Comprehensive

Review

Profiling Dihydropyrimidine Dehydrogenase Deficiency in Patients with Cancer Undergoing 5-Fluorouracil/Capecitabine Therapy Cédric Mercier,1 Joseph Ciccolini2 Abstract Fluoropyrimidine drugs such as 5-fluorouracil (5-FU) and capecitabine are a mainstay in the treatment of numerous solid tumors, including colorectal cancers, alone or as part of combination therapies. Cytotoxic drugs such as 5-FU and oral capecitabine display narrow therapeutic indexes combined with high interpatient pharmacokinetic variability. As a result, severe toxicities often limit or delay the administration of successive, optimal chemotherapeutic courses, leading to unfavorable clinical outcome in patients with cancer. Catabolism and deactivation of fluoropyrimidine drugs depend on a single and exclusive enzymatic step driven by dihydropyrimidine dehydrogenase (DPD). Dihydropyrimidine dehydrogenase is prone to marked circadian rhythms, drug-drug interactions, and genetic polymorphisms; influence of its erratic activity on 5-FU pharmacokinetics and toxicity profile has been extensively investigated, and it is now well known that DPD deficiency leads to severe toxicities with 5-FU or possibly capecitabine exposure. With the everincreasing number of patients with cancer likely to be treated with fluoropyrimidines, predicting and preventing the occurrence of such toxicities is now a major issue in clinical oncology. Early determination of DPD status in patients with cancer would allow identification of those at risk and help in subsequent dose adjustment or selection of other treatment modalities. Numerous methods, either genotypic or phenotypic, have been proposed to achieve this goal.This review covers a wide range of techniques available to establish DPD status in patients with cancer.

Clinical Colorectal Cancer, Vol. 6, No. 4, 288-296, 2006 Key words: Fluoropyrimidines, Genetic polymorphisms, Pharmacokinetics, Phenotypic polymorphisms

Detoxifying Fluoropyrimidine Drugs: The Dihydropyrimidine Dehydrogenase Gateway 5-fluorouracil (5-FU) metabolism has been extensively studied for decades. As a pyrimidine derivative, 5-FU is converted within tumor cells to active nucleotides, following 2 different routes known as the RNA and the DNA pathways. The role of orotate phosphoribosyl transferase,1,2 uridine phosphorylase,3,4 thymidine phosphorylase,5-8 and thymidine kinase9 in the activation process of fluoropyrimidine drugs has been extensively investigated, and so far, the exact way 5-FU is metabolized after tumoral 1EA3286,

Medical Oncology Unit, La Timone University Hospital Pharmacokinetics Laboratory, Université de la Méditerranée Marseille, France 2EA3286,

Submitted: Sep 6, 2006; Revised: Oct 27, 2006; Accepted: Nov 1, 2006 Address for correspondence: Cédric Mercier, MD, Service d’Oncologie Médicale, CHU Timone Adultes, 264, rue Saint Pierre, 13385 Marseille Cedex 05, France Fax: 33-491-835-667; e-mail: [email protected]

uptake remains far from being easily predictable.10 In contrast, the 5-FU elimination pattern is univocal. The drug displays an extensive first-pass metabolism, because > 95% of an administered dose of 5-FU is quickly dehydrogenated in the liver by dihydropyrimidine dehydrogenase (DPD) to dihydrofluorouracil (5-FU-H2). Dihydrofluorouracil is subsequently converted to β-fluoro-β-ureido-propionic acid, then fluoro-β alanine (FβAL) by dihydropyrimidinase and β-ureidopropionase, respectively, and conjugated FβAL derivatives are eventually eliminated in urine.11-14 As a result, terminal half-life of 5-FU rarely exceeds 15 minutes in most patients. The rate-limiting role of DPD in the catabolism of 5-FU has been extensively studied. It has been shown that DPD activity was slightly lower in women (< 15%) than in men,15 an observation fully consistent with the fact that 5-FU clearance is lower in women.16 Besides intersex variations, DPD displays marked circadian rhythms, with interpatient and intrapatient variabilities.17,18 Controversial evidence has emerged from the studies of ethnic influence on DPD activity. Variations in DPD activities ranged from 1 to 5 in white patients, 1 to 8 in Kenyan patients,

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and 1 to 13 in Asian patients but with nonsignificant different medians between the groups.19-22 As a result, population studies of DPD activity revealed extremely heterogeneous values,15 ranging from 65 pmol/min/mg to 559 pmol/min/mg (ratio, 9; n = 185), with unimodal or multimodal distributions.23-25 Besides circadian variations, genetic factors might partly explain this interpatient variability. The DPYD gene is highly polymorphic with > 40 mutations being identified so far,26 plus a possible methylation of the promoter leading to reduced activity,27 coupled with epigenetic regulations.28 Admittedly, respective frequencies of partial and total DPD deficiencies linked with a gene polymorphism syndrome are 3%-5% and 0.5%.15 Recent studies have suggested that black patients would be more prone to DPYD gene polymorphism than white patients,29 an observation that requires further investigations to be confirmed. Finally, intrapatient and interpatient differences in DPD activity can be caused by drug interactions. Any pyrimidine derivative that acts as a substrate for DPD can interact with fluoropyrimidine drugs in a competitive manner, such as the antiviral sorivudine.30 Conversely, anticancer drugs such as raltitrexed proved to upregulate DPD in several experimental models, with an increase in enzymatic activity and messenger RNA (mRNA) levels.31 Because of its pivotal role in 5-FU and capecitabine elimination patterns, it is not surprising that any change in DPD activity has dramatic impact on clinical outcome of patients with cancer. Admittedly, 30%-40% of the severe toxicities upon 5-FU exposure could be attributed to a diminished DPD activity32; however, recent studies suggest that DPD impairment is responsible for 60%-70% of the treatmentrelated toxicities.33

Dihydropyrimidine Dehydrogenase Deficiency and 5-Fluorouracil Pharmacokinetics: The Less You Have, the More You Get Regarding the major role played by fluoropyrimidine drugs in the treatment of numerous cancer types, alone or in combination, extensive pharmacokinetic studies of 5-FU and capecitabine have been undertaken for years, and all showed marked variations between individuals.34 Doses, routes of administration,35,36 and demographic data, such as sex or age,15,16,37,38 are the most commonly reported factors influencing 5-FU pharmacokinetic profile, although clinical studies sometimes have conflicting data. Because 5-FU elimination follows a single enzymatic pattern driven by liver DPD, many studies have focused on the impact of DPD erratic activity on 5-FU pharmacokinetic parameters.39-41 Although no relation was established between overall hepatic function and the pharmacokinetic profile of 5-FU,42,43 data have shown that DPD impairment leads to profound alteration of 5-FU pharmacokinetic profile, with reduced clearance and increased half-life.24,44,45 Consequently, patients with deficient DPD activity given the standard dosage are likely to be dramatically overexposed. This hypothesis has been fully confirmed by some case reports showing 5-FU area under the curve increasing from 9.2 (control population) to 24.7 hours per µg/mL in a patient with deficient activity46 and by a recent study showing that patients with impaired DPD displayed 5-FU plasma

concentrations up to 15-times higher than usually observed.47 Besides 5-FU, influence of DPD activity on the pharmacokinetics of oral capecitabine has been investigated and confirmed using physiologically based pharmacokinetic models.48

Dihydropyrimidine Dehydrogenase Activity and Clinical Outcome: The Lower the Activity, the Deeper the Impact Since the late 1980s, numerous studies have investigated the clinical consequences of 5-FU and capecitabine administration in patients with impaired DPD activity.44-47,49 It is well known now that reduced activity leads to sharp overexposure to 5-FU when standard dosages are administered, with subsequent sharp increase in the occurrence of drug-related toxicities. Admittedly, more than one third of the severe toxicities reported with 5-FU could be attributed to partial or total alteration in DPD activity.32 Recent studies have reconsidered this scoring on the rise, and impaired DPD function could be finally suspected in > 60%-70% of 5-FU unanticipated toxicities.33,50,51 In the most extreme cases, total DPD deficiency can lead to toxic death,52 although some studies have shown that fatal outcome can also be observed in only partially deficient patients.53 Similarly, such a dramatic outcome has been reported in 18 Japanese patients with abrogated DPD activity after the intake of sorivudine, an anti–herpes zoster drug that interacted with tegafur, one of the oral 5-FU prodrugs, in a competitive manner.30,54 Digestive and hematologic toxicities are the most frequently reported side effects observed in DPD-deficient patients treated with standard dosages of 5-FU.17,46,47,55-57 Such severe toxicities not only greatly affect patient quality of life, in some instances when not directly life-threatening, they also coerce patients to postpone or cancel the forthcoming administrations, with subsequent loss of successive and optimal courses in a row. The impact of DPD deficiency on the clinical outcome of patients with cancer upon capecitabine treatment is highly controversial. As a triple oral prodrug, capecitabine has been normally designed to yield specifically 5-FU in tumors, and in this respect, its pharmacokinetic profile should not be heavily affected by erratic DPD activity in the liver. As the final activating enzymes of capecitabine, the cytidine deaminase thymidine phosphorylase is expressed in hepatocytes, and unscheduled synthesis of 5-FU can occur in the liver, thus putting DPD-deficient patients at risk.10 This has been suggested by clinical reports describing toxicities with capecitabine in patients with impaired DPD activity.58 This has finally been confirmed by the first toxic death case reported in a patient with DPYD gene polymorphism and severe deficiency undergoing a capecitabine/oxaliplatin protocol.49

Determining Dihydropyrimidine Dehydrogenase Status in Patients with Cancer: To Be or Not to Be Deficient, That is the Question Regarding the dramatic impact of DPD impairment on 5-FU pharmacokinetic profile and subsequent occurrence of treatmentrelated toxicities, DPD status is unanimously considered a critical Clinical Colorectal Cancer November 2006 • 289

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Profiling Dihydropyrimidine Dehydrogenase Deficiency determinant for predicting clinical outcome in patients treated with fluoropyrimidine drugs.26 Because millions of patients with cancer receive fluoropyrimidines each year, detecting DPD deficiency has been an increasing concern since the early 1990s. Several methods (either genotypic or phenotypic, direct or indirect, or focusing on DPD protein expression levels) have been proposed to achieve this goal. As of now, none of them have stood out as a standard because of a wide range of bias, technical limitations, or inadequacies with routine clinical practice requiring simple, rapid, and inexpensive detection methods.

Methods for Gene Polymorphism of Dihydropyrimidine Dehydrogenase Gene polymorphism of DPD has been well characterized since the mid-1990s as an autosomal recessive disease, with 0.5% and 5% of the white patients being subsequently affected by total and partial deficiencies, respectively.15,59 Inherited complete DPD deficiency at birth causes a condition called thymine uraciluria that can be asymptomatic or associated with several neurologic disorders.60-62 The 150 kb DPYD gene is located on chromosome 1p22 and consists of 23 exons, ranging in size from 69 pb to 1404 pb, with 1-20 kb introns.63,64 The DPYD gene is highly polymorphic, with > 40 mutations described so far.26,65,66 The IVS14+1G>A splice-site mutation is reported as being the most common mutation in Europeans.32,50,55,59,67-70 Beside this canonical polymorphism, several deletions (298delTCAT, 812delT, 1897delC) and other missense mutations (85 T>C, 496 A>G, 703 T>C, 775 A>G, 1601 G>A, 1627 A>G, 1679 T>G, 2194 G>A, and 2846 A>T, to name but a few) have been described as well, with possible decreased expression and low DPD activity in patients.67,68,71-74 Some point mutations at positions 62 (G>A), 74 (A>G), 1003 (G>T), 1156 (G>T), and 1714 (C>G) and deletion (1812delT) have been found more specifically in Asian patients, such as Taiwanese and Japanese.19,75,76 More recently, methylation of the DPYD promoter has been related to decreased activity27 but with a probable tissue specificity.77 In addition to promoter hypermethylation, epigenetic gene silencing through histone modification could play a role in DPD impairment, at least in some tumor tissues.28 Several direct sequencing and polymerase chain reaction (PCR)–based genotypic methods59,71,78-81 and denaturing high performance liquid chromatography (HPLC) techniques74,82,83 have been published to detect the most frequent mutations on the DPYD gene. Extracting DNA from formalin-fixed paraffin-embedded tissue or blood sample is a standard, simple, and automatable procedure. The most frequently used methods for detecting single nucleotide polymorphisms are based upon real-time (RT)–PCR approaches or PCR–restriction fragment length polymorphism assays using appropriate restriction enzymes. Real time–PCR is based on different melting temperatures of fluorescent-labeled oligonucleotide hybridization probes using a single-step assay that combines fluorescence PCR and melting curve analysis. Although more complex, denaturing HPLC is capable of detecting all known sequence variations but can also identify unknown polymorphisms and promoter

hypermethylations.27,83 This method discriminates different sequence variations in the same DNA fragment because of specific elution patterns under the optimized buffer gradient conditions and calculated melting temperatures of different melting domains inherent in a given DNA sequence. Whatever method is used, the prevalence of most commonly reported mutations such as the exon-14 skipping remains controversial, with allele frequencies ranging from 0 to 2.7% according to the population studied.19,55,71,76,84-86

Methods for Phenotypic Gene Polymorphism of Dihydropyrimidine Dehydrogenase Based on enzymatic nature, developing functional testing of DPD has been proposed for years for directly or indirectly establishing deficient/nondeficient status in patients with cancer. Dihydropyrimidine dehydrogenase activity can be evaluated after 2 alternative approaches: determining enzymatic activity through ex vivo DPD direct assays or indirect evaluations performed in patients based on the monitoring of endogenous or exogenous substrates of DPD as surrogate markers. Finally, a test dose of 5-FU has been proposed as well as a possible strategy to determine DPD status in patients with cancer.

Dihydropyrimidine Dehydrogenase Direct Assays Although most markedly expressed in the liver, DPD is a ubiquitous enzyme, and several reports have shown that DPD activity measured in surrogate tissues such as lymphocytes or fibroblasts could be used as a marker for the actual liver activity.87,88 Precise measurement of DPD activity ex vivo from blood mononuclear cells was the first method proposed in the late 1980s to evaluate overall DPD functionality.23 This method was derived from the radiometric assay reported by Naguib et al for tumor biopsies.89 Dihydropyrimidine dehydrogenase activity was assessed using a radio-HPLC method measuring the conversion of radiolabeled 5-FU into 5-FU-H2. Adapted and optimized methods used tritium-labeled 5-FU, radioactive carbon–labeled (14C) 5-FU, 14C thymine, or 14C uracil as substrates, with limit of detection as low as 5 pmol/min/mg protein, down to 0.4 pmol.17,87,90-94 In addition to HPLC, a thin layer chromatography method has been developed based on the use of radiolabeled uracil.95 More recently, HPLC methods without the use of radiolabeled substrates have been proposed, thus rendering this approach cheaper and easier to adapt in most laboratories.96,97 Admittedly, peripheral blood mononuclear cell DPD levels < 100-150 pmol/min/mg protein are associated with a partial deficiency syndrome, whereas patients displaying DPD < 50-80 pmol/min/mg protein are considered profoundly deficient.24,91,98-100

Indirect Evaluations An estimation of the DPD functionality can be achieved by monitoring physiologic substrates used as probes. Plasma uracil level was proposed as a surrogate marker but showed poor correlation with DPD activity or 5-FU clearance, thereby showing limited relevance.24 Expression of levels of DPD substrate and its catabolite as a ratio was proposed to yield

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Cédric Mercier, Joseph Ciccolini more relevant information regarding the enzymatic activity. Sumi et al first theorized the utility of determining uracil-dihydrouracil ratios as a potential surrogate for DPD status in patients with cancer.101 Numerous methods have monitored uracil or, more frequently, uracil and dihydrouracil, sometimes along with thymine and dihydrothymine102 in urine103,104 or, more frequently, plasma,33,105-111 using UVHPLC105,108,109 and gas chromatography–mass spectrometry102,103,111 or liquid chromatography–mass spectrometry/mass spectometry104,112-114 systems. Clear correlations have been demonstrated between uracil-dihydrouracil values and 5-FU pharmacokinetic parameters108,113 or between ratio values and the occurrence of severe/lethal toxicities of 5-FU or capecitabine.33,49,51 More recently, a method known as the Uracil Breath Test has been proposed; this test is based on the ingestion of a stable isotope of uracil (13C uracil) and monitoring of expired 13C carbon dioxide.98,115,116 Following the same principle of exogenous uracil intake before monitoring in vivo catabolism, a simpler method that does not require labeled uracil or a specific analytic device gave promising preliminary results.117

5-Fluorouracil Test Dose Reduced test dose strategies with subsequent sampling for pharmacokinetic evaluation could provide valuable information on patients with impaired DPD and increased risk of iatrogeny upon 5-FU administration. Monitoring of 5-FU and 5-FU catabolites such as dihydrofluorouracil97,118 or FβAL119 in plasma has been proposed as a marker for DPD function. Precisely, it has been shown that FβAL plasma levels demonstrated good correlation with DPD activity in lymphocytes, thus suggesting it could be used to detect patients with deficient activity; however, this approach requires ≥ 1 dose of 5-FU to be administered before determining in real time the DPD status. A recently demonstrated pharmacokinetic-based test to prevent severe toxicities upon 5-FU administration showed that using a reduced 5-FU test dose with 5-FU/5-FU-H2 monitoring permitted one to detect approximately 2% of patients with marked alterations in 5-FU pharmacokinetic profiles. These patients were subsequently selected for alternative treatments without fluoropyrimidine drugs, thus preventing life-threatening toxicities.120 Another simpler approach consists of administration of low-dose 5-FU without pharmacokinetic support, with careful monitoring of toxicities during the first cycle of treatment and a possible dose escalation in the subsequent courses. Although rather empirical, this would ensure some safety for patients with possible DPD impairment, provided the patient remains hospitalized for monitoring.

Other Methods: Determining Dihydropyrimidine Dehydrogenase Genetic/Protein Expression Several methods for genetic or protein DPD measurement have been reported, essentially to determine DPD levels in tumors as a putative marker of response to 5-FU. Intratumoral thymidylate synthase and DPD expression has been extensively investigated and was a predictive marker of response

to 5-FU in many cancer types.121-123 Most of these methods evaluate the DPD expression through its mRNA with RT-PCR assays59,81,95,124 or directly by Western blot13,91,95,125 or immunohistochemistry methods.126 Importantly, all these methods have been developed to predict the sensitivity to 5-FU, with little application for detecting DPD-deficient patients at risk from fluoropyrimidine drugs. Although most of these methods provide good correlation between Western blot, DPD activity, and mRNA expression, they only provide semiquantitative data.91,95 More recently, fully quantitative enzyme-linked immunosorbent assays for DPD have been reported.127-131 This approach displays excellent correlations with enzyme activity,128,129 but to date, this approach has been developed and validated only in tumor biopsies of surrounding tissues as a marker of response to 5-FU.

Dihydropyrimidine Dehydrogenase Profiling: Pros and Cons Preliminary determination of DPD status in patients with cancer eligible for treatment with 5-FU or capecitabine should reduce the occurrence of severe/lethal toxicities. This would lead to optimizing clinical outcome through appropriate dosing, using DPD status as a covariate to be taken into account to customize dosage.132 In an ideal world, every patient should benefit from this approach. To achieve such a routine, largescale, systematic prescreening, one should use a rapid, simple, and inexpensive method that yields unambiguous data regarding the deficient/nondeficient status of patients with cancer. There are now a wide range of methods and strategies available to determine DPD status in patients with cancer likely to be given fluoropyrimidine drugs. Because mutations affecting the DPYD gene have been extensively investigated, genotypic studies might look attractive, because most of them are based upon simple, rapid, and widely used methods, such as RT-PCR technology, using µg quantities of patients’ genomic DNA. In addition, DPYD is highly polymorphic, and the fact that all mutations are probably not yet documented might yield false-negative results. Use of new DNA-binding dye such as LCGreen® should overcome this drawback in the near future through the generation of high-resolution melting curves for the detection of subtle alterations indicative of change in the hybridization status in the PCR product, but to date, no such methods are available for DPD genotyping. Besides, of the 40 mutations described, only the canonical IVS14+1A>G and the less frequent 1679 T>G polymorphisms have been frequently associated with decreased DPD activity,52,133-135 yet the actual clinical relevance of the detected single nucleotide polymorphisms remains unclear. For instance, the mutation reported as the most critical is the IVS14+1G>A polymorphism located on the exon 14, which results in a truncated protein.50 This has often been linked to the most severe toxicities of 5-FU treatment.26,52 However, recent studies have suggested that the implication of this exon-14 skipping might be less frequent than initially presumed in the occurrence of treatment-related side effects.86 In a retrospective 10-year Clinical Colorectal Cancer November 2006 • 291

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Profiling Dihydropyrimidine Dehydrogenase Deficiency study, 142 of 144 patients (98.6%) with severe toxicities with 5-FU did not display the IVS14+1G>A mutation,57 leading to serious concerns about its relevance as a predictive marker of severe toxicities with fluoropyrimidine drugs. In another study screening for exon 14 skipping in 105 patients with severe toxicities after 5-FU or capecitabine intake, none of them showed this mutation, including the 6 patients with fatal outcome.33 However, in the first case of toxic death reported after capecitabine intake, retrospective phenotypic determination of the DPD status showed marked DPD deficiency but without being associated with the IVS14+1G>A polymorphism. Further investigations instead revealed a heterozygosity for the 1896 C>T mutation located in exon 14 of the DPYD gene affecting the codon for F632, which could alter DPYD mRNA and, subsequently, protein expression.49 This strongly suggests that the canonical IVS14+1G>A polymorphism might not be a reliable, predictive marker of DPD deficiency and might be involved in subsequent increased risk of toxicities after 5-FU or capecitabine-based chemotherapies. Similar concerns have emerged from the study of other mutations such as the 85 T>C or 74 A>G polymorphisms on DPD activity and clinical outcome.76 The allelic regulations of the DPYD gene could ensure a proper enzymatic activity despite heterozygosity for sequence variations, with a risk of wrongly diagnosing deficiency syndrome in patients with restored DPD function.86 Finally, patients at risk after 5-FU or 5-FU–derivative intake from DPD deficiency caused by exogenous factors such as drug interactions30,54,136 will not be identified as deficient using any genotypic methods. Taken together, this strongly suggests that a limited number of patients with cancer could benefit from such genotypic screening, with an increased risk of precluding the proper diagnosis in routine clinical settings. In many respects, considering DPD activity as an ultimate endpoint to detect patients with impaired DPD, regardless of causes, might look safer. Several direct enzymatic assays in peripheral blood mononuclear cells (PBMCs) or fibroblasts have been proposed for 20 years now, but all require large (up to 60 mL) volumes of blood and time-consuming multistep sample treatment, and most are based on the use of radiolabeled substrates, even if a couple of recent methods are based on nonradioactive material.96 Besides these technical limitations, several biases hinder these methods. Isolating PBMCs is a long procedure that can hardly be automated, DPD activity can vary according to the subfraction of monocytes,72,90 and only a weak link was found between DPD activity in PBMCs and 5-FU clearances.24,100,118,120 Besides significant relationships between liver and PBMCs, DPD activity showed mild correlation (r² < 0.3),87 thus hampering proper diagnosis, especially in patients with intermediate deficiencies.90 Simpler and cheaper methods using surrogate markers as an index for DPD status is the most attractive approach for detecting DPD deficiencies in patients with cancer. Monitoring physiologic uracil and dihydrouracil in plasma as a ratio is a well-established strategy that combines simple sample treatment and analytical procedures such as basic UV-HPLC. Retrospective

studies have proven that 70% of the severe toxicities and 80% of the toxic deaths caused by 5-FU/capecitabine treatment would have been detected using this approach,33,47,51 yet interpretation of the ratios can vary from one institute to another based on the methods used, and most laboratories have performed local reference-population studies before setting their own cutoff indicative of a DPD deficiency. Indeed, uracil-dihydrouracil ratios are generated from calibration curves built from the original biologic matrix,106,109,113 water,105,107,108 or from a simple signal/signal basis,33,47,49 with subsequent large variations in the values reported between institutes. This is illustrated by the fact that mean molar ratios observed for dihydrouracil/uracil levels in urine ranged from 0.42 to 4.8 according to the populations studied.112 Finally, it is still not clear which of the ratios measured in plasma or urine would give the most relevant information on DPD status in patients with cancer. Higher concentrations of uracil and dihydrouracil found in urine would theoretically allow a better analytic determination while being noninvasive, but urine levels of uracil are more likely to be influenced by food intake, thus leading to a possible bias in the determination of the ratio. Recently, the Uracil Breath Test has been proposed as an alternative test to detect DPD deficiency in patients. The advantage of this noninvasive test is that it is expedited by the use of exogenous 2-13C uracil as a probe. However, subjects are asked to spend up to 3 hours blowing into dozens of bags every 5-10 minutes,98 a “patient-unfriendly” requirement compared with a single 3-mL blood sample to perform most uracil-dihydrouracil ratio determinations in plasma. Besides, this test requires specific equipment such as an infrared spectrophotometer breath analyzer, whereas dihydrouracil/uracil monitoring can be performed with basic HPLC already widely available in most hospitals. Finally, a major drawback of this method is the amount of 2-13C uracil to be ingested by the patients (6 mg/kg), which renders this procedure extremely costly, especially for large-scale systematic screening. The oral uracil challenge test requiring a nonlabeled probe, single blood sample, and UV-HPLC procedure could be an attractive alternative, although only preliminary results are available so far.137 Still, whatever is the phenotypic method chosen, one will have to deal with the issue of circadian regulation of DPD activity. Most tests are performed empirically in the morning as an attempt to limit the influence of intrapatient variability, although no data support this hypothesis, and a more significant circadian pattern has been evidenced when DPD function is expressed as uracil-dihydrouracil ratios, compared with the actual activity in PBMCs.114 Based on these drawbacks, administering test doses of fluoropyrimidine drugs, with pharmacokinetic support, would appear as the less biased approach for detecting DPD impairment in patients with cancer. The major advantage of this strategy is that actual drug administration is monitored in real time, without the experimental bias encountered in prescreening methods. Still, the reduced dose administered must be high enough to allow pharmacokinetic monitoring and, therefore, would lead to life-threatening toxicities in patients with totally abrogated

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Cédric Mercier, Joseph Ciccolini DPD activity. Furthermore, even if no pharmacokinetics are required, patients must be hospitalized for careful monitoring of toxicities, thus excluding the ones treated with ambulatory therapies such as capecitabine.

Conclusion Choosing a method for identifying DPD-deficient patients at risk with fluoropyrimidine drugs remains an uneasy task. No method has stood out as a standard that would meet all the requirements (eg, time- and cost-effectiveness, availability, and relevance) of large-scale screening. However, whatever method is eventually chosen, it is still a better option than sticking with the blind administration of standardized dosages of 5-FU performed regardless of the DPD status of patients with cancer. Because of the countless reports demonstrating the relationships between DPD deficiency and severe toxicities with 5-FU/capecitabine, specific sensitive screening methods for detecting DPD expression are warranted.

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