The Pharmacogenomics Journal (2013) 13, 389–395 & 2013 Macmillan Publishers Limited All rights reserved 1470-269X/13 www.nature.com/tpj REVIEW Evaluation of predictive tests for screening for dihydropyrimidine dehydrogenase deficiency MC van Staveren1, H Jan Guchelaar2, ABP van Kuilenburg3, H Gelderblom4 and JG Maring5 5-Fluorouracil (5-FU) is rapidly degraded by dihyropyrimidine dehydrogenase (DPD). Therefore, DPD deficiency can lead to severe toxicity or even death following treatment with 5-FU or capecitabine. Different tests based on assessing DPD enzyme activity, genetic variants in DPYD and mRNA variants have been studied for screening for DPD deficiency, but none of these are implemented broadly into clinical practice. We give an overview of the tests that can be used to detect DPD deficiency and discuss the advantages and disadvantages of these tests. The Pharmacogenomics Journal (2013) 13, 389–395; doi:10.1038/tpj.2013.25; published online 16 July 2013 Keywords: dihyropyrimidine dehydrogenase; 5-fluorouracil; genotyping; pharmacokinetics; phenotyping; uracil INTRODUCTION Pyrimidinemia’, ‘uracil’, ‘capecitabine’, ‘dihydrouracil dehydrogenase The fluoropyrimidine 5-fluorouracil (5-FU) and its prodrug (nadp)’, ‘dihydrouracil dehydrogenase’, ‘dihydropyrimidine dehydrogen- ase’, ‘dihydrouracil dehydrogenase (nad þ )’ and ‘dpd’. The search resulted capecitabine, are the cornerstone of treatment of numerous types in 397, 378, 568 and 28 hits for MEDLINE, EMBASE, Web of Science and of cancer. The use of fluoropyrimidines is associated with Cochrane, respectively. The unique hits collected from these databases numerous side effects, such as myelosuppression, hand-foot were selected and further limited to English language papers from 1980 to syndrome, mucositis, diarrhea and occasionally cardiac toxicity. 1 January 2012. Papers describing studies aimed at testing DPD activity After parenteral administration of 5-FU, 70–90% of the parent and/or DPD deficiency in volunteers or patients were selected. Cross drug is degraded by dihydropyrimidine dehydrogenase (DPD).1–5 references were identified from bibliographies from the selected studies. A small proportion of patients develop extreme toxicity after Only articles that describe the complete performance of the test were administration of a fluoropyrimidine due to a partial or complete included; reviews and papers describing in vitro studies including DPD DPD deficiency and hence a strongly reduced capacity to degrade activity in cancer cells or studies in animals were neglected. The collected publications were divided into three categories, describing tests aimed 5-FU.6–10 In case of complete DPD deficiency, 5FU treatment may 11 at assessing DPD enzyme activity (38 articles), genetic variants in DPYD even result in a lethal outcome. It was initially estimated that in (20 articles) and mRNA variants (3 articles). 3–5% of Caucasians the activity of DPD is strongly reduced due to (epi)genetic variations in the gene encoding DPD.4,12 However, this percentage has been disputed as there is still no consensus Tests aimed at assessing (surrogates for) DPD enzyme activity on the definition of DPD deficiency, and therefore the incidence Several tests have been described to assess the activity of the enzyme of DPD deficiency reported in numerous studies is strongly DPD. dependent on the method used to assess DPD deficiency13 and the cutoff level chosen to define DPD deficiency.5 DPD activity in peripheral blood mononuclear cells (PBMCs). The majority of DPD is reported to be in the liver,1,14 but DPD activity in other tissues such Prospective testing for DPD deficiency in patients might as lymphocytes contribute to metabolism of fluoropyrimidines as well.15 prevent DPD-deficient patients from severe toxicity or even death. The liver DPD activity in patients revealed a strong correlation with DPD In this review, we describe current methods for determination of activity in PBMCs.16 The mean DPD activity in PBMCs of patients with a DPD deficiency. We discuss the potential and limitations of these partial DPD deficiency is approximately 48% to that observed in the tests for routine clinical use. In addition, we have defined normal population due to heterozygosity for a pathological mutation.17 recommendations that can help successful implementation of a The methodology of the test includes incubating isolated lymphocytes pre-emptive testing strategy to predict fluoropyrimidine-related with radioactive labeled 5-FU or thymine after which the degradation toxicity. products are measured by high-performance liquid chromatography (HPLC) with a radioisotope flow detector.2,18–20 A HPLC-electrospray tandem mass spectrometry (HPLC MS/MS) method has also been developed.17,21 The use of a porous graphitic carbon column22 results in METHODS a HPLC process that is highly pH stable compared with the reversed-phase To identify studies describing diagnostic tests to detect DPD deficiency, C-18 column, and the detection limit was at least similar to the C-18 a systematic MEDLINE, EMBASE, Web of Science and Cochrane search columns with considerably shorter analysis time. This method was was conducted using the following combination of MESH terms:, ‘dihydro- validated (Table 1). Evaluation of the stability of DPD in PBMCs indicated pyrimidine dehydrogenase deficiency’, ‘dpd deficiency’, ‘Familial that the DPD activity decreased approximately 50% upon freezing but was 1Department of Pharmacy, Scheper Hospital Emmen and Ro¨pcke Zweers Hospital Hardenberg, Emmen, The Netherlands; 2Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands; 3Laboratory Genetic Metabolic Diseases, Academic Medical Center Amsterdam, University of Amsterdam, Amsterdam, The Netherlands; 4Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands and 5Department of Pharmacy, Diaconessen Hospital Meppel and Bethesda Hospital Hoogeveen, Meppel, The Netherlands. Correspondence: MC van Staveren, Department of Pharmacy, Scheper Hospital Emmen and Ro¨pcke Zweers Hospital Hardenberg, Boermarkeweg 60, Emmen 7824 AA, The Netherlands. E-mail: [email protected] Received 7 November 2012; revised 22 May 2013; accepted 29 May 2013; published online 16 July 2013 390 The Pharmacogenomics Journal (2013), 389 – 395 Table 1. Validation parameters of the diagnostic tests at the enzyme level to screen for DPD deficiency Inter-assay Study n Reference method Recovery Sensitivity Specificity variability Linear Intra-assay variability 24 Mattison et al. 58 volunteers DPD in PBMC ND 100% 96% ‘Reproducible’ ND o5% Genotyping with DHPLC Mattison et al.25 23 volunteers, 8 cancer patients DPD in PBMC ND ND ND ND ND ND Mattison et al.26 258 volunteers DPD in PBMC ND 86% 99% ND ND ND 19 Johnson et al. 100 volunteers, 80 cancer NA 495% ND ND o8% CV Yes o6.5% CV patients 22 Liem et al. 132 volunteers NA 99±2%, 3% CV ND ND o4% CV Yes o3% Lostia et al.21 39 cancer patients HPLC-UV ND ND ND 0.7–5.6% CVa Yes 8.3–1.4% CVa b c van Kuilenburg 28 cancer patients, 1 patient Radioactive 94.4–102.4 99.4–101.6% ND ND 1.7–4.7% CV Yes 1.0–3.2% CV Predictive tests for DPD et al.17 with verified DPYD mutation thymine by HPLC MC van Staveren MS/MS van Kuilenburg 2 cancer patients, unknown NA ND ND ND 9% CV Yes 5% CV et al.18 number healthy volunteers Bi et al.29 4 patients participating in a NA (Relative recovery ND ND 6.73, 6.95, 5.96% ND 7.04, 5.40, 1.24% (RSD phase 1 trial compared with water) (RSD 0.27, 25 and 0.27, 25 and 250 mM) 95.8±2.0 1 mM 95.5±10.9% 250 mM) et al 100 mM Garg et al.34 23 cancer patients, blank plasma NA 93–100 Uracil d, 95–99% ND ND 0.2–7% RSD Yes 0.8–7% RSD uracile, healthy volunteers UH2 Uracil 1.2–9.9% 2.6–9.7% RSD UH2 RSD UH2 Kristensen et al.37 68 CRC patients, 100 healthy IVS þ 1G4A ND 87% 93% ND ND ND controls mutation 5-FU plasma levels Ciccolini et al.28 30 blank plasma samples, one U/UH2 ratio 90% ND ND 10.6, 1212.8% Yes 12, 2.4, 3.3% case report 20 ng ml À 1, 20 ng ml À 1, 75 ng ml À 1, 75 ng ml À 1, 375 ng ml À 1 5-FU 375 ng ml À 1 5-FU 42 Beumer et al. 156 plasma samples obtained LC-MS/MS 93–104% ND ND o2% CV Yes 2.1–3.9% CV from patients; Plasma pool samples di Paolo et al.39 25 CRC patients DPD in PBMC 85 5-FU; 81% 5-FDHU ND ND 5.28–9.44 5-FU; Yes 4.91–6.05% 5-FU; 5.00–9.03 5-FDHU 4.55–6.99% 5-FDHU Remaud et al.36 8 volunteers, blank plasma NA 73±2% Uracil 67±2% ND ND 0.9–2.3% U; Yes 1.3–5.3% U; 1.3–7.1% & UH2; 82±3FUH2% 0.7–5.6% UH2 UH2 2013 Macmillan Publishers Limited van Kuilenburg 30 cancer patients, 18 controls 5-FU loading dose ND 100%f 90%f ND Yes ND et al.8 De´ porte-Fe´ty 1 volunteer, 29 cancer patients DPD in PBMC with ND ND ND 2.5–8.7% Yes 9.5–3.2% et al.40 radiolabeled 5-FU Abbreviations: CRC, colorectal cancer; CV, coefficient of variation; DHPLC, denaturing high-performance liquid chromatography; DPD, dihydropyrimidine dehydrogenase; 5-FDHU, 5-fluoro-5,6-dihydrouracil; FUH2, dihydrofluorouracil; 5-FU, 5-fluorouracil; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NA, not applicable; ND, not determinated; PBMC, peripheral blood mononuclear cell; RSD, relative standard variation; U, uracil; UH2, dihydrouracil; UV, ultraviolet. a5-FU concentration range of 0.156, 0.195, 078 and 1.95 mgmlÀ 1. bInter-assay recovery at low, normal and high concentration of dihydrothymine.