identification of patients with severe toxicity were comparable. CONCLUSIONS: The [UH2]/[U] metabolic ratios in plasma, fresh saliva and DSS were significantly associated with 5FU systemic exposure and toxicity degree. This study also demonstrated the applicability of DSS as alternative sampling for evaluating DPD activity.

Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation.

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.

Evaluation of 5-fluorouracil pharmacokinetics in cancer patients with a c.1905+1G>A mutation in DPYD by means of a Bayesian limited sampling strategy.

BACKGROUND AND OBJECTIVE: Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme in the catabolism of 5-fluorouracil (5FU) and DPD deficiency is an important pharmacogenetic syndrome. So far, only very limited information is available regarding the pharmacokinetics of 5FU in patients with a (partial) DPD deficiency and no limited sampling models have been developed taking into account the non-linear pharmacokinetic behaviour of 5FU. The aim of this study was to evaluate the pharmacokinetics of 5FU and to develop a limited sampling strategy to detect decreased 5FU elimination in patients with a c.1905+1G>A-related DPD deficiency. METHODS: Thirty patients, heterozygous for the c.1905+1G>A mutation in DPYD, and 18 control patients received a dose of 5FU 300 mg/m2 and/or 5FU 450 mg/m2, followed by pharmacokinetic analysis of the 5FU plasma levels. A population pharmacokinetic analysis was performed in order to develop a compartmental pharmacokinetic model suitable for a limited sampling strategy. Clinical aspects of treating DPD-deficient patients with 5FU-based chemotherapy were assessed from the retrospectively collected clinical data. RESULTS: In a two-compartment model with Michaelis-Menten elimination, the mean maximum enzymatic conversion capacity (V(max)) value was 40% lower in DPD-deficient patients compared with controls (p < 0.001). Using a limited sampling strategy, with V(max) values calculated from 5FU concentrations at 30 or 60 minutes, significant differences were observed between DPD-deficient patients and controls at both dose levels (p < 0.001). The positive predictive value and negative predictive value for V(max), calculated from 5FU levels at 60 minutes, were 96% and 88%, respectively, in patients treated with a single dose of 5FU 300 mg/m2. All seven DPD-deficient patients (two males and five females) who had been genotyped prior to initiation of standard 5FU-containing chemotherapy developed grade 3-4 toxicity, with one case of lethal toxicity in a female patient. No grade 4 toxicity or lethal outcome was observed in 13 DPD-deficient patients treated with reduced doses of 5FU. The average dose of 5FU in DPD-deficient patients with mild toxicity (grade ≤2) was 61 ± 16% of the normal 5FU dose (n = 10). CONCLUSIONS: Profound differences in the elimination of 5FU could be detected between DPD-deficient patients and control patients. Pharmacokinetic 5FU profiling, using a single 5FU concentration at 60 minutes, may be useful for identification of DPD-deficient patients in order to reduce severe toxicity. Furthermore, treatment of DPD-deficient patients with standard 5FU-containing chemotherapy was associated with severe (lethal) toxicity.