Lethal Outcome of a Patient with a Complete Dihydropyrimidine Dehydrogenase (DPD) Deficiency after Administration of 5-Fluorouracil: Frequency of the Common IVS1411G>A Mutation Causing DPD Deficiency

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk from developing a severe 5FU-associated toxicity. In this study, we demonstrated that a lethal toxicity after a treatment with 5FU was attributable to a complete deficiency of DPD. Analysis of the DPD gene for the presence of mutations showed that the patient was homozygous for a G3A mutation in the invariant GT splice donor site flanking exon 14 (IVS1411G>A). As a consequence, no significant residual activity of DPD was detected in peripheral blood mononuclear cells. To determine the frequency of the IVS1411G>A mutation in the Dutch population, we developed a novel PCR-based method allowing the rapid analysis of the IVS1411G>A mutation by RFLP. Screening for the presence of this mutation in 1357 Caucasians showed an allele frequency of 0.91%. In our view, the apparently high prevalence of the IVS1411G>A mutation in the normal population, with 1.8% heterozygotes, warrants genetic screening for the presence of this mutation in cancer patients before the administration of 5FU. INTRODUCTION 5FU remains one of the most widely used chemotherapeutic agents for the systemic treatment of cancers of the gastrointestinal tract, breast, and head and neck. As a single drug, 5FU has only limited efficacy. To improve the clinical response of 5FU, optimal administration schedules as well as the combination of 5FU with other drugs, which should increase its antitumor activity or decrease the host toxicity, have been investigated. It has been shown that long-term continuous i.v. infusion of 5FU is superior to bolus injections of 5FU in terms of response rate, however, only a small increase in median survival was observed (1), and high interand intrapatient variations in the plasma concentrations of 5FU have been observed during prolonged infusion of the drug (2). In addition, a relationship between the 5FU dose intensity and the therapeutic response, as well as toxicity, has been noted (3, 4). An important determinant in predicting the toxicity as well as the efficacy of 5FU might be the activity of DPD. DPD is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, but also of the pyrimidine analogue 5FU. It has been reported that .80% of the administered 5FU is catabolized by DPD (5). Furthermore, a correlation has been observed between the pretreatment activity of DPD in PBM cells and the systemic clearance of 5FU in cancer patients (6). The pivotal role of DPD in chemotherapy using 5FU has been shown in cancer patients with a complete or near-complete deficiency of this enzyme. These patients suffered from severe toxicity, including death, after the administration of 5FU (7–11). It was shown that a number of these patients were genotypically heterozygous for a mutant DPD allele (9–12). To date, 17 variant DPYD alleles have been identified in pediatric patients suffering from a complete or near-complete DPD deficiency, or in tumor patients with decreased DPD activity (12–15). Analysis of the prevalence of the various mutations among DPD patients has shown that the splice-site mutation, IVS1411G.A, was by far the most common one (52%; Ref. 13). To date, the frequency of the splice site mutation IVS1411G.A in the normal population is not known. On the basis of population analysis of the DPD activity, the frequency of heterozygotes has been estimated to be as high as 3% (6). Such individuals might be at risk of developing severe toxicity after the administration of 5FU. Furthermore, only a small number of cases have been reported regarding lethal toxicity after the administration of 5FU. In none of these cases have the Received 8/16/00; revised 1/23/01; accepted 1/25/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the “Stichting Kinder Oncologisch Centrum Amsterdam.” 2 To whom requests for reprints should be addressed, at Academic Medical Center, Laboratory Genetic Metabolic Diseases, F0-224, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Phone: 31-205665958; Fax: 31206962596; E-mail: [email protected]. 3 The abbreviations used are: 5FU, 5-fluorouracil; DPD, dihydropyrimidine dehydrogenase; PBM, peripheral blood mononuclear. 1149 Vol. 7, 1149–1153, May 2001 Clinical Cancer Research Research. on July 15, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from molecular mechanisms underlying the 5FU-induced death been resolved. In this paper, we describe a simple genotyping procedure to test for the presence of the IVS1411G.A mutation. Furthermore, we describe the first patient with lethal toxicity, after the administration of 5FU, who proved to be homozygous for the IVS1411G.A mutation. MATERIALS AND METHODS Chemicals. [4-C]-thymine (1.85–2.22 GBq/mmol) was obtained from Moravek Biochemicals (Brea, CA). Lymphoprep (specific gravity, 1.077 g/ml; 280 mOsm) was obtained from Nycomed Pharma AS (Oslo, Norway). LeucoSep tubes were supplied by Greiner (Frickenhausen, Germany). FCS was obtained from BioWhittaker (Walkersville, MD). HAM-F10 medium with 20 mM HEPES was obtained from Life Technologies, Inc. (Breda, the Netherlands). AmpliTaq Taq polymerase BigDye-Terminator-Cycle-Sequencing-Ready Reaction kits were supplied by Perkin-Elmer Corp. (Foster City, CA). Restriction endonuclease NdeI was obtained from Roche Diagnostics Nederlands B.V. (Almere, the Netherlands). A Qiaquick Gel Extraction kit was obtained from Qiagen (Hilden, Germany). All other chemicals used were of analytical grade. Analysis of Pyrimidine Bases. The concentrations of the pyrimidine bases, uracil and thymine in plasma, were determined using reversed-phase HPLC combined with diode-array detection, as described before (16). Culture Conditions of Human Fibroblasts. Fibroblasts were cultured from skin biopsies obtained from controls and the index patient. Biopsies were incubated at 37°C in HAM-F10 medium, supplemented with 20 mM HEPES and 15% (v/v) FCS in 25-cm cell-culture flasks until an adequate number of proliferating cells was obtained. Subsequently, cells were cultured in HAM-F10 medium supplemented with 20 mM HEPES and 10% (v/v) FCS. Fibroblasts were harvested with 0.25% (w/v) trypsin, and after washing the cells once with PBS and twice with 0.9% (w/v) NaCl, the cells were collected by centrifugation (175 3 g at 7°C for 5 min), and the supernatant was discarded. The pellets were stored at 280°C. Isolation of Human PBM Cells and Granulocytes. PBM cells were isolated from 15 ml EDTA-anticoagulated blood by centrifugation over Lymphoprep, and the cells from the interface were collected and treated with ice-cold NH4Cl to lyse the contaminating erythrocytes, as described before (17). The pellet of the centrifugation step over Lymphoprep containing the granulocytes and erythrocytes was diluted with 7 ml of supplemented PBS [9.2 mM Na2HPO4, 1.3 mM NaH2PO4, 140 mM NaCl, 0.2% (w/v) BSA, 13 mM sodium citrate, and 5 mM glucose (pH 7.4)] and centrifuged at 800 3 g at room temperature for 10 min. To lyse the erythrocytes, the pellet was resuspended in 7 ml of ice-cold ammonium chloride solution (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) and kept on ice for 5 min. After the addition of 10 ml of ice-cold supplemented PBS, the solution was centrifuged at 250 3 g at 4°C for 10 min. The pellet was collected and subjected to another lysis step as described above. The pellet containing the granulocytes was washed once more with supplemented PBS, and the final cell pellet was frozen in liquid nitrogen and stored at 280°C until further analysis. Determination of the DPD Activity. The activity of DPD was determined in a reaction mixture containing 35 mM potassium phosphate (pH 7.4), 2.5 mM MgCl2, 1 mM DTT, 250 mM NADPH, and 25 mM [4-C]-thymine (17). Separation of radiolabeled thymine from radiolabeled dihydrothymine was performed isocratically [50 mM NaH2PO4 (pH 4.5) at a flow rate of 2 ml/min] by high-performance liquid chromatography on a reversed-phase column (Alltima C18; 250 3 4.6 mm; 5-mm particle size; Alltech Associates Inc., Deerfield, IL) and protected by a guard column (Supelguard LC-18-S; 5-mm particle size; 20 3 4.6 mm; Supelco, Bellefonte, PA) with online detection of the radioactivity, as described before (17). Protein concentrations were determined with a copper-reduction method using bicinchoninic acid, essentially as described by Smith et al. (18). PCR Amplification of Exon 14. DNA was isolated from purified granulocytes by standard procedures. PCR amplification of exon 14 and its flanking intronic regions was carried out using the primer sets DPD14f and DPD14r as specified in Table 1. Amplification of exon 14 was carried out in a 50-ml reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 10 pmol each primer, 200 mM each deoxynucleotide triphosphate, and 2 units of Taq polymerase. After initial denaturation for 5 min at 95°C, amplification was carried out for 35 cycles (1 min 95°C, 1 min 55°C, 1 min 72°C). The PCR product was separated on 1% agarose gels, visualized with ethidium bromide, and purified using a Qiaquick Gel Extraction kit or used for direct sequencing. RFLP of Exon 14. PCR amplification of exon 14 and its flanking 59 donor intronic region was carried out using the primer sets NDEf and NDEr as specified in Table 1. Amplification of exon 14 was carried out in a 25-ml reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 5 pmol each primer, 200 mM each deoxynucleotide triphosphate and 2 units of Taq polymerase. After initial denaturation for 5 min at 96°C, amplification was carried out for 35 cycles (0.5 Table 1 Oligonucleotides used for genomic PCR of exon 14 of the DPYD gene Primer Sequence Direction Position DPD14f 59-TCCTCTGCAAAAATGTGAGAAGGGACC-39 Sense 451–477 DPD14r 59-TCACCAACTTATGCCAATTCTC-39 Antisense 762–783 NDEf 59-ATCAGGACATTGTGACATATGTTTC-39 b Sense 565–589 NDEr 59-CTTGTTTTAGATGTTAAATCACACATA-39 b Antisense 736–762 a Numbering according to the intron sequences flanking exon 14 of DPYD as published by Vreken et al. (19). b The NdeI restriction sites introduced by site-directed mutagenesis (primers NDEf and NDEr) are underlined. The single-base mismatches introducing the Nde1 restriction site are depicted in boldface. 1150 DPD Deficiency and 5-Fluorouracil Toxicity Research. on July 15, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from min 96°C, 0.5 min 60°C, and 1 min 72°C). Restriction analysis of the PCR products was performed in a 20-ml reaction mixture containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM dithioerythitol, 1 ml of PCR product, and 10 units of restriction endonuclease NdeI. The mixture was incubated overnight at 37°C. The DNA fragments were subsequently separated on a 3% agarose gel and visualized with ethidium