Decreased Dihydropyrimidine Dehydrogenase Activity Research Articles

Rapid determination of uracil in biological fluids at mercury thin film electrode for early detection of potential 5-fluorouracil toxicity due to dihydropyrimidine dehydrogenase deficiency

Determination of plasma uracil was reported as a method for evaluation of Dihydropyrimidine dehydrogenase (DPD) activity that is highly demanded to ensure the safe administration of 5-fluorouracil (5-FU)-based therapies to cancer patients. This work reports the development of a simple electroanalytical method based on adsorptive stripping square wave voltammetry (AdSWV) at mercury film-coated glassy carbon electrode (MF/GCE) for the highly sensitive determination of uracil in biological fluids that can be used for diagnosis of decreased DPD activity. Due to the formation of the HgII–Uracil complex at the electrode surface, the accuracy of the measurement was not affected by the complicated matrices in biological fluids including human serum, plasma, and urine. The high sensitivity of the developed method results in a low limit of detection (≈1.3 nM) in human plasma samples, falling below the practical cut-off level of 15 ng mL−1 (≈0.14 μM). This threshold concentration is crucial for predicting 5-FU toxicity, as reported in buffer, and ≤1.15% in biological samples), and accuracy (recovery percentage close to 100%).

Dihydropyrimidine dehydrogenase phenotype in peripheral blood mononuclear cells is related to adverse events of fluoropyrimidine-therapy.

The primary objective of this study was to determine if dihydropyrimidine dehydrogenase (DPD) activity measured in peripheral blood mononuclear cells (PBMCs) is related to adverse events during fluoropyrimidine therapy. A retrospective cohort study was conducted. The study population included 481 patients who received fluoropyrimidine treatment and for whom relevant patient characteristics were known and adverse events were noted in the electronic health records. Factors besides DPD phenotype that could affect the incidence of adverse events were corrected for using log regression. These log regression models were used to identify an association between the DPD phenotype measured in PBMCs and adverse events. Patients with a decreased DPD activity measured in PBMCs suffered more adverse events. Results from log regression data show that this effect remains significant after correcting for dosage, chemotherapy regimen and relevant patient characteristics. A significant correlation was found between reduced DPD enzyme activity in PBMCs and adverse events. The findings in this paper support further exploring DPD phenotyping as a method for preventing fluoropyrimidine-related adverse events. Further assessment of DPD phenotyping will require clinical validation in a prospective study.

Potential added value of combined DPYD/DPD genotyping and phenotyping to prevent severe toxicity in patients with a DPYD variant and decreased dihydropyrimidine dehydrogenase enzyme activity.

To investigate if dihydropyrimidine dehydrogenase phenotyping has added value when combined with DPYD genotyping in predicting fluoropyrimidine-related toxicity. Retrospective cohort study in which treatment and toxicity data were collected of 228 patients genotyped for four DPYD variants and phenotyped using an ex vivo peripheral blood mononuclear cell assay. Severe toxicity occurred in 25% of patients with a variant and normal dihydropyrimidine dehydrogenase activity, in 21% of patients without a variant and with decreased dihydropyrimidine dehydrogenase activity, and in 29% of patients without a variant and with normal dihydropyrimidine dehydrogenase activity (controls). The majority of patients with a variant or a decreased dihydropyrimidine dehydrogenase activity received an initial dose reduction (68% and 53% vs 19% in controls) and had a lower mean dose intensity (75% and 81% vs 91% in controls). Fifty percent of patients with a variant and decreased enzyme activity experienced severe toxicity, despite the lowest initial dose and whole treatment dose intensity. They also experienced more grade 4/5 toxicities. Our results indicate that a combined genotype-phenotype approach could be useful to identify patients at increased risk for fluoropyrimidine-associated toxicity (e.g. patients with a variant and decreased dihydropyrimidine dehydrogenase activity). Because the group sizes are too small to demonstrate statistically significant differences, this warrants further research in a prospective study in a larger cohort.

Abnormal Dihydropyrimidine Dehydrogenase Activity as an Indicator of Potential 5-Fluorouracil Linked Cardiotoxicity in Colorectal Cancer Patients: Are Toxic Events Inevitable?

Colorectal cancer (CRC) treatment can be limited to surgical resection for low stages of the disease while subsequent chemotherapy is the preferred treatment for the higher-stage disease. This chemotherapy relies heavily on fluoropyrimidine: 5-fluorouracil (5-FU) and capecitabine, a role played for decades. Fluoropyrimidine-linked treatment can present important and even lethal toxic events at the cardiac level like acute coronary syndrome, arrhythmias, and death.The production of these toxic events depends on the capacity of a subject to metabolize the fluoropyrimidines adequately, and this depends on the activity of the enzyme dihydropyrimidine dehydrogenase (DPD). Any change that affects the quantity or quality of this enzyme will compromise its capacity to metabolize the fluoropyrimidines. The resultant abnormal enzyme activity exposes the patient to continuously high levels of the chemotherapeutic agent or its catabolites. Consequently, the patient becomes more susceptible to pyrimidine-linked toxic adverse events.Genetic testing of patients for potential decreased DPD activity before subjecting them to fluoropyrimidine-based chemotherapy will help identify subjects at greater risk of increased cardiotoxicities, the possibility of prompt intervention, should these appear, and a multidisciplinary strategy aimed at managing these cases. Potential cases of cardiotoxicity in CRC patients, candidates to fluoropyrimidine toxicities, can be anticipated by pretreatment screening of DPD activity. Pretreatment screening will reduce many hospitalizations with a consequent decrease in costs both to the patients and the healthcare system.This review article will examine the 5-FU linked cardiotoxicity, known correlated risk factors, clinical manifestations, management strategy, and the role of genetic testing in identifying high-risk patients.

Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation.

Indirect assessment of dihydropyrimidine dehydrogenase activity in cats

Use of 5-fluoropyridimine antimetabolite drugs, specifically 5-fluorouracil (5-FU), has been discouraged in cats because of adverse events including neurotoxicity and death. Causes of toxicity have never been elucidated. In humans, toxicity has been associated with ineffective metabolism secondary to deficiencies in dihydropyrimidine dehydrogenase (DPD). Direct assessment of DPD activity is challenging; determination of uracil:dihydrouracil (U:UH2 ) in plasma using high performance liquid chromatography (HPLC) has been reported as an indirect measurement. U:UH2 was measured in the plasma of 73 cats. Mean U:UH2 for all cats was 1.66 ± 0.11 (median 1.53, range 0.24-7.00). Seventeen (23%) cats had U:UH2 >2, a value associated with decreased DPD activity in humans. Spayed female cats had significantly lower U:UH2 as compared with intact females, and age and U:UH2 were weakly but significantly negatively correlated (r = -0.26). Studies correlating U:UH2 and 5-FU tolerability are required to further determine the validity and use of this test in cats.

Abstract 5451: High-throughput detection of dihydropyrimidine dehydrogenase gene (DPYD) variants using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)

Abstract Catabolism of fluoropyrimidine drugs such as 5-fluorouracil (5-FU) and its prodrug capecitabine depends on an exclusive enzymatic step, dihydropyrimidine dehydrogenase (DPD), which results in drug inactivation. DPD exhibits wide interindividual variation, with partial to complete DPD deficiency occurring in approximately 3 to 5% of the general population. Patients with less pronounced, but decreased DPD activity may also be unable to efficiently inactivate 5-FU, such that this may contribute to 5-FU toxicity. Though numerous tests have been developed to phenotypically assess the DPD enzyme, they remain labor intensive and time consuming with difficulty in defining threshold values for separating DPD deficient patients from those with normal DPD enzyme activity. To date, attempts to develop genotypic assays have been complicated by the size and complex structure of the gene encoding the DPD enzyme (DPYD). Numerous sequence variants have been reported with varying frequencies across different populations. Unfortunately many DPYD variants have not been examined to determine whether they are associated with loss of function in the DPD enzyme. Current genotypic methods are limited due to the inability to rapidly assess multiple variants in a large number of samples at one time. In the current study, we have developed a rapid, high-throughput genotypic method using 6 multiplexed PCR assays covering 115 DPYD variants analyzed with the Sequenom matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Selected variants included previously identified and functionally characterized SNPs as well as newly identified mutations within the coding region from a previous mutation discovery analysis in Coriell cell lines and patient samples. Due to the potential importance of variants in the untranslated regions (UTRs), we have included known variants from the 5′- and 3′- UTRs (9 and 16 respectively) within our plexes. A total of 95 DNA samples derived from 4 colorectal cancer cell lines, 76 Coriell cell lines (40 Caucasian and 36 African American), and 7 Caucasian patients with clinically observed 5-FU toxicity and/or decreased DPD activity were utilized for initial assay testing. To assess “calling” efficiency and presence of false positives, 8 Coriell DNA samples plated in duplicate and 1 negative control were included. Our results showed that identified variants were 100% in agreement across duplicated samples, and only four variants within the 6 different plexes were removed due to PCR/extension failure. In summary, this method provides a comprehensive and rapid analysis of 111 DPYD variants within 6 different plexes with both sensitive and accurate detection suitable for high-throughput patient screening. Functional studies in a large patient population are underway (CA116964). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2011-5451

Uracil pharmacokinetics in a DPD-deficient patient with a novel DPYD mutation compared to volunteers with normal DPD activity.

e13134 Background: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the metabolism of 5-fluorouracil (5-FU). Patients with a partial or complete DPD deficiency are at risk to develop severe toxicity after 5-FU administration. Uracil (U) is degraded in dihydrouracil (DHU) in a similar way as 5-FU. An oral uracil test dose might be useful to determine the systemic DPD activity by measuring uracil and its metabolite dihydrouracil in plasma. DPD deficiency is hypothesized to result in higher uracil levels and a reduced turnover of uracil into dihydrouracil. Methods: Uracil (500 mg/m2) was administered to 11 healthy volunteers with normal DPD activity (≥ 6 nmol/mg/hour) and 1 patient with colorectal carcinoma with a novel DPYD mutation and decreased DPD activity (4.6 nmol/mg/hour). DPD activity was measured in PBMC, as determined as described earlier. Blood samples were taken at t= 15, 30, 45, 60, 80, 100, 120, 150, 180, and 220 or 240 min after oral uracil intake. U and DHU pla...

Altered Dihydropyrimidine Dehydrogenase Activity Associated with Mild Toxicity in Patients Treated with 5-Fluorouracil Containing Chemotherapy

Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5-fluorouracil (5FU). In patients treated with capecitabine or 5FU combined with other chemotherapeutic drugs, DPD activity in peripheral blood mononuclear cells was increased in patients experiencing grade I/II neutropenia. In contrast, decreased DPD activity proved to be associated with grade I/II dermatological toxicity, including hand-foot syndrome. Thus, patients with a low-normal or high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-based chemotherapy, demonstrating the important role of DPD in the etiology of toxicity associated with 5FU and the catabolites of 5FU.

Use of Pyrosequencing to Detect Clinically Relevant Polymorphisms in Dihydropyrimidine Dehydrogenase

Common human diseases, such as cancer, have been associated with multiple types of variation in the genome, including sequence repeats and deletions and single-nucleotide polymorphisms (SNPs) (1). Of these, SNPs are the most abundant in the human genome (2)(3). As a result of the efforts of many groups (4), an estimated 5 million SNPs are now deposited in public databases (5), providing a resource for determining how genomic variation affects human biology. Building on this work, many groups have shown that drug response is also influenced by genomic variation (6). Multiple SNPs have been identified that have a major impact on response to chemotherapy (7)(8)(9)(10)(11); it is therefore necessary to have rapid and efficient SNP evaluation techniques to analyze genes that influence chemotherapy response. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the three-step degradation of uracil to β-alanine (12). This is the only endogenous pathway for production of the neurotransmitter β-alanine (13). DPD also degrades >80% of the anticancer agent 5-fluorouracil (5-FU), a pyrimidine analog used to treat colorectal cancer, and limits the oral absorption of the drug (13). DPD activity is found to be highest in liver and mononuclear cells, but it is also present in most other human tissues (14). Variation in DPD activity can lead to nonbeneficial physiologic conditions. A complete absence of DPD has been associated with the hereditary metabolic disorder thymine-uraciluria (9). This is characterized by mental retardation and is sometimes accompanied by other neurologic disorders, such as microcephaly, motor retardation, and autism (9). In addition, decreased DPD activity leads to severe toxicity from 5-FU even when typical doses are administered. This is often accompanied by severe diarrhea, neutropenia, and sometimes, neurotoxicity (9). The DPYD gene located on chromosome 1p20 …

Pharmacogenetic and clinical aspects of dihydropyrimidine dehydrogenase deficiency.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). A deficiency of DPD is increasingly being recognized as the cause of an important pharmacogenetic syndrome. The importance of DPD deficiency in the aetiology of unexpected severe 5FU toxicity has been demonstrated by the fact that, in 39-59% of cases, decreased DPD activity could be detected in peripheral blood mononuclear (PBM) cells. It was observed that 55% of the patients with a decreased DPD activity suffered from grade IV neutropenia compared with 13% of the patients with a normal DPD activity (P = 0.01). Furthermore, toxicity developed significantly earlier in patients with low DPD activity than in patients with normal DPD activity (10.0 +/- 7.6 versus 19.1 +/- 15.3 days, P < 0.05). In patients suffering from severe 5FU-associated toxicity, 11 mutations have been identified in DPYD, including one splice-site mutation (IVS14 + 1G-->A), one nonsense mutation (E386X), four missense mutations (M166V, V335L, I560S, D949V) and five polymorphisms (C29R, R21Q, S534N, I543V, V732I). Considering the common use of 5FU in the treatment of cancer patients, the severe 5FU-related toxicities in patients with a low DPD activity and the high prevalence of the IVS14 + 1G-->A mutation, analysis of the DPD activity in PBM cells or screening for the IVS14 + 1G-->A mutation should be routinely carried out prior to the start of treatment with 5FU.

High prevalence of the IVS14 + 1G>A mutation in the dihydropyrimidine dehydrogenase gene of patients with severe 5-fluorouracil-associated toxicity.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU) and a DPD deficiency is increasingly being recognized as an important pharmacogenetic factor in the aetiology of severe 5FU-associated toxicity. In this study, we evaluated the DPD activity and the prevalence of the common splice site mutation IVS14 + 1G>A in tumour patients suffering from severe grade 3-4 toxicity after the administration of 5FU. DPD activity was measured with a radiochemical assay and screening for the presence of the IVS14 + 1G>A mutation was performed by restriction fragment length polymorphism. A decreased DPD activity could be detected in peripheral blood mononuclear cells in 60% of the cases. Furthermore, a high prevalence of the IVS14 + 1G>A mutation was noted as 28% of all patients were heterozygous or homozygous for this mutation. In patients with a low DPD activity, 42% were heterozygous and one patient (3%) was homozygous for the IVS14 + 1G>A mutation. In contrast, the IVS14 + 1G>A mutation could be detected in only one out of 24 (4%) patients with a normal DPD activity. Our study demonstrates that a DPD deficiency is the major determinant of 5FU-associated toxicity. The apparently high prevalence of the IVS14 + 1G>A mutation warrants genetic screening for this mutation in cancer patients before the administration of 5FU.

Increased risk of grade IV neutropenia after administration of 5-fluorouracil due to a dihydropyrimidine dehydrogenase deficiency: high prevalence of the IVS14+1g>a mutation.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk of developing severe 5-FU-associated toxicity. We evaluated the importance of DPD deficiency, gender and the presence of the IVS14+1G>A mutation in the etiology of 5-FU toxicity. In 61% of cases, decreased DPD activity could be detected in peripheral blood mononuclear cells. Furthermore, the number of females (65%) in the total group of patients appeared to be higher than the number of males (35%) (p = 0.03). Patients with partial DPD deficiency appeared to have a 3.4-fold higher risk of developing grade IV neutropenia than patients with normal DPD activity. Analysis of the DPYD gene of patients suffering from grade IV neutropenia for the presence of the IVS14+1G>A mutation showed that 50% of the patients investigated were heterozygous or homozygous for the IVS14+1G>A mutation. Adopting a threshold level for DPD activity of 70% of that observed in the normal population, 14% of the population is prone to the development of severe 5-FU-associated toxicity. Below this threshold level, 90% of individuals heterozygous for a mutation in the DPYD gene can be identified. Considering the common use of 5-FU in the treatment of cancer, the severe 5-FU-related toxicities in patients with low DPD activity and the apparently high prevalence of the IVS14+1G>A mutation, screening of patients at risk before administration of 5-FU is warranted.

Severe 5-fluorouracil-induced toxicity due to decreased dihydropyrimidine dehydrogenase activity: report of a patient survival and urinary pyrimidine and dihydropyrimidine levels

We report a Japanese woman who survived severe 5-fluorouracil (5-FU) toxicity after receiving 5′-deoxy-5-fluoro-uridine (5′DFUR) for the treatment of breast cancer (pt2n1m1); the toxicity was due to decreased dihydropyrimidine dehydrogenase (DPD) activity. Pharmacokinetic investigation showed very high and prolonged plasma 5-FU concentrations after termination of the oral administration of 5′DFUR. The DPD activity in all plasma samples was below 30 pmol/min per mg protein. Her urinary thymine and uracil levels were high, and the ratios of urinary dihydrothymine/thymine and dihydrouracil/uracil were very low compared with the ratios in healthy people. These findings suggest that determination of urinary pyrimidine and dihydropyrimidine levels would be an appropriate urinary screening sytem that may be useful for evaluating DPD activity before 5-FU treatment is initiated.