5-Fluorouracil-induced acute leukoencephalopathy in the absence of dihydropyrimidine dehydrogenase deficiency: A case report.

Dihydropyrimidine dehydrogenase (DPD) deficiency is prevalent in 3-5% of the Caucasian population; however, the frequency of this pharmacogenetic syndrome in the Indian population and other racial and ethnic groups remains to be elucidated. We describe an Indian patient who presented to clinic for the treatment of gastric adenocarcinoma with 5-flurouracil (5-FU) therapy who subsequently was diagnosed with DPD deficiency by using the peripheral blood mononuclear cell (PBMC) DPD radioassay. This observation prompted us to examine the data generated from healthy (cancer-free) Indian subjects who were enrolled in a large population study to determine the sensitivity and specificity of the uracil breath test (UraBT) in the detection of DPD deficiency. Thirteen Indian subjects performed the UraBT. UraBT results were confirmed by PBMC DPD radioassay. The Indian cancer patient demonstrated reduced DPD activity (0.11 nmol/min/mg protein) and severe 5-FU toxicities commonly associated with DPD deficiency. Of the 13 Indian subjects [ten men and three women; mean age, 26 years (range: 21-31 years)] enrolled in the UraBT, 12 Indian subjects demonstrated UraBT breath profiles and PBMC DPD activity within the normal range; one Indian subject demonstrated a reduced breath profile and partial DPD deficiency. DPD deficiency is a pharmacogenetic syndrome which is also present in the Indian population. If undiagnosed, the DPD deficiency can lead to death. Future epidemiological studies would be helpful to determine the prevalence of DPD deficiency among racial and ethnic groups, allowing for the optimization of 5-FU chemotherapy.

BackgroundPretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive.MethodsAmong a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated.ResultsWhen [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a “wild-type” genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant.ConclusionsFrequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

To the editor, With interest we read the article by Dr Cubero and colleagues, in which they evaluated the safety of tegafur-uracil (UFT®) in five cases with partial dihydropyrimidine dehydrogenase (DPD) deficiency [Cubero et al. 2012]. Based on our previous experience [Deenen et al. 2010], however, we would like to express our concern about their conclusion that UFT is a safe alternative for the treatment of patients with partial DPD deficiency. Cubero and colleagues make the erroneous and unproven statement that the presence of uracil in UFT creates an artificial DPD deficiency, and that the DPD activity in patients with normal DPD activity would then be similarly as low as in DPD-deficient patients. This assumption, however, is incorrect. As uracil is a competitive inhibitor of DPD, it competes with 5-fluorouracil (5-FU) for DPD-mediated metabolism. This does not mean that the activity of DPD is depleted, as suggested by Cubero and colleagues, in contrast, its activity is fully utilized, as well as for the metabolism of uracil, as for the metabolism of 5-FU. We would like to caution that treating patients with partial DPD deficiency with the standard dose of UFT may unnecessarily lead to severe, potentially lethal toxicity. Unlike the cases described by Cubero and colleagues, we could previously describe four cases presenting with comparable severe toxicity profiles upon treatment with UFT as had previously occurred during treatment with capecitabine or 5-FU. In all subjects an underlying partial DPD deficiency was identified by genotype and phenotype analyses [Deenen et al. 2010]. Furthermore, there are several pharmacological lines of argument that support our clinical observation, i.e. that the standard dose of UFT is not safe in (partial) DPD-deficient patients. First, pharmacokinetic studies have shown that DPD remains essential for the metabolism of UFT, with significantly longer half-lives of 5-FU after administration of UFT compared with 5-FU administered intravenously [Ho et al. 1998]. This is due to the presence of uracil in UFT. Since DPD-deficient patients already have longer half-lives of 5-FU than other patients [Mattison et al. 2006], presence of uracil increases its half-life even further. This in turn leads to prolonged and elevated circulating levels of 5-FU, with a subsequently increased risk of 5-FU-induced severe toxicity. Another argument underscoring the importance of normal DPD function in the safe application of UFT, is the experience with S-1. S-1 is another drug combination of tegafur, consisting of tegafur, 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium oxonate in a molar ratio of 1:0.4:1. CDHP inhibits DPD 200-fold more potently than does uracil [Shirasaka et al. 1996a, 1996b]. Even after administration of S-1, the primary 5-FU metabolite formed by DPD is observed in significant concentrations in plasma [Kim et al. 2007]. Thus, DPD remains an essential detoxification enzyme of 5-FU, even when its activity is strongly inhibited. The ultimate proof of theory is the occurrence of 18 treatment-related deaths in patients with cancer and herpes zoster given UFT plus the antiviral drug sorivudine [Pharmaceutical Affairs Bureau, 1994]. Subsequent studies in rats showed that a metabolite of sorivudine, (E)-5-(2-bromovinyl)uracil, instantly and irreversibly inactivates DPD by covalent binding, which has been identified as the underlying mechanism of these toxic deaths [Ogura et al. 1998; Okuda et al. 1998]. It is for these arguments that the Summary of Product Characteristics of UFT notes a known DPD deficiency as a contra-indication [Merck Serono, 2011]. The fact that the patients described by Cubero and colleagues did not develop significant toxicity might be due to patient selection, the slightly decreased dose intensity of 90%, or despite their DPYD*2A genotype a DPD enzyme activity within the (lower) range of normal. We are not aware of this, because DPD enzyme activity was not determined in these patients. In summary, we would like to state that standard-dose UFT is not a safe treatment in (partial) DPD-deficient patients. Instead, dose reductions of on average 50% of either capecitabine, 5-FU or UFT with careful monitoring of safety and further dose titration are proposed as the standard of care [Deenen et al. 2011].

Capecitabine, an oral prodrug of 5-fluorouracil (5-FU) is extensively used to treat many solid tumors, particularly breast and colorectal cancers. Neurotoxicity of capecitabine has been rarely reported as peripheral neuropathy, cerebellar syndrome, and multifocal leukoencephalopathy. Although very little is known about the pathogenetic mechanisms responsible for this toxicity, dihydropyrimidine dehydrogenase (DPD) deficiency has been found in few of these patients. TYMS gene encodes for the human thymidylate synthase, and is considered a candid factor for toxicity and efficacy of 5-FU and capecitabine. However, TYMS polymorphism has been associated previously with capecitabine-induced neurotoxicity. We report here a 31-year-old patient with metastatic colorectal cancer undergoing chemotherapy consisting of oxaliplatin and capecitabine who developed acute cerebellar syndrome during cycle 5. MRI did not show any abnormalities. We performed pharmacogenetic studies related to capecitabine including DPD deficiency and TYMS polymorphism. DPD gene mutation analysis was negative for the IVS14+1G>A mutation in the DPD gene, which accounts for 50% of the DPD deficiency alleles. However, the patient was found to have 3RG/3RC genotype and Del/Del genotype of TYMS 3'-untranslated region. Withdrawal of capecitabine improved his neurotoxicity in 9 days. No re-challenge was given to this patient but he was able to tolerate irinotecan, oxaliplatin, and bevacizumab without any toxicities. To the best of our knowledge, this is the first patient in the literature who developed acute cerebellar syndrome following capecitabine and was found to have mutations of TYMS. Patients on fluoropyrimidines, including capecitabine with new neurological symptoms must be investigated for a rare but real central neurotoxicity. Though the treatment of 5-FU neurotoxicity is supportive care but use of uridine triacetate may be indicated in few patients, especially with overdose.

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the delta C1897 frame-shift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency.

Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might down-regulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients.

Is Capecitabine Safe in Patients with Gastrointestinal Cancer and Dihydropyrimidine Dehydrogenase Deficiency?

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 (13)CO(2) metabolite formation, plasma [2-(13)C]dihydrouracil formation, [2-(13)C]uracil clearance, and DPD activity. An aqueous solution of [2-(13)C]uracil (6 mg/kg) was orally administered to 23 healthy volunteers and 8 cancer patients. Subsequently, breath (13)CO(2) concentrations and plasma [2-(13)C]dihydrouracil and [2-(13)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. 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 (13)CO(2) DOB(50) significantly correlated with PBMC DPD activity (r(p) = 0.78), plasma [2-(13)C]uracil area under the curve (r(p) = -0.73), [2-(13)C]dihydrouracil appearance rate (r(p) = 0.76), and proportion of [2-(13)C]uracil metabolized to [2-(13)C]dihydrouracil (r(p) = 0.77; all Ps < 0.05). UraBT breath (13)CO(2) pharmacokinetics parallel plasma [2-(13)C]uracil and [2-(13)C]dihydrouracil pharmacokinetics and are an accurate measure of interindividual variation in DPD activity. These pharmacokinetic data further support the future use of the UraBT as a screening test to identify DPD deficiency before 5-fluorouracil-based therapy.

AimsTreatment with dihydropyrimidines poses a significant risk of serious adverse reactions for patients with dihydropyrimidine dehydrogenase (DPD) deficiency. This study seeks to analyse the correlation between DPD deficiency and plasmatic...

Dihydropyrimidine dehydrogenase (DPD) deficiency with a defect of the pyrimidine catabolic pathway has recently become the focus of considerable attention, due to the severe 5-fluorouracil (5-FU) toxicities occurring in DPD deficiency patients. Studies also suggest that 5-FU toxicities could occur in another pyrimidine metabolic disorder, dihydropyrimidinuria (DHPuria). This study shows that urinary dihydrothymine (DHT) and thymine (THY) are useful indexes for detection of DPD deficiency and DHPuria. We measured urinary DHT and THY in 276 Japanese adults to establish reference ranges. When males and females were compared, both DHT and THY levels were found to be significantly higher in females. The reference ranges (mean +/- SD with logarithmic values) for males were found to be 1.56-5.70 micromol/g of creatinine for DHT and 0.40-1.47 micromol/g of creatinine for THY. The reference ranges for females were found to be 1.89-8.33 micromol/g of creatinine for DHT and 0.58-2.30 micromol/g of creatinine for THY. In addition to this study we analyzed a DPD deficiency case and a DHPuria case. In the DPD deficiency case, the THY concentrations of all urine samples were out of the reference range. However, uracil levels in most of the samples were within the normal range. The DHPuria case excreted large amounts of DHT and dihydrouracil, both out of the normal range.

521 Background: DPD deficiency is a pharmacogenetic syndrome associated with dose-limiting toxicity to fluoropyrimidines. Oncologists are expected to recognize and diagnose this syndrome, as toxicities could be fatal. Over 40 single nucleotide polymorphisms (SNPs) and deletions have been identified within the DPYD gene. IVS14+1G&gt;A (DPYD*2A) is the most common (40-50%) and best studied of these SNPs. Yet, it showed a median sensitivity of 30% and is absent in Japanese, Korean and African Americans. Overall, the data on DPYD testing is insufficient to provide enough guidance to diagnose DPD deficiency. Herein we describe our experience with germline pharmacogenomics in patients with DPD deficiency. Methods: Between 2011 and 2015, 35 patients with gastrointestinal malignancies were tested for DPYD mutations; 17 were tested after developing toxicities to treatment and 18 were tested prior to treatment. IVS14+1G&gt;A (DPYD*2A) was tested in all patients. DPYD c.85T&gt;C (DPYD*9A), DPYD c.1679T&gt;G (DPYD*13A), DPYD c.-1590T&gt;C, and DPYD c.2846A&gt;T were tested in 24 patients (69%) only. We explored the association between DPYD mutations and fluoropyrimidine-related toxicity using Fisher’s exact test. Results: Median age was 60 years, 43% were male, 80% were Caucasian and 20% were African American. Capecitabine-based regimens (71%) and 5-Fluorouracil-based regimens (29%). 14 out of 35 patients (40%) had DPYD mutations. Grade 3 toxicities were encountered in 64% of patients with DPYD mutation and 48% of patients with no DPYD mutation. In patients who received full dose fluoropyrimidines (57% of patients with DPYD mutation and 81% of patients with no DPYD mutation), DPYD mutations were associated with a significantly higher rate of grade 3 diarrhea (p=0.026). In patients with DPYD mutation, 2 (14%) had DPYD*A2 mutation and 12 (86%) had DPYD*9A mutation. Conclusions: In patients treated with fluoropyrimidines, the rate of grade 3 diarrhea was significantly higher in patients with mutated DPYD gene. Testing for DPYD*2A alone to diagnose DPD deficiency is suboptimal. Testing for other DPYD mutation variants including DPYD*9A provides a more comprehensive approach. These data should further be validated in prospective clinical trials.

1618 Background: DPD deficiency is the most important risk factor for developing fluoropyrimidine-related adverse events. Genetic variants causing DPD deficiency are found in 6-8% of Caucasian patients. However, there is limited information on their prevalence in underrepresented ethnic groups, such as Hispanics and Latinos, and testing for these variants is not routinely recommended in Latin America. Our goal was to assess the allele frequency of clinically actionable dihydropyrimidine dehydrogenase ( DPYD) risk variants defined by the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the European Medicines Agency (EMA) among admixed Mexican patients with GI malignancies. Methods: Patients with recently diagnosed GI cancer candidates for fluropyrimidine therapy were recruited from a single institution in Mexico City. After providing informed consent, a blood sample and clinical characteristics were collected. We utilized the Illumina Infinium Global Screening Array (GSA)-to genotype 34 DPYD variants, six of which are known to lead to an increased risk of fluoropyrimidine toxicity and are considered clinically actionable. Results: Two hundred and eight patients with a mean age of 62 years (SD 13.2) were included. 47% were female. The most common type of cancer was colorectal (38%) followed by pancreas (22%) and biliary tract (18%). DNA samples from 192 patients passed quality control, of which 156 (62%) received fluoropyrimidines during follow-up. Only 2 patients (1%) were heterozygous for actionable DPYD intermediate metabolizer risk variant alleles: one with c.2846A &gt; T ( rs67376798 , D949V) and one with c.1129–5923C &gt; G [ rs75017182; HapB3 SNP c.1236G &gt; A; rs56038477]. No patients were found to have other CPIC-listed DPYD risk variants . Additionally, we investigated the allele frequencies of other 30 DPYD variants and observed low-frequency variation (between 0.260 and 0.0032) in rs56038477, rs1801160, rs17376848, rs1801159, rs1801158, rs45589337, rs2297595, rs200562975, and rs1801265. Several of these may be related to decreased DPYD activity and warrant further analysis regarding their impact on adverse drug reactions. Conclusions: In contrast with reports from Caucasic populations, we found a very low allele frequency of DPYD actionable variants. Our findings highlight the limitation of current pharmacogenomic testing recommendations and panels, which may not be appropriate for admixed ethnic populations such as Hispanics/Latinos due to disparities in representation. There is a need to study the role of other DPYD variants in larger patient samples to understand their role in the toxicity risk of admixed populations in Mexico and Latin America, to explore the use of novel techniques such as Next Generation Sequencing, and to investigate the effect of other related genes on toxicity risk.

Prevention of 5-fluorouracil-induced early severe toxicity by pre-therapeutic dihydropyrimidine dehydrogenase deficiency screening: Assessment of a multiparametric approach

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases and it catalyzes the reduction of uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine, respectively. In children, a deficiency of DPD is often accompanied by a neurological disorder but a considerable variation in the clinical presentation among these patients has been reported1. In these patients, a large accumulation of uracil and thymine has been detected in urine, blood and in cerebrospinal fluid whereas no activity of DPD could be detected in fibroblasts and mononuclear cells . The detection of more than 30 patients of various nationalities with a (partial) DPD deficiency within 15 years in The Netherlands alone suggest that this type of inborn error is less rare than previously assumed 1, 2 The recent cloning of the cDNA coding for human DPD and the sequence of the entire human DPD gene3 (DPYD) has allowed the detection of the defects at the molecular level. Identification of disease-causing mutations in the DPD gene will allow rapid pre-screening of patients at risk.

e14019 Background: Severe dihydropyrimidine dehydrogenase (DPD) deficiency can be lethal in 0.5-3.0% of patients receiving fluoropyrimidines. Unfortunately, there is no routine test in medical practice to identify high-risk patients. Here, we evaluated the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratio and DPYD genotyping, as a means to identify patients with DPD deficiency and fluoropyrimidine toxicity. In addition, we report on a functional test using UH2/U metabolic ratio in dried saliva spots (DSS). Methods: Prior to fluoropyrimidine therapy, plasma and saliva samples were obtained from 60 patients with GI cancer. U and UH2 levels were measured by LC-MS/MS in plasma and saliva. Patients were also genotyped for DPYD (*7/*2A/*13/Y186C). WHO grading were used to report treatment toxicity. Results: In 21 patients (35%) toxicity was documented. For those, no variant allele carrier for DPYD was identified. The UH2/U metabolic ratios were 0.1-26.7 in plasma and 0.1-24.0 in saliva, with a higher correlation with toxicity grade in saliva as compared to plasma (rs 0.52 vs 0.28). Median metabolic ratios were lower in patients with severe toxicity as compared to those with no toxicity (0.59 vs 2.83 saliva; 1.62 vs 6.75 plasma, P &lt; 0.01). A cut-off of 1.16 for the salivary UH2/U ratio was set (AUC 0.84) with 86% sensitivity and 77% specificity for the identification of grade 3-4 toxicity. A plasma cut-off of 4.0 (AUC 0.75) revealed a 71% sensitivity and 76% specificity. Moreover, saliva of 21 patients were applied to filter paper to obtain DSS and sent to the laboratory by regular mail. U and UH2 were stable in DSS stored at 45°C up to 7 days. In this set of patients, grade 3-4 toxicity was documented in 3/21 cases (14%), all three cases had metabolic ratios below 1.16 in DSS, confirming our prior results. Conclusions: DPYD genotyping failed to identify severe DPD deficiency, but the UH2/U metabolic ratios in saliva showed enough sensitivity and specificity to deserve further evaluation. DSS samples allowed medical oncologists working at distant sites to send us samples by post, with results available within a week. This test is being validated in a larger sample population.