Dihydropyrimidinase Deficiency and Severe 5-Fluorouracil Toxicity

Vol. 9, 4363–4367, October 1, 2003 Clinical Cancer Research 4363

Andre´B. P. van Kuilenburg,1 Rutger Meinsma, important role in the determination of toxicity as well as the Bernard A. Zonnenberg, Lida Zoetekouw, efficacy toward 5FU (1–3). It has been reported that Ͼ80% of Frank Baas, Koichi Matsuda, Nanaya Tamaki, the administered 5FU is catabolized by three consecutive enzymes of the pyrimidine degradation pathway (4). and Albert H. van Gennip DPD catalyzes the conversion of 5FU to FUH2, which is Academic Medical Center, University of Amsterdam, Emma the initial and rate-limiting step in this catabolism (Fig. 1). Children’s Hospital, and Departments of Clinical Chemistry and ␤ FUH2 can be additionally degraded to fluoro- -ureidopropi- Neurogenetics, 1100 DE Amsterdam, the Netherlands [A. B. P. v. K., ␤ ␤ R. M., L. Z., F. B., A. H. v. G.]; University Medical Center Utrecht, onate and subsequently to fluoro- -alanine by DHP and -urei- Department of Medical Oncology, 3508 GA Utrecht, the Netherlands dopropionase, respectively. The pivotal role of DPD in chemo- [B. A. Z.]; and Kobe Gakuin University Igawadani-cho, Kobe, Japan therapy using 5FU has been shown in cancer patients with a [K. M., N. T.] complete or partial deficiency of this enzyme. These patients suffered from severe toxicity, including death, after the admin- ABSTRACT istration of 5FU (5–8). A number of these patients proved to be heterozygous or homozygous for a mutant DPYD allele (5–9). Dihydropyrimidinase (DHP) is the second enzyme in It has also been suggested that patients with a deficiency of the catabolism of 5-fluorouracil (5FU), and it has been sug- DHP are at risk of developing severe 5FU-associated toxicity gested that patients with a deficiency of this enzyme are at (10–12). However, no studies have been reported describing the risk from developing severe 5FU-associated toxicity. In this analysis of the DHP gene for the presence of mutations in study, we demonstrated for the first time that in one patient patients with severe 5FU toxicity. Here, we describe the first the severe toxicity, after a treatment with 5FU, was attrib- patient with severe 5FU-associated toxicity who proved to be utable to a partial deficiency of DHP. Analysis of the DHP heterozygous for a missense mutation in the DHP gene. gene showed that the patient was heterozygous for the missense mutation 833G>A (G278D) in exon 5. Heterologous expression of the mutant enzyme in Escherichia coli showed MATERIALS AND METHODS that the G278D mutation leads to a mutant DHP enzyme Analysis of Dihydropyrimidines. The concentrations of without residual activity. An analysis for the presence of this dihydrouracil and dihydrothymine in plasma were determined mutation in 96 unrelated Dutch Caucasians indicates that using reversed-phase HPLC combined with electrospray tandem the allele frequency in the normal population is <0.5%. Our mass spectrometry (13). results show that a partial DHP deficiency is a novel phar- Determination of the DPD and DHP Activity. The macogenetic disorder associated with severe 5FU toxicity. activity of DPD in peripheral blood mononuclear cells was determined using a radiochemical assay with subsequent sepa- INTRODUCTION ration of radiolabelled thymine from radiolabelled dihydrothy- 5FU2 is one of the most commonly used chemotherapeutic mine with reversed-phase HPLC (14). The activity of DHP was agents for the systemic treatment of cancers of the gastrointes- determined in an assay mixture containing 100 mM Tris-HCl 14 tinal tract, breast, head, and neck. Recent advances in our (pH 8.0), 1 mM DTT, and 500 ␮M [2- C]-dihydrouracil (1.85– understanding of the metabolism of 5FU, and the key enzymes 2.22 GBq/mmol; Moravek Biochemicals, Brea, CA). Separation involved in the activation and degradation of 5FU have led to an of radiolabelled dihydrouracil from N-carbamyl-␤-alanine was increased awareness that the catabolic route of 5FU plays an performed isocratically [50 mM NaH2PO4 (pH 4.5) at a flow rate of 1 ml/min] by reversed-phase HPLC on a Supelcosil LC-18-S column (250 ϫ 4.6 mm; 5 ␮m particle size) with on-line detection of radioactivity (15). Protein concentrations were determined with a copper-reduction method using bicinchoninic Received 3/21/03; revised 5/21/03; accepted 5/27/03. acid, essentially as described by Smith et al. (16). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked PCR Amplification of Coding Exons. DNA was isolated advertisement in accordance with 18 U.S.C. Section 1734 solely to from granulocytes by standard procedures. The PCR amplification indicate this fact. of all nine of the coding exons and flanking intronic regions was 1 To whom requests for reprints should be addressed, at Academic carried out using the primer sets as described previously (17). Medical Center, Laboratory Genetic Metabolic Diseases, F0–224, However, for exon 5, the sequence of the reverse primer was Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Phone: 31- Ј Ј Ј 205665958; Fax: 31-206962596; E-mail: a.b.vankuilenburg@amc. 5 -GGATCCAGATGGGAGGAC-3 . Forward primers had a 5 - uva.nl. TGTAAAACGACGGCCAGT-3Ј extension, whereas reverse 2 The abbreviations used are: 5FU, 5-fluorouracil; DHP, dihydropyrim- primers had an 5Ј-CAGGAAACAGCTATGACC-3Ј extension at idinase; DHPLC, denaturing high performance liquid chromatography; their 5Ј-ends. These sequences were complementary to the labeled DPD, dihydropyrimidine dehydrogenase; DRP, dihydropyrimidinase- -21M13 and M13 reversed primers used in the dye-primer se- related protein; FUH2, fluoro-5,6-dihydrouracil; HPLC, high-performance liquid chromatography; TBS, Tris-buffered saline; CMF, cyclo- quence reaction. Amplification of all of the exons was carried out phosphamide, methotrexate, and fluorouracil. in 50-␮l reaction mixtures containing 10 mM Tris-HCl (pH 8.3), 50

potassium phosphate (pH 7.4) and complete; Roche Diagnos- tics, Almere, the Netherlands] and resuspended in 500 ␮lof isolation buffer. The cell suspension was frozen for 16 h at Ϫ20°C, thawed on ice, and lysed by sonication. The crude lysate was centrifuged at 18,000 ϫ g for 15 min, and the resulting supernatant was stored at Ϫ80°C. Western Blot Analysis. Cell extracts (2.5 ␮g) were frac- tionated on a 7.5% (w/v) SDS-polyacrylamide gel and trans- ferred to a nitrocellulose filter. Blocking of the membrane was performed for 16 h with TBS [25 mM Tris, 137 mM NaCl, and 2.7 mM KCl (pH 7.4)] containing 5% (w/v) nonfat dry milk. Subsequently, the membrane was incubated for 1 h with a 1:1000 dilution of rabbit antirat DHP polyclonal antibody in TBS, supplemented with 0.05% (v/v) Tween 20. The mem- branes were washed three times (5 min each) with TBS containing 0.05% (v/v) Tween 20 and incubated for 45 min with TBS containing 0.05% (v/v) Tween 20, 5% (w/v) nonfat dry milk, and a 1:5000 dilution of a pig antirabbit secondary antibody conjugated to horseradish peroxidase (Dako, Copenhagen, Denmark). After rinsing the membrane three times (5 min each) with TBS containing 0.05% Tween 20, detection of DHP was performed with enhanced chemiluminescence (Amersham Phar- macia Biotech, Buckinghamshire, United Kingdom). DHPLC Analysis of the 833G>A Mutation. PCR fragments containing exon 5 were analyzed on a Agilent 1100-DHPLC system (Agilent Technologies Netherlands B.V., Amstelveen, the Netherlands). The buffer system for the DHPLC consisted of buffer Fig. 1 The catabolic route of 5FU. DPD catalyses the conversion of ␤ A [0.1 M triethylammonium acetate and 1 mM EDTA (pH 7.0)] and 5FU to FUH2. FUH2 can be additionally degraded to fluoro- -ureido- propionate (FUPA) and subsequently to fluoro-␤-alanine (FBAL) by buffer B [buffer A supplemented with 25% (v/v) acetonitrile]. DHP and ␤-ureidopropionase, respectively. Before injection, PCR fragments were denatured at 95°C followed by slow renaturation. PCR fragments (5 ␮l) were loaded in 40% buffer B on a Zorbax ds DNA column (Agilent Technologies Netherlands B.V.) and subsequently eluted, within 6 min at 65°C, ␮ mM KCl, 1.5–2.5 mM MgCl2, 10 pmol of each primer, 200 M of with a gradient from 55–65% buffer B. each dNTP, and 2 units of Taq polymerase (Promega Benelux Sequence Analysis. Sequence analysis of genomic frag- B.V., Leiden, the Netherlands). After initial denaturation for 5 min ments amplified by PCR and expression plasmids was carried at 96°C, amplification was carried out for 35 cycles (0.25 min out on an Applied Biosystems model 3100 automated DNA 96°C, 0.5 min 60°C, and 0.5 min 72°C). PCR products were sequencer using the dye-terminator and the dye-primer method separated on 1% agarose gels, visualized with ethidium bromide, (Perkin-Elmer Corp., Foster City, CA), respectively. and purified using a Qiaquick Gel Extraction kit (Qiagen, Hilden, Germany) or used for direct sequencing. Expression of DHP in E. coli. An expression plasmid RESULTS containing the wild-type human DHP cDNA (pSE420-DHP) Clinical Evaluation. The patient was female, born in was constructed by subcloning the EcoRI-BamHI insert of a 1943, and had a history of an appendectomy, middle ear surgery, plasmid, containing the complete coding region of the cDNA bilateral removal of cervical ribs, multiple cysts, and abscesses encoding human DHP, into the pSE420 vector (18). The in the vagina and a hysterectomy. In addition, she suffered from 833GϾA mutation (G278D) was introduced into the human obesity and diabetes mellitus. In 1991, the patient underwent a

DHP sequence using the megaprimer technique and subse- mastectomy for a ductal carcinoma (pT2N1M0) combined with quently subcloned in a pSE420 vector (pSE420-DHP-G278D). five courses of CMF chemotherapy (60 mg methotrexate i.v., The resulting clones were sequenced completely to verify the 900 mg 5FU i.v. on days 1 and 8, and p.o. 150 mg cyclophos- presence of the mutation and to exclude the presence of random phamide from days 1 to 14). The chemotherapy with CMF mutations introduced by PCR artifacts. Expression plasmids caused considerable mucosal toxicity and, therefore, the planned were introduced into the E. coli strain BL21. A 5-ml Luria- sixth course with CMF was cancelled. In 1994, the patient Bertani broth culture, supplemented with 50 ␮g/ml ampicillin, developed skeletal metastasis for which she was treated succes- was inoculated with 100 ␮l of an overnight preculture grown in sively with tamoxifen, aminogluthetimide, and Re-186 etidr- Luria-Bertani broth. Cells were grown for3hat37°C, and onate. In 1997, lung metastasis was observed, and therapy was induction was performed by the addition of isopropyl-␤-D- changed to chemotherapy with doxorubicine and cyclophos- thiogalactoside to a final concentration of 1 mM. Cells were phamide. In April 1998, because of progression, she received sedimented after 3 h, washed with an isolation buffer [35 mM radiotherapy and docetaxel. Because of disease progression,

Fig. 2 Genomic organization and con- servation of amino acid sequences of the DHP gene. The DHP gene consists of 10 exons encoding an open reading frame of 1560 bp (top panel). The different mutations identified in patients with a deficiency of DHP are indicated, numbers correspond to the cDNA position. The bottom panel shows the alignment of the amino-acid sequences of DHP, DRP, and Hydantoinase (Hy) from Agrobacterium radiobacter (Ar), Arabidopsis thaliana (A), Bacillus stearothermophilus (Bs), bovine (b), Burkholderia pickettii (bp), Caenorhabditis elegans (Ce), Chicken (c), Dictyostelium discoideum (Dd), Dro- sophila melanogaster (Dm), Human (h), Mouse (m), Pseudomonas aeruginosa (Pa), Pseudomonas putida (Pp), Rat (r), Saccharomyces kluyveri (Sk), Streptomy- ces coelicolor (Sc), and Xenopus laevis (x). The GenBank accession numbers are depicted in parentheses.InSaccharomy- ces kluyveri, the inserted sequences were removed before alignment (24). Con- served amino-acid sequences are boxed, and the arrow indicates the position of the mutation G278D in human DHP.

palliative treatment commenced with continuous infusion of Sequence Analysis of the DHP Gene. Analysis of the 5FU (300 mg/m2/day). During the first week of treatment she genomic sequences of exons 1–9 of the DHP gene showed that the noticed increased pain while swallowing, and after 14 days, a tumor patient was heterozygous for a missense mutation 833GϾA skin rash was observed that developed to skin ulcers on the in exon 5, leading to the amino acid substitution G278D (Fig. 2). In thorax, palms of the hands, and soles of both feet. Her condition addition, the patient proved to be homozygous for a -1TϾC mu- worsened, and she became confused and was hospitalized with tation and heterozygous for a silent mutation 216CϾT (F72F) in leucopenic fever and sepsis. At this stage, treatment with 5FU exon 1. No missense mutations could be detected in the DHP gene was aborted. Antibiotic treatment with floxapen and gentamicin of 22 other patients, with normal DPD activity, but who suffered was administered and she recovered. However, after 2 weeks, nevertheless from severe 5FU associated-toxicity. her mental state worsened again and brain metastases were Alignment of various eukaryotic DHP, DRP and prokary- suspected. The patient declined additional diagnostic procedures otic (D-hydantoinase) protein sequences revealed that the gly- and returned home for terminal care. She died 5 months later. cine at position 278 is conserved in DHP from mammals, DPD Activity and Dihydropyrimidines in Plasma. A Drosophila melanogaster, Dictyostelium discoideum, and Sac- normal activity of DPD was detected in peripheral blood mono- charomyces kluyveri, as well as in DRP1–4 (Fig. 2). In Cae- nuclear cells of the patient (13.1 nmol/mg/h; controls 9.9 Ϯ 2.8; norhabditis elegans and Arabidopsis thaliana, the glycine is n ϭ 54), thus excluding a (partial) deficiency of DPD as the replaced by a similar alanine and the hydrophobic residue iso- underlying cause of the severe toxicity, after the administration leucine, respectively. However, in bacteria, significant sequence of 5FU. To investigate whether the severe toxicity might have variation exists in the region around G278 when compared with been caused by a complete deficiency of DHP, plasma was DHP and DRP1–4 in mammals, and G278 was replaced by collected to determine the levels of 5,6-dihydrouracil and 5,6- either hydrophobic or hydrophilic residues. This might indicate dihydrothymine. A normal concentration of 5,6-dihydrouracil that significant structural differences exist around residue 278 in (3.9 ␮M; controls 2.2 Ϯ 3.1 ␮M; n ϭ 97) and a high-normal eukaryotic DHP and the hydantoinases from bacteria. concentration of 5,6-dihydrothymine (1.44 ␮M; controls 0.7 Ϯ Expression Analysis of the G278D Mutation. To inves- 0.4 ␮M; n ϭ 97) were detected in plasma. The concentration of tigate the effect of the mutation G278D on the activity of DHP, 5,6-dihydrothymine was at the upper limit of the 95% distribu- the mutation was introduced into the pSE420-DHP vector by tion range of the control population. site-directed mutagenisis and expressed in E. coli. No endoge-

Fig. 3 DHP activity and immunoblot analysis of wild-type and mutant DHP expressed in E. coli. The activity of DHP was assayed in E. coli Fig. 4 DHPLC screening for the 833GϾA mutation. PCR fragments of lysates. The results represent the mean of four independent measure- exon 5 of the DHP gene containing the wild-type sequence (----) and ments; bar, Ϯ 1SD(left panel). For immunoblot analysis, equal from the patient heterozygous for the 833GϾA mutation (OOO) were amounts of protein (2.5 ␮g) were separated by 7.5% SDS-PAGE and separated at 65oC with DHPLC. analyzed on immunoblot with DHP-specific antibodies (right panel).

in 60% of the patients suffering from severe 5FU-associated Ͻ nous DHP activity ( 0.01 nmol/mg/h) could be detected in the toxicity (5, 8, 9). To date, the underlying mechanisms for the E. coli strain used for the expression of the constructs. Intro- observed increased sensitivity toward 5FU, in patients with a duction of the wild-type DHP construct increased the DHP normal DPD activity, is unknown. Ͼ activity 61,000-fold above the background. Expression of the To investigate the role of DHP, the second enzyme of the DHP construct containing the G287D mutation yielded no de- 5FU catabolic pathway, in the etiology of 5FU toxicity, we have tectable activity of DHP (Fig. 3). analyzed the DHP gene of 23 tumor patients with normal DPD To exclude the fact that the lack of enzyme activity was the activity but, nevertheless, suffering from severe toxicity for the result of an inability to produce the mutant protein in E. coli,we presence of mutations. One of these patients proved to be analyzed the expression level by immunoblotting. Fig. 3 shows heterozygous for a 833GϾA mutation in exon 5, and heterolo- that the mutant protein carrying the G287D mutation was ex- gous expression of this mutation showed that the mutant DHP pressed in comparable amounts as the wild-type protein. Fur- protein bore no residual activity. thermore, no DHP protein could be detected in mock-transfected DHP deficiency is an autosomal recessive disease character- cells. Thus, the lack of DHP activity in E. coli transfected with ized by dihydropyrimidinuria and has been associated with a vari- the pSE420-DHP-G278D construct is not because of rapid deg- able clinical phenotype (10–12, 17). Loading studies with uracil, in radation of the mutant DHP protein in the E. coli lysates. patients suffering from a complete DHP deficiency, showed Population Screening for the 833G>A Mutation. strongly elevated levels and prolonged retention of uracil and Ͼ Screening of individuals for the presence of the 833G A mutation dihydrouracil in serum, with Ͼ80% of the administered dose being was performed with DHPLC. Fig. 4 shows that there is a clear excreted either unchanged or in the form of dihydrouracil during separation between heteroduplex DNA from homoduplex DNA. the first 24 h after the load (10, 11). In individuals heterozygous for Therefore, distinctive chromatographic patterns were obtained for a mutant DHP allele, the urinary concentration of dihydrouracil Ͼ PCR fragments containing the 833G A mutation or the wild-type was several fold higher compared with that observed in controls, Ͼ sequence. An analysis for the presence of the 833G A mutation in after loading with uracil (12). Under normal conditions, a low DHP 96 Dutch Caucasians did not identify individuals either heterozy- activity is probably sufficient to maintain dihydrouracil and dihy- gous or homozygous for this mutation. Thus, the allele frequency drothymine homeostasis as heterozygotes do not excrete elevated Ͼ Ͻ of the 833G A mutation in the normal population is 0.5%. levels of dihydropyrimidines. After the loading of such patients with uracil, the accumulation of dihydrouracil in urine increased DISCUSSION several fold compared with normal individuals, indicating a de- Although originally synthesized over 4 decades ago, 5FU creased capacity of heterozygotes to degrade dihydropyrimidines remains one of the most commonly prescribed anticancer agents (12). In this respect, it is worthwhile to note that the coadministra-

for the treatment of several common human malignancies, in- tion of FUH2 with 5FU attenuated the antitumor activity and cluding those of the gastrointestinal tract, breast, head, and neck. increased the toxicity of 5FU (20). Furthermore, in the presence of Adverse drug reactions are a major clinical problem, and a elevated concentrations of dihydropyrimidines, the reverse reac- meta-analysis involving 1219 patients with colorectal cancer tion, catalyzed by DPD, toward the pyrimidine bases is stimulated

showed that grade 3 to 4 toxicity was encountered in 31–34% of (10, 21). Thus, the decreased capacity to degrade FUH2 and 5FU, the patients receiving 5FU with 0.5% of the patients experienc- because of a decreased DHP activity, might be directly responsible ing lethal toxicity (19). It is likely that a significant proportion for the observed toxicity in the study patient. In this respect, it is of these adverse drug reactions are because of genetically based worthwhile to note that neutropenia has also been associated with differences in the response to 5FU. In this respect, it has been a partial deficiency of DPD (5, 7, 8). shown that a (partial) deficiency of DPD is an important phar- The identification of genetic factors predisposing patients macogenetic syndrome and responsible for the observed toxicity for development of severe 5FU-associated toxicity is increas-

ingly being recognized as an important field of study. It has been deficiency: high prevalence of the IVS14ϩ1GϾA mutation. Int. J. shown recently that the C677T polymorphism in the methyl- Cancer, 101: 253–258, 2002. enetetrahydrofolate reductase gene might be responsible for the 9. Van Kuilenburg, A. B. P., Meinsma, R., Zoetekouw, L., and van ϩ Ͼ severe toxicity observed in some breast cancer patients receiv- Gennip, A. H. High prevalence of the IVS14 1G A mutation in the dihydropyrimidine dehydrogenase gene of patients with severe 5-flu- ing adjuvant treatment with cyclophosphamide, methotrexate, orouracil-associated toxicity. Pharmacogenetics, 12: 555–558, 2002. and 5FU (22). In addition, it has been suggested that a poly- 10. Duran, M., Rovers, P., de Bree, P. K., Schreuder, C. H., Beukenhorst, morphism in the enhancer region of the thymidylate synthase H., Dorland, L., and Berger, R. Dihydropyrimidiuria: a new inborn error of gene promoter is associated with toxicity toward 5FU (23). pyrimidine metabolism. J. Inher. Metab. Dis., 14: 367–370, 1991. Our results indicate that a partial DHP deficiency is not a 11. Hayashi, K., Kidouchi, K., Sumi, S., Mizokami, M., Orito, E., major determinant in the etiology of 5FU toxicity. To date, only Kumada, K., Ueda, R., and Wada, Y. Possible predicition of adverse reactions to pyrimidine chemotherapy from urinary pyrimidine levels 9 individuals suffering from a complete DHP deficiency have and a case of asymptomatic adult dihydropyrimidinuria. Clin. Cancer been reported, which, to some extent, may be because of the Res., 2: 1937–1941, 1996. lack of specific and efficient methods in most laboratories to 12. Sumi, S., Imaeda, M., Kidouchi, K., Ohba, S., Hamajima, N., detect the dihydropyrimidines. In fact, the prevalence of a DHP Kodama, K., Togari, H., and Wada, Y. Population and family studies of deficiency in Japan has been estimated to be 1 in 10,000, which dihydropyrimidinuria: prevalence, inheritance moe and risk of fluorou- is comparable with the estimated frequency of patients with a racil toxicity. Am. J. Med. Genet., 78: 336–340, 1998. DPD deficiency in the Netherlands (6). Therefore, a DHP defi- 13. Van Lenthe, H., van Kuilenburg, A. B. P., Ito, T., Bootsma, A. H., van Cruchten, A., Wada, Y., and van Gennip, A. H. Defects in Pyrim- ciency might be less rare than generally assumed. idine degradation identified by HPLC-electrospray tandem mass spectrometry of urine specimens or urine-soaked filter paper strips. Clin. ACKNOWLEDGMENTS Chem., 46: 1916–1922, 2000. 14. Van Kuilenburg, A. B. P., Van Lenthe, H., Tromp, A., Veltman, We thank Eva Beke and Dr. Doreen Dobritzsch for assistance with P. C. J., and Van Gennip, A. H. Pitfalls in the diagnosis of patients with the sequence analysis, and Dr. Hans R. Waterham and Fiona Ward for a partial dihydropyrimidine dehydrogenase deficiency. Clin. Chem., 46: critical reading of the manuscript. 9–17, 2000. 15. Van Kuilenburg, A. B. P., van Lenthe, H., and van Gennip, A. H. REFERENCES Radiochemical assay for determination of dihydropyrimidinase activity 1. Salonga, D., Danenberg, K. D., Johnson, M., Metzger, R., Groshen, using reversed-phase high-performance liquid chromatography. 729: S., Tsao-Wei, D. D., Lenz, H-J. Leichman, C. G., Leichman, L., Diasio, J. Chrom. B, 307–314, 1999. R. B., and Danenberg, P. Colorectal tumors responding to 5-fluorouracil 16. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., have low gene expression levels of dihydropyrimidine dehydrogenase, Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., thymidylate synthase, and thymidine phosphorylase. Clin. Cancer Res., Olson, B. J., and Klenk, D. C. Measurement of protein using bicincho- 6: 1322–1327, 2000. ninic acid. Anal. Biochem., 150: 76–85, 1985. 2. Ichikawa, W., Uetaka, H., Shirota, Y., Yamada, H., Nishi, N., Nihei, 17. Hamajima, N., Kouwaki, M., Vreken, P., Matsuda, K., Sumi, S., Z., Sugihara, K., and Hirayama, R. Combination of dihydropyrimidine Imaeda, M., Ohba, S., Kidouchi, K., Nonaka, M., Sasaki, M., Tamaki, dehydrogenase and thymidylate synthase gene expressions in primary N., Endo, Y., De Abreu, R., Rotteveel, J., van Kuilenburg, A., van tumors as predictive parameters for the efficacy of fluoropyrimidine- Gennip, A. H., Togari, H., and Wada, Y. Dihydropyrimidinase defi- based chemotherapy for metastatic colorectal cancer. Clin. Cancer Res., ciency: structural organisation, chromosomal localisation, and mutation 9: 786–791, 2003. analysis of the human dihydropyrimidinase gene. Am. J. Hum. Genet., 63: 3. Van Kuilenburg, A. B. P., De Abreu, R. A., and Van Gennip, A. H. 717–726, 1998. Pharmacogenetic and clinical aspects of dihydropyrimidine dehydrogen- 18. Hamajima, N., Matsuda, K., Sakata, S., Tamaki, N., Sasaki, M., and ase deficiency. Ann. Clin. Biochem., 40: 41–45, 2003. Nonaka, M. A Novel gene family defined by human dihydropyrimidi- 4. Heggie, G. D., Sommadossi, J-P., Cross, D. S., Huster, W. J., and nase and three related proteins with differential tissue distribution. Gene, 188: Diasio, R. B. Clinical pharmacokinetics of 5-fluorouracil and its metab- 157–163, 1996. olism in plasma, urine, and bile. Cancer Res., 47: 2203–2206, 1987. 19. Meta-Analysis Group in Cancer. Toxicity of fluorouracil in patients 5. Van Kuilenburg, A. B. P., Haasjes, J., Richel, D. J., Zoetekouw, L., with advanced colorectal cancer: effect of administration schedule and 16: Van Lenthe, H., De Abreu, R. A., Maring, J. G., Vreken, P., and van prognostic factors. J. Clin. Oncol., 3537–3541, 1998. Gennip, A. H. Clinical implications of dihydropyrimidine dehydrogen- 20. Spector, T., Cao, S., Rustum, Y. M., Harrington, J. A., and Porter, ase (DPD) deficiency in patients with severe 5-fluorouracil-associated D. J. T. Attenuation of the antitumor activity of 5-fluorouracil by toxicity: identification of new mutations in the DPD gene. Clin. Cancer (R)-5-fluoro-5, 6-dihydrouracil. Cancer Res., 55: 1239–1241, 1995. Res., 6: 4705–4712, 2000. 21. Porter, D. J. T., Harrington, J. A., Almond, M. R., Lowen, G. T., 6. Van Kuilenburg, A. B. P., Muller, E. W., Haasjes, J., Meinsma, R., and Spector, T. (R)-5-Fluorouracil-5, 6-dihydrouracil: kinetics of oxi- Zoetekouw, L., Waterham, H. R., Baas, F., Richel, D. J., and van dation by dihydropyrimidine dehydrogenase and hydrolysis by dihydro- Gennip, A. H. Lethal outcome of a patient with a complete dihydropy- pyrimidine aminohydrolase. Biochem. Pharmacol., 48: 775–779, 1994. rimidine dehydrogenase (DPD) deficiency after administration of 5-flu- 22. Toffoli, G., Veronesi, A., Boiocchi, M., and Crivellari, D. MTHFR orouracil: frequency of the common IVS14ϩ1GϾA mutation causing gene polymorphism and severe toxicity during adjvant treatment of DPD deficiency. Clin. Cancer Res., 7: 1149–1153, 2001. early breast cancer with cyclophosphamide, methotrexate, and fluorou- 7. Raida, M., Schwabe, W., Ha¨usler, P., Van Kuilenburg, A. B. P., Van racil (CMF). Ann Oncol., 11: 373–375, 2000. Gennip, A. H., Behnke, D., and Ho¨ffken, K. Prevalence of a common 23. Pullarkat, S. T., Stoehlmacher, J., Ghaderi, V., Xiong, Y-P., Ingles, point mutation in the dihydropyrimidine dehydrogenase (DPD) gene S. A., Sherrod, A., Warren, R., Tsao-Wei, D., Groshen, S., and Lenz, within the 5Ј-splice donor site of intron 14 in patients with severe H-J. Thymidylate synthase gene polymorphism determines response and 5-fluorouracil (5-FU)-related toxicity compared to controls. Clin. Can- toxicity of 5-FU chemotherapy. Pharmacogenomics J., 1: 65–70, 2001. cer Res., 7: 2832–2839, 2001. 24. Gojkovic, Z., Rislund, L., Andersen, B., Sandrini, M. P. B., Cook, 8. Van Kuilenburg, A. B. P., Meinsma, R., Zoetekouw, L., and van P. F., Schnackerz, K. D., and Pisˇkur, J. Dihydropyrimidine amidohy- Gennip, A. H. Increased risk of grade IV neutropenia after the admin- drolases and dihydroorotases share the same origin and several enzy- istration of 5-fluorouracil due to a dihydropyrimidine dehydrogenase matic properties. Nucl. Acids Res., 31: 1–9, 2003.

André B. P. van Kuilenburg, Rutger Meinsma, Bernard A. Zonnenberg, et al.

Clin Cancer Res 2003;9:4363-4367.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/9/12/4363

Cited articles This article cites 23 articles, 11 of which you can access for free at: http://clincancerres.aacrjournals.org/content/9/12/4363.full#ref-list-1

Citing articles This article has been cited by 7 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/9/12/4363.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/9/12/4363. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.