[Show abstract][Hide abstract] ABSTRACT: 5-fluorouracil (5-FU) is used to treat many aggressive cancers, such as those of the colon, breast, and head &neck. The responses to 5-FU, both toxicity and efficacy, vary between racial groups, potentially due to variability in enzyme activity of dihydropyrimidine dehydrogenase (DPD, encoded by DPYD). In the present study, the genetic associations between DPYD variations and circulating mononuclear cell DPD enzyme activity were evaluated in 94 African American and 81 European American volunteers. The DPYD-Y186Cvariantwasunique to individuals of African ancestry, and DPD activity was 46% reduced in carriers compared tonon-carriers (279±35 compared to 514±168 pmol 5-FU min(-1) mg(-1); P=0.00029).26% of the African Americans with reduced DPD activityin this studycarried Y186C. In the African American cohort, following exclusion of Y186C carriers,homozygous carriers of C29R showed 27% higher DPD activity compared to non-carriers (609±152 and 480±152 pmol 5-FU min(-1) mg(-1), respectively; P=0.013).Clinical Pharmacology & Therapeutics (2013); accepted article preview online 3 April 2013 doi:10.1038/clpt.2013.69.
[Show abstract][Hide abstract] ABSTRACT: Breath tests (BTs) represent a safe non-invasive alternative strategy that could provide valuable diagnostic information in conditions like fat malabsorption, carbohydrate (lactose and fructose) malabsorption, liver dysfunction, impaired gastric emptying, abnormal small bowel transit time, small intestinal bacterial overgrowth and Helicobacter pylori infection. To date, despite the availability of a number of breath tests, only three have gained approval by the FDA for application in a clinical setting (13C-urea breath test for the detection of H. pylori; NO breath test for monitoring asthma and alkane breath test for heart transplant rejection). Unfortunately, none of these tests investigate cancer patients or response to cancer chemotherapy. Several years ago it was realized that the presence of a reliable non-invasive approach could assist in the detection of patients at risk of developing severe life-threatening toxicities prior to the administration of fluoropyrimidines (e.g. 5-FU) or related cancer chemotherapy. 5-FU toxicity results mainly from deficient uracil catabolism. This review discusses the development of a BT that utilizes an orally administered pyrimidine ([2-13C]-uracil) which is metabolized via the same catabolic pathway as 5-FU. This ([2-13C]-uracil) breath test could provide a valuable addition to the patients' standard of care.
Journal of Breath Research 11/2009; 3(4):047002. DOI:10.1088/1752-7155/3/4/047002 · 3.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Approximately 30-40% of grade III-IV toxicity to 5-FU has been associated with partial or profound deficiency in dihydropyrimidine dehydrogenase (DPD), the first of three enzymes in the catabolic pathway of fluoropyrimidines. There remains, however, a subset of patients presenting with 5-FU-associated toxicity despite normal DPD activity, suggesting possible deficiencies in enzymes downstream of DPD: dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. Previously, we reported the identification of inactivating mutations in the DPYS gene that could potentially alter the uracil catabolic pathway in healthy individuals with normal DPD enzyme activity. This study investigates the possible role of UPB1 genetic variations in the regulation of the uracil catabolic pathway in individuals presenting with a deficient uracil breath test (13C-UraBT) despite normal DPD enzyme activity.
This study included 219 healthy asymptomatic volunteers with known DPD enzyme activity and [2-(13)C]-uracil breath test (UraBT). All samples were genotyped for sequence variations in the UPB1 gene using denaturing high performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation of detected sequence variants by direct sequencing.
Seven novel and six previously reported sequence variations were identified, including one nonconservative mutation, which demonstrated 97.3% reduction in BUP-1 activity when expressed in the RKO cell line.
Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD and/or DHP deficiency and that inactivating mutations in the UPB1 gene might impair uracil catabolism.
Pharmacogenetics and Genomics 02/2008; 18(1):25-35. DOI:10.1097/FPC.0b013e3282f2f134 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dihydropyrimidine dehydrogenase (DPD) deficiency accounts for approximately 43% of grade 3-4 toxicity to 5-fluorouracil. There, however, remain a number of patients presenting with 5-fluorouracil-associated toxicity despite normal DPD enzyme activity, suggesting possible deficiencies in dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. This study investigates the role of DPYS sequence variations in individuals with unexplained molecular basis of altered uracil catabolism.
This study included 219 asymptomatic healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles. All samples were genotyped for sequence variations in the DPYS gene using denaturing high-performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation by direct sequencing. Site-directed mutagenesis and expression analysis were performed to determine the effect of the identified nonconservative mutations on DHP enzyme activity.
Seven previously reported and 11 novel sequence variations were identified, including three nonconservative mutations; two of which (L7V and 1635delC) demonstrated decreased DHP activity when expressed in the RKO cell line (P=0.25). The P values were not significant due to the small sample size (n=3); however, a modified [2-13C]-uracil breath test, the 13C-dihydrouracil breath test, was administered to four volunteers to confirm that the 1635delC mutation does in fact reduce in-vivo DHP activity.
Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD deficiency, and that inactivating mutations in DHP might impair uracil catabolism in cases of normal DPD activity.
Pharmacogenetics and Genomics 12/2007; 17(11):973-87. DOI:10.1097/FPC.0b013e3282f01788 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: 5-Fluorouracil (5-FU) is an integral part of treatment of GI malignancies. While normal DPD enzyme activity is rate limiting in 5-FU catabolism, its deficiency could increase concentrations of bioavailable 5-FU anabolic products leading to 5-FU related toxicity syndrome. METHODOLOGY: Twenty-three patients were tested for DPD deficiency after excessive toxicities from 5-FU and/or capecitabine. DPD activity was evaluated by Peripheral Blood Mononuclear Cell (PBMC) radioassay, genotyping of DPYD gene by Denaturing High Performance Liquid Chromatography (DHPLC), or 2-(13)C uracil breath test (UraBT). RESULTS: Of 23 patients with excessive toxicities from 5-FU and/or capecitabine, 7 (30%) were DPD deficient with a median age of 66 years, M:F ratio = 1.3:1 and ethnicities included Caucasian (71%), African-American (14%) and South-Asian (14%). DPD activity ranged from 0.064 - 0.18nmol/min/mg. Three patients were treated with bolus 5-FU/LV, two with capecitabine, and two with high dose bolus 5-FU with 2', 3', 5'-tri-O-acetyluridine. Toxicities included mucositis (71%), diarrhea (43%), skin rash (43%), memory loss/altered mental status (43%), cytopenias (43%), nausea (29%), hypotension (14%), respiratory distress (14%) and acute renal failure (14%) Re-challenge with capecitabine in one patient after the Mayo regimen caused grade 3 hand-foot syndrome. Genotypic analysis of the DPYD gene in one patient with severe leucopenia demonstrated a heterozygous mutation (IVS14+1 G>A, DPYD). The UraBT in two patients of 112.8; PDR of 49.4%) and borderline normal values revealed 1 to be DPD-deficient (DOB(50) of 130.9; PDR of 52.5%) in a second patient. There were 2 toxicity-related deaths among (DOB(50) DPD-deficient patients (28%). CONCLUSIONS: DPD deficiency was observed in several ethnicities. Akin to 5-FU, capecitabine can also lead to severe toxicities in DPD-deficient patients. Screening patients for DPD deficiency prior to administration of 5-FU or capecitabine using UraBT could potentially lower risk of toxicity. Future studies should validate this technique.
[Show abstract][Hide abstract] ABSTRACT: Peripheral neuropathy secondary to 5-flourouracil and capecitabine (Xeloda) has been reported. We report the first case of exacerbation of peripheral neuropathy related to topical 5-flourouracil (Efudex). A 70-year-old Caucasian male with a history of actinic keratosis for 15 years was treated intermittently with topical application of 5-flourouracil. He also developed sensory peripheral neuropathy around the same time, but extensive work-up disclosed no clear etiology. In early 2005, he developed an exacerbation of his peripheral neuropathy following a 21-day course of topical 5-flourouracil for actinic keratosis, especially pain and parasthesias. Dihydropyrimidine dehydrogenase activity was evaluated in the peripheral mononuclear cells both by radioassay and by [2-C] uracil breath test. Dihydropyrimidine dehydrogenase activity was within the normal range by both methods. Stopping topical 5-flourouracil resolved the symptoms to baseline. Instead of topical 5-flourouracil, topical imiquimod was used which did not exacerbate his neuropathy. He was not re-challenged with topical 5-flourouracil. Topical 5-flourouracil has been known to cause mainly dermatological adverse effects, but systemic effects because of absorption are possible, especially in dihydropyrimidine dehydrogenase-deficient patients. As our patient had no other cause responsible for his neuropathy, the onset of symptoms coincided historically with topical application of 5-flourouracil and the 5-flourouracil usage preceded an exacerbation of sensory symptoms, we conclude that this drug was responsible for his polyneuropathy.
[Show abstract][Hide abstract] ABSTRACT: African-American patients with colorectal cancer were observed to have increased 5-fluorouracil (5-FU)-associated toxicity (leukopenia and anemia) and decreased overall survival compared with Caucasian patients. One potential source for this disparity may be differences in 5-FU metabolism. Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme of 5-FU catabolism, has previously been shown to have significant interpatient variability in activity. Several studies have linked reduced DPD activity to the development of 5-FU toxicity. Although the distribution of DPD enzyme activity and the frequency of DPD deficiency have been well characterized in the Caucasian population, the distribution of DPD enzyme activity and the frequency of DPD deficiency in the African-American population are unknown.
Healthy African-American (n=149) and Caucasian (n=109) volunteers were evaluated for DPD deficiency using both the [2-(13)C]uracil breath test and peripheral blood mononuclear cell DPD radioassay.
African-Americans showed significantly reduced peripheral blood mononuclear cell DPD enzyme activity compared with Caucasians (0.26+/-0.07 and 0.29+/-0.07 nmol/min/mg, respectively; P=0.002). The prevalence of DPD deficiency was 3-fold higher in African-Americans compared with Caucasians (8.0% and 2.8%, respectively; P=0.07). African-American women showed the highest prevalence of DPD deficiency compared with African-American men, Caucasian women, and Caucasian men (12.3%, 4.0%, 3.5%, and 1.9%, respectively).
These results indicate that African-Americans, particularly African-American women, have significantly reduced DPD enzyme activity compared with Caucasians, which may predispose this population to more 5-FU toxicity.
Clinical Cancer Research 10/2006; 12(18):5491-5. DOI:10.1158/1078-0432.CCR-06-0747 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Chemotherapy and Pharmacology 10/2006; 58(3):396-401. DOI:10.1007/s00280-005-0174-5 · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Clinical Cancer Research 02/2006; 12(2):549-55. DOI:10.1158/1078-0432.CCR-05-2020 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Dihydropyrimidine dehydrogenase (DPD) deficiency leads to life-threatening 5-Fluorouracil toxicity. We developed a 2-13C-uracil (13C-Ura) breath test (BT) to rapidly screen cancer patients. Deficient patients have low breath 13CO2 concentrations due to reduced catabolism of 13C-Ura to 13C-dihydrouracil (13C-DHU) and 13CO2.Aim: Develop a pharmacokinetic (PK) model to simultaneously describe 13C-Ura and catabolite concentrations in plasma and breath from normal and DPD deficient subjects.Methods: Blood and breath samples were collected for 3h after oral 13C-Ura (6 mg/kg) administration to 19 normal and 10 DPD deficient subjects. Plasma 13C-Ura, 13C-DHU, and breath 13CO2 concentrations were quantified. ADAPT II simultaneously fitted parent, metabolite, and breath concentration-time data. Akaike's Information Criterion was used to select the model.Results: A one compartment linear absorption/elimination parent/metabolite model with cumulative breath13CO2 elimination described concentration-time data of 13C-Ura, 13C-DHU, and 13CO2. Significant differences in modeled PK parameters between normal and DPD deficient subjects were observed.Conclusion: This is the first model to simultaneously describe orally administered 13C-Ura disposition in normal and DPD deficient subjects. Bayesian parameter estimation and simulation studies will be used to identify an optimal limited sampling strategy to detect DPD deficiency in patients who aren't candidates for BT screening. (See Table)
[Show abstract][Hide abstract] ABSTRACT: 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.
Clinical Cancer Research 01/2006; 11(24 Pt 1):8699-705. DOI:10.1158/1078-0432.CCR-05-1520 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dihydropyrimidine dehydrogenase (DPD)-deficient cancer patients have been shown to develop severe toxicity after administration of 5-fluorouracil. Routine determination of DPD activity is limited by time-consuming and labor-intensive methods. The purpose of this study was to develop a simple and rapid 2-(13)C-uracil breath test, which could be applied in most clinical settings to detect DPD-deficient cancer patients.
Fifty-eight individuals (50 "normal," 7 partially, and 1 profoundly DPD-deficient) ingested an aqueous solution of 2-(13)C-uracil (6 mg/kg). (13)CO(2) levels were determined in exhaled breath at various time intervals up to 180 min using IR spectroscopy (UBiT-IR(300)). DPD enzyme activity and DPYD genotype were determined by radioassay and denaturing high-performance liquid chromatography, respectively.
The mean (+/-SE) C(max), T(max), delta over baseline values at 50 min (DOB(50)) and cumulative percentage of (13)C dose recovered (PDR) for normal, partially, and profoundly DPD-deficient individuals were 186.4 +/- 3.9, 117.1 +/- 9.8, and 3.6 DOB; 52 +/- 2, 100 +/- 18.4, and 120 min; 174.1 +/- 4.6, 89.6 +/- 11.6, and 0.9 DOB(50); and 53.8 +/- 1.0, 36.9 +/- 2.4, and <1 PDR, respectively. The differences between the normal and DPD-deficient individuals were highly significant (all Ps <0.001).
We demonstrated statistically significant differences in the 2-(13)C-uracil breath test indices (C(max), T(max), DOB(50), and PDR) among healthy and DPD-deficient individuals. These data suggest that a single time-point determination (50 min) could rapidly identify DPD-deficient individuals with a less costly and time-consuming method that is applicable for most hospitals or physicians' offices.
Clinical Cancer Research 05/2004; 10(8):2652-8. DOI:10.1158/1078-0432.CCR-03-0374 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A prominent example of the potential application of pharmacogenomics and pharmacogenetics to oncology is the study of dihydropyrimidine dehydrogenase (DPD) in 5-fluorouracil (5-FU) metabolism. 5-FU is currently one of the most widely administered chemotherapeutic agents used for the treatment of epithelial cancers. DPD is the rate-limiting enzyme in the catabolism and clearance of 5-FU. The observation of a familial linkage of DPD deficiency from a patient exhibiting 5-FU toxicity suggested a possible molecular basis for variations in 5-FU metabolism. Molecular studies have suggested there is a relationship between allelic variants in the DPYD gene (the gene that encodes DPD) and a deficiency in DPD activity, providing a potential pharmacogenetic basis for 5-FU toxicity. In the last decade, studies have correlated tumoral DPD activity with 5-FU response, suggesting it may be a useful pharmacogenomic marker of patient response to 5-FU-based chemotherapy. This article reviews the basis and discusses the challenges of pharmacogenetic and pharmacogenomic testing of DPD for the determination of 5-FU efficacy and toxicity.
[Show abstract][Hide abstract] ABSTRACT: A pharmacogenetic syndrome caused by molecular defects in the dihydropyrimidine dehydrogenase gene (DPYD ) results in partial to complete loss of dihydropyrimidine dehydrogenase (DPD) enzyme activity with patients exhibiting life-threatening toxicity following administration of routine doses of 5-fluorouracil. To date, more than 19 reported mutations have been putatively associated with DPD deficiency with 16 occurring within the open reading frame of the cDNA. The purpose of this study was to examine the conservation of functional domains (including the uracil, flavine adenine dinucleotide and NADPH binding sites) across three phyla (Chordata, Arthropoda and Nematoda) and the conservation of regions corresponding to the previously reported mutations. Comparative analysis of the uracil and NADPH binding sites in mammals and invertebrates demonstrated 100% amino acid identity between mammals and Drosophila melanogaster. Caenorhabditis elegans demonstrated 89% and 88% identity in these domains, respectively. The mammalian sequences demonstrated 100% identity in two iron sulphur motifs (amino acids 953-964 and 986-997) with significant conservation in D. melanogaster (92% and 92% identity, respectively) and C. elegans (100% and 92% identity, respectively). Comparative amino acid analysis revealed non-conservation in the loci of four DPYD mutations [DPYD*12 (R21Q), DPYD*5 (I543V), DPYD*6 (V732I), DPYD*9A (C29R)]. Seven mutations occurred in highly conserved regions [M166V, DPYD*8 (R235W), DPYD*11 (V335l), DPYD*4 (S534N), DPYD*9B (R886H), D949V, DPYD*10 (V995F)]. In summary, this comparative analysis identified conserved regions which may be critical to enzyme structure and/or function. The conservation of loci where DPYD mutations occur was also examined to evaluate their functional significance on DPD enzyme activity. These data should prove useful in the evaluation of newly discovered mutations in the DPYD gene.