Lori K. Mattison

Implications of dihydropyrimidine dehydrogenase on 5-fluorouracil pharmacogenetics and pharmacogenomics

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.

Increased Prevalence of Dihydropyrimidine Dehydrogenase Deficiency in African-Americans Compared with Caucasians

Purpose: 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. Experimental Design: Healthy African-American (n = 149) and Caucasian (n = 109) volunteers were evaluated for DPD deficiency using both the [2-13C]uracil breath test and peripheral blood mononuclear cell DPD radioassay. Results: 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). Conclusion: 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.

Methylation of the DPYD promoter: an alternative mechanism for dihydropyrimidine dehydrogenase deficiency in cancer patients.

Purpose: 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-egulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. Experimental Design: 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. Results: 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. Discussion: 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.

The Uracil Breath Test in the Assessment of Dihydropyrimidine Dehydrogenase Activity: Pharmacokinetic Relationship between Expired 13CO2 and Plasma [2-13C]Dihydrouracil

Purpose: Dihydropyrimidine dehydrogenase (DPD) deficiency is critical in the predisposition to 5-fluorouracil dose-related toxicity. We recently characterized the phenotypic [2-13C]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 13CO2 metabolite formation, plasma [2-13C]dihydrouracil formation, [2-13C]uracil clearance, and DPD activity. Experimental Design: An aqueous solution of [2-13C]uracil (6 mg/kg) was orally administered to 23 healthy volunteers and 8 cancer patients. Subsequently, breath 13CO2 concentrations and plasma [2-13C]dihydrouracil and [2-13C]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. Results: 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 13CO2 DOB50 significantly correlated with PBMC DPD activity (rp = 0.78), plasma [2-13C]uracil area under the curve (rp = −0.73), [2-13C]dihydrouracil appearance rate (rp = 0.76), and proportion of [2-13C]uracil metabolized to [2-13C]dihydrouracil (rp = 0.77; all Ps < 0.05). Conclusions: UraBT breath 13CO2 pharmacokinetics parallel plasma [2-13C]uracil and [2-13C]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.

Genetic regulation of dihydropyrimidinase and its possible implication in altered uracil catabolism

Objective 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 &bgr;-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. Methods 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. Results 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. Conclusion 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.

A DPYD variant (Y186C) in individuals of african ancestry is associated with reduced DPD enzyme activity.

5‐Fluorouracil (5‐FU) is used to treat many aggressive cancers, such as those of the colon, breast, and head and neck. The responses to 5‐FU, with respect to both toxicity and efficacy, vary among racial groups, potentially because of variability in the activity levels of the enzyme dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene). In this 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‐Y186C variant was unique to individuals of African ancestry, and DPD activity was 46% lower in carriers as compared with noncarriers (279 ± 35 vs. 514 ± 168 pmol 5‐FU min−1 mg−1; P = 0.00029). In this study, 26% of the African Americans with reduced DPD activity were carriers of Y186C. In the African‐American cohort, after excluding Y186C carriers, homozygous carriers of C29R showed 27% higher DPD activity as compared with noncarriers (609 ± 152 and 480 ± 152 pmol 5‐FU min−1 mg−1, respectively; P = 0.013).

Genetic regulation of ??-ureidopropionase and its possible implication in altered uracil catabolism

Objective 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 &bgr;-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. Methods This study included 219 healthy asymptomatic volunteers with known DPD enzyme activity and [2-13C]-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. Results 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. Conclusion 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.

LBOVI-B-2Describing the catabolism of orally administered 2-13C-uracil in normal and dihydropyrimidine dehydrogenase deficient subjects using a parent/metabolite/breath model

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.