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 F 0.07 and 0.29 F 0.07 nmol/min/mg, respectively; P = 0.002). The prevalence of DPD deficiency was 3-fold higher in AfricanAmericans 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.5-Fluorouracil (5-FU) and its fluoropyrimidine derivatives (e.g., capecitabine) are widely prescribed in oncologic practice to treat gastrointestinal malignancies and are often used in the management of breast and head and neck cancer (1 -4). However, despite its widespread use, f31% of patients with advanced colorectal cancer who receive bolus 5-FU regimens experience grades 3 to 4 hematologic toxicities (5). The pharmacogenetic syndrome, dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) deficiency, has been shown to predispose cancer patients to severe 5-FU toxicity (6 -9). In particular, it is estimated that 40% to 60% of patients with cancer who present with severe 5-FU toxicity are 11).Several studies show the pivotal role of DPD in 5-FU metabolism and response. Earlier biochemical studies showed that DPD, the initial and rate-limiting enzyme of the pyrimidine catabolic pathway, degrades uracil, thymine, and 5-FU to dihydrouracil, dihydrothymine, and 5-fluoro-dihydrouracil, respectively (12, 13). Pharmacokinetic evaluation has further shown that DPD catabolizes >80% of an administered dose of 5-FU, thereby determining the amount of 5-FU available for anabolism (7). Furthermore, data from combined pharmacokinetic/pharmacodyna...

Purpose: 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 DPDdeficient cancer patients.Experimental Design: 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.Results: The mean (؎SE) C max , T max , ␦ 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).Conclusions: 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 DPDdeficient individuals. These data suggest that a single time-point determination (50 min) could rapidly identify DPD-deficient individuals with a less costly and timeconsuming method that is applicable for most hospitals or physicians' offices.

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-Y186C variant was unique to individuals of African ancestry, and DPD activity was 46% reduced in carriers compared to non-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 activity in this study carried 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).

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 downegulate 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.Dihydropyrimidine dehydrogenase (DPD) enzyme deficiency, a known pharmacogenetic syndrome detected in 3% to 5% of the population (1), has been associated with toxicity to 5-fluorouracil (5-FU) cancer chemotherapy and death in some cases with profound deficiency of the enzyme (2). DPD is the first enzyme in a three-step catabolic pathway responsible for the degradation of f85% of administered 5-FU. Genotypic studies have identified >32 sequence variants in the DPYD gene (1, 3). Expression analysis of these variants showed that many were polymorphisms with no obvious functional significance (4), with the exception of a few mutations. One example commonly associated with DPD deficiency and severe toxicity to 5-FU is an intronic sequence variation (IVS14 + 1 G > A, DPYD*2A), which results in a truncated protein that lacks 55 amino acids due to the skipping of exon 14 (5). A second example is a less common mutation (1679T > G, I560S, DPYD*13), which is associated with decreased DPD activity and 5-FU toxicity (6) due to a nonconservative amino acid change from isoleucine to serine at codon 560 (I560), which is 100% conserved among human, mouse, rat, bovine, and pig species, suggesting its imp...