Semi-automated radioassay for determination of dihydropyrimidine dehydrogenase (DPD) activity screening cancer patients for DPD deficiency, a condition associated with 5-fluorouracil toxicity

Johnson, Martin R.; Yan, Jieming; Shao, Lingning; Albin, Nicolas; Diasio, Robert B.

doi:10.1016/s0378-4347(97)00253-3

Cited by 81 publications

(70 citation statements)

References 14 publications

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“…PBMC DPD activity was determined for all subjects as described previously (11,12). To minimize interassay variation in enzyme activity, 60 mL whole blood was collected into heparinized vacutainers at f12 p.m. on the day of testing and processed within 10 minutes of collection.…”

Section: Methodsmentioning

confidence: 99%

“…The concentration of cytosolic protein was quantified by the Bradford method (13). A reaction mixture containing 250 Ag cytosolic protein, NADPH, buffer A, and [6][7][8][9][10][11][12][13][14] C]-5-FU was incubated for 30 minutes. Every 5 minutes, 130 AL aliquots were removed and added to an equal volume of ice-cold ethanol.…”

Section: Methodsmentioning

confidence: 99%

“…C]-5-FUH 2 were separated and quantified using a previously described reverse-phase high-performance liquid chromatography method (11,12). The amount of [6-14 C]-5-FUH 2 formed at each time point (Y axis) was plotted against time (X axis).…”

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confidence: 99%

“…Linear regression analysis was used to calculate the equation of the line and determine the formation rate of [6][7][8][9][10][11][12][13][14] C]-5-FUH 2 . DPD enzyme activity was calculated by dividing the formation rate of [6][7][8][9][10][11][12][13][14] C]-5-FUH 2 by the amount of protein used in the reaction mixture (i.e., nmol/min/ mg protein). Subjects were considered to be partially DPD deficient by radioassay when their fresh PBMC DPD activity was <0.18 nmol/min/ mg protein (11).…”

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confidence: 99%

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The Uracil Breath Test in the Assessment of Dihydropyrimidine Dehydrogenase Activity: Pharmacokinetic Relationship between Expired 13CO2 and Plasma [2-13C]Dihydrouracil

Mattison

Fourie

Hirao

et al. 2006

Clinical Cancer Research

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Purpose: Dihydropyrimidine dehydrogenase (DPD) deficiency is critical in the predisposition to 5-fluorouracil dose-related toxicity. We recently characterized the phenotypic [2-13 C]uracil breath test (UraBT) with 96% specificity and 100% sensitivity for identification of DPD deficiency. In the present study, we characterize the relationships among UraBT-associated breath C]uracil concentrations were determined over 180 minutes using IR spectroscopy and liquid chromatography-tandem mass spectrometry, respectively. Pharmacokinetic variables were determined using noncompartmental methods. Peripheral blood mononuclear cell (PBMC) DPD activity was measured using the DPD radioassay. Results: The UraBT identified 19 subjects with normal activity, 11 subjects with partial DPD deficiency, and 1 subject with profound DPD deficiency with PBMC DPD activity within the corresponding previously established ranges. UraBT breath Dihydropyrimidine dehydrogenase (EC 1.3.1.2, DPD) is the rate-limiting enzyme in uracil and 5-fluorouracil (5-FU) catabolism, converting >80% of an administered dose of 5-FU to inactive metabolites (1, 2). The initial step of catabolism is mediated by DPD converting 5-FU to 5-dihydrofluorouracil, with subsequent catabolism by dihydropyrimidinase and h-ureidopropionase enzymes to ultimately produce fluoroh-alanine, ammonia, and CO 2 . The latter final metabolic endproducts are excreted in the urine and breath (3).The pharmacogenetic syndrome of complete and partial DPD deficiency is prevalent in f0.1% and 3% to 5% of the general population, respectively (4). DPD deficiency is a significant pharmacogenetic factor in the predisposition of cancer patients to increased risk of altered 5-FU pharmacokinetics and associated toxicity. Specifically, 60% of patients presenting with severe 5-FU-related hematologic toxicity showed reduced DPD activity (5).Recent studies have investigated the predictive value of the ratio of plasma dihydrouracil area under the curve (AUC) to uracil AUC (DUUR) for the assessment of DPD activity and potential individualization of 5-FU therapy. Specifically, 5-FU dose optimization may be based on the plasma DUUR observed before 5-FU administration (6). Jiang et al. have also showed that the pre-5-FU treatment DUUR may be a good index of DPD activity (7,8).Our laboratory recently reported the rapid noninvasive phenotypic [2-13 C]uracil breath test (UraBT) for assessment of DPD activity with 96% specificity and 100% sensitivity (9). Application of the UraBT to a large population of cancer-free subjects (n = 255) showed an observed 86% sensitivity (with 12 of 14 DPD-deficient subjects identified as DPD deficient) and

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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The Uracil Breath Test in the Assessment of Dihydropyrimidine Dehydrogenase Activity: Pharmacokinetic Relationship between Expired 13CO2 and Plasma [2-13C]Dihydrouracil

Mattison

Fourie

Hirao

et al. 2006

Clinical Cancer Research

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“…PBMCs are used as a surrogate for the determination of DPD activity. Radio-assays with 14C-fluorouracil have been developed [49,50].The mean value of DPD activity in cancer patients was estimated to be (189-222 pmol/min/mg) [49,51].…”

Section: Phenotyping Strategiesmentioning

confidence: 99%

Individualization of 5-Fluorouracil in the Treatment of Colorectal Cancer

Alnaim¹

2010

SRX Pharmacology

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Chemotherapeutic agents are generally characterized by a large inter-individual pharmacokinetic variability. The balance of efficacy and toxicity is critical and the imbalance can have devastating effects on patients. Standard dosing methods are inadequate in optimizing systemic exposure .Therapeutic drug monitoring (TDM) has the potential to improve the clinical use of chemotherapy agents. TDM has been successfully applied to optimize a few anticancer treatments including carboplatin, methotrexate, and 6-mercaptopurine. 5-Flurouracil (5-FU) is considered the backbone in treatments of advanced CRC. Toxic side effects remain a significant problem despite increased safety of newer regimens. Molecular mechanisms that control DPD-activity are complex and have not been fully elucidated and cannot be used effectively to individualize Fluorouracil dosing as there is no established standard genetic test for DPD deficiency. Current phenotypic methods to measure DPD-activity are cumbersome. A reliable relationship between DPD genotype and 5-FU toxicity phenotype has not been established. Standard dosing of 5FU is by body surface area (BSA). Low correlation exists between exposure and BSA. A better predictor of total exposure is the area under the curve (AUC). Toxicity and efficacy have been correlated to AUC with target of 24-30 (mg.h/l). The therapeutic window is very narrow and difficult to attain by clinical follow-up alone. TDM has been shown effective in adjusting the dose based on AUC.

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Complete DPYD genotyping combined with dihydropyrimidine dehydrogenase phenotyping to prevent fluoropyrimidine toxicity: A retrospective study

De Metz,

Hennart,

Aymes

et al. 2024

Cancer Medicine

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IntroductionIn April 2019, French authorities mandated dihydropyrimidine dehydrogenase (DPD) screening, specifically testing uracilemia, to mitigate the risk of toxicity associated with fluoropyrimidine‐based chemotherapy. However, this subject is still of debate as there is no consensus on a standardized DPD deficiency screening test. We conducted a real‐life retrospective study with the aim of assessing the impact of DPD screening on the occurrence of severe toxicity and exploring the potential benefits of complete genotyping using next‐generation sequencing.MethodsAll adult patients consecutively treated with 5‐fluorouracil (5‐FU) or its oral prodrug at six cancer centers between March 2018 and February 2019 were considered for inclusion. Dihydropyrimidine dehydrogenase deficiency screening included gene encoding DPD (DPYD) genotyping using complete genome sequencing and DPD phenotyping (uracilemia or dihydrouracilemia/uracilemia ratio) or both tests. Associations between each DPD screening method and (i) severe (grade ≥3) early toxicity and (ii) fluoropyrimidine dose reduction in the second chemotherapy cycle were evaluated using multivariable logistic regression analysis. Furthermore, we assessed the concordance between DPD genotype and phenotype using Cohen's kappa.ResultsA total of 551 patients were included. Most patients were tested for DPD deficiency (86%) including DPYD genotyping only (6%), DPD phenotyping only (8%), or both (72%). Complete DPD deficiency was not detected in the study population. Severe early toxicity events were observed in 73 patients (13%), with two patients (0.30%) presenting grade 5 toxicity. Despite the numerically higher toxicity rate in untested patients, the occurrence of severe toxicity was not significantly associated with the DPD screening method (p = 0.69). Concordance between the DPD genotype and phenotype was weak (Cohen's kappa of 0.14).ConclusionDue to insufficient numbers, our study was not able to demonstrate any added value of DPYD genotyping using complete genome sequencing to prevent 5‐FU toxicity. The optimal strategy for DPD screening before fluoropyrimidine‐based chemotherapy requires further clinical evaluation.

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Semi-automated radioassay for determination of dihydropyrimidine dehydrogenase (DPD) activity screening cancer patients for DPD deficiency, a condition associated with 5-fluorouracil toxicity

Cited by 81 publications

References 14 publications

The Uracil Breath Test in the Assessment of Dihydropyrimidine Dehydrogenase Activity: Pharmacokinetic Relationship between Expired 13CO2 and Plasma [2-13C]Dihydrouracil

The Uracil Breath Test in the Assessment of Dihydropyrimidine Dehydrogenase Activity: Pharmacokinetic Relationship between Expired 13CO2 and Plasma [2-13C]Dihydrouracil

Individualization of 5-Fluorouracil in the Treatment of Colorectal Cancer

Complete DPYD genotyping combined with dihydropyrimidine dehydrogenase phenotyping to prevent fluoropyrimidine toxicity: A retrospective study

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