Quantitative Contribution of rs75017182 to Dihydropyrimidine Dehydrogenase mRNA Splicing and Enzyme Activity.

Dihydropyrimidine dehydrogenase (DPD; DPYD gene) variants have emerged as reliable predictors of adverse toxicity to the chemotherapy agent 5-fluorouracil (5-FU). The intronic DPYD variant rs75017182 has been recently suggested to promote alternative splicing of DPYD. However, both the extent of alternative splicing and the true contribution of rs75017182 to DPD function remain unclear. In the present study we quantified alternative splicing and DPD enzyme activity in rs75017182 carriers utilizing healthy volunteer specimens from the Mayo Clinic Biobank. Although the alternatively spliced transcript was uniquely detected in rs75017182 carriers, canonically spliced DPYD levels were only reduced by 30% (P = 2.8 × 10-6 ) relative to controls. Similarly, DPD enzyme function was reduced by 35% (P = 0.025). Carriers of the well-studied toxicity-associated variant rs67376798 displayed similar reductions in DPD activity (31% reduction). The modest effects on splicing and function suggest that rs75017182 may have clinical utility as a predictor of 5-FU toxicity similar to rs67376798.

Novel Deleterious Dihydropyrimidine Dehydrogenase Variants May Contribute to 5-Fluorouracil Sensitivity in an East African Population.

Clinical studies have identified specific genetic variants in dihydropyrimidine dehydrogenase (DPD; DPYD gene) as predictors of severe adverse toxicity to the commonly used chemotherapeutic 5-fluorouracil (5-FU); however, these studies have focused on European and European-American populations. Our laboratory recently demonstrated that additional variants in non-European haplotypes are predictive of 5-FU toxicity. The objective of this study was to identify potential risk variants in an understudied East African population relevant to our institution's catchment area. The DPYD protein-coding region was sequenced in 588 individuals of Somali or Kenyan ancestry living in central/southeast Minnesota. Twelve novel nonsynonymous variants were identified, seven of which significantly decreased DPD activity in vitro. The commonly reported toxicity-associated variants, *2A, D949V, and I560S, were not detected in any individuals. Overall, this study demonstrates a critical limitation in our knowledge of pharmacogenetic predictors of 5-FU toxicity, which has been based on clinical studies conducted in populations of limited diversity.

Epigenetic mechanisms of protein tyrosine phosphatase 6 suppression in diffuse large B-cell lymphoma: implications for epigenetic therapy.

Protein tyrosine phosphatases such as PTPN6 can be downregulated in various neoplasms. PTPN6 expression by immunohistochemistry in 40 diffuse large B-cell lymphoma (DLBCL) tumors was lost or suppressed in 53% (21/40). To elucidate the molecular mechanisms of PTPN6 suppression, we performed a comprehensive epigenetic analysis of PTPN6 promoter 2 (P2). None of the DLBCL primary tumors (0/37) had PTPN6 hypermethylation on the CpG1 island using methylation-specific PCR, pyrosequencing, and high-resolution melting assays. However, hypermethylation in 57% (21/37) of cases was found in a novel CpG island (CpG2) in P2. PTPN6 gene suppression was reversed by 5-aza-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor, and the histone deacetylase inhibitor (HDACi) LBH589. LBH589 and 5-Aza in combination inhibited DLBCL survival and PTPN6 hypermethylation at CpG2. The role of histone modifications was investigated with a chromatin-immunoprecipitation assay demonstrating that PTPN6 P2 is associated with silencing histone marks H3K27me3 and H3K9me3 in DLBCL cells but not normal B cells. 3-Deazaneplanocin A, a histone methyltransferase inhibitor, decreased the H3K27me3 mark, whereas HDACi LBH589 increased the H3K9Ac mark within P2 resulting in re-expression of PTPN6. These studies have uncovered novel epigenetic mechanisms of PTPN6 suppression and suggest that PTPN6 may be a potential target of epigenetic therapy in DLBCL.

Clinical Pharmacogenetics Implementation Consortium guidelines for dihydropyrimidine dehydrogenase genotype and fluoropyrimidine dosing.

The fluoropyrimidines are the mainstay chemotherapeutic agents for the treatment of many types of cancers. Detoxifying metabolism of fluoropyrimidines requires dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene), and reduced or absent activity of this enzyme can result in severe, and sometimes fatal, toxicity. We summarize evidence from the published literature supporting this association and provide dosing recommendations for fluoropyrimidines based on DPYD genotype (updates at http://www.pharmgkb.org).

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).

Prognostic significance of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase protein expression in colorectal cancer patients treated with or without 5-fluorouracil-based chemotherapy.

BACKGROUND: Low tumour expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) have been linked with improved outcome for colorectal cancer (CRC) patients treated with 5-fluorouracil (5-FU). It is unclear whether this occurs because such tumours have better prognosis or they are more sensitive to 5-FU treatment. PATIENTS AND METHODS: Associations between TS, DPD and TP levels, determined by tissue microarrays and immunohistochemistry, and survival was evaluated in 945 CRC patients according to treatment status. RESULTS: Low TS and DPD expression associated with worse prognosis in stage II [hazard ratio (HR) = 1.69, 95% confidence interval (CI) (1.09-2.63) and HR = 1.92 (95% CI 1.23-2.94), respectively] and stage III CRC patients treated by surgery alone [HR = 1.39 (95% CI 0.92-2.13) and HR = 1.49 (95% CI 1.02-2.17), respectively]. Low TS, DPD and TP associated with trends for better outcome in stage III patients treated with 5-FU [HR = 0.81 (95% CI 0.49-1.33), HR = 0.70 (95% CI 0.42-1.15) and HR = 0.66 (95% CI 0.39-1.12), respectively]. CONCLUSION: Low TS and DPD expression are prognostic for worse outcome in CRC patients treated by surgery alone, whereas low TS, DPD and TP expression are prognostic for better outcome in patients treated with 5-FU chemotherapy. These results provide indirect evidence that low TS, DPD and TP protein expression are predictive of good response to 5-FU chemotherapy.

Role of matrix metalloproteinases in colorectal carcinogenesis.

OBJECTIVE: To measure coexpression of matrix metalloproteinase (MMP)-2, MMP-7, and MMP-9 genes by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in benign and malignant phases of colorectal carcinogenesis. SUMMARY BACKGROUND DATA: Matrix metalloproteinases degrade and remodel the extracellular matrix and have been implicated in facilitating carcinoma cells to invade and metastasize. MMP-2, MMP-7, and MMP-9 have been shown to be overexpressed in various carcinomas; however, simultaneous examination of these enzymes in human normal mucosa, adenoma, and carcinoma has not been performed to date. METHODS: Between January 1, 1998, and June 15, 2000, 40 patients underwent colectomy and harvest and snap-freezing of normal mucosa, adenoma, and carcinoma. Five patients had adenoma and carcinoma in the same specimen; 35 had either adenoma (n = 6) or carcinoma (n = 29). Taqman qRT-PCR methodology was used to measure MMP gene copy number and normalized to beta-actin RNA expression. RESULTS: The mean age was 62 +/- 4 years, with 22 men and 18 women. One fifth of the adenomas exhibited severe dysplasia. MMP-7 gene expression was significantly increased in adenomas (43 times normal mucosa) but did not increase further in carcinomas (50 times normal mucosa). MMP-2 and MMP-9 were not different in adenomas (1.8 and 1.4 times normal mucosa, respectively) but were elevated in carcinomas (2.2 and 1.8 times normal mucosa, respectively). There was no correlation between size or dysplasia in adenomas or AJCC stage in carcinomas and MMP gene expression. CONCLUSIONS: Overexpression of MMP-7 is an early event in the adenoma-to-carcinoma pathway, and expression does not appear to increase further in carcinomas. MMP-2 and MMP-9 appear to be primarily overexpressed in carcinomas. This may be one mechanism by which adenoma cells gain the ability to invade and carcinoma cells to metastasize.

Current status of oral chemotherapy for colorectal cancer.

The treatment of advanced colorectal cancer over the past 4 decades has required the use of intravenous chemotherapy, most typically fluorouracil (5-FU). The possibility of providing an alternative to intravenous delivery while at the same time improving the quality of life of patients who require fluorouracil for advanced or adjuvant therapy has provided the stimulus for the development of oral fluoropyrimidine drugs. Five oral fluoropyrimidine drugs have recently entered clinical trials in the United States. These include capecitabine (Xeloda), UFT (uracil and tegafur) or UFT/leucovorin (Orzel), eniluracil (ethynyluracil), S-1, and BOF A-2. At least two of these drugs have demonstrated survival equivalent to the standard intravenous fluorouracil and leucovorin regimens used to treat advanced colorectal cancer. This, together with less severe toxicity and potential increased quality of life, should lead to approval of one or more of these oral agents in the near future. Based on both patient and physician acceptance of oral fluoropyrimidines, other oral drugs from classes other than fluoropyrimidines will likely be developed in the near future.

Clinical implications of dihydropyrimidine dehydrogenase on 5-FU pharmacology.

Dihydropyrimidine dehydrogenase (DPD) is the initial rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), accounting for catabolism of over 85% of an administered dose of 5-FU. DPD plays an important role in regulating the availability of 5-FU for anabolism. DPD also accounts for much of the variability observed with the therapeutic use of 5-FU. This includes variable 5-FU levels over 24 hours during a continuous infusion; the widely reported variability in the pharmacokinetics of 5-FU; the observed variable bioavailability that led to the recommendation that 5-FU not be administered as an oral agent; and lastly, the observed variability in both toxicity and drug response (resistance) after identical 5-FU doses. Knowledge of the DPD level, as well as the levels of other potentially important molecular markers (e.g., thymidylate synthase), may permit adjustments or modulation of the 5-FU dose that can result in an increase in the therapeutic efficacy of 5-FU.

Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells.

Severe 5-fluorouracil (5-FU) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase (DPD) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5-FU to 5'-6'-dihydrofluorouracil, which is further metabolized to fluoro-beta-alanine. We demonstrate here that overexpression of human DPD confers resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and in human CD34+-enriched hematopoietic progenitor cells. An SFG-based dicistronic retroviral vector containing human DPD cDNA, an internal ribosomal entry site (IRES), and the neomycin phosphotransferase (Neo) gene was constructed (SFG-DPD-IRES-Neo). Transduced NIH3T3 cells demonstrated a 2-fold (ED50) increase in resistance to a 4-hour exposure of 5-FU in comparison to nontransduced cells. Expression of DPD was confirmed by Northern and Western blot analyses, and DPD enzyme activity was detectable only in transduced cells. Infection of mouse bone marrow cells with this retroviral construct resulted in an increased number of 5-FU-resistant CFU-GM colonies, compared to mock-transduced bone marrow in both 4-hour and 12- to 14-day exposures. Infection of human CD34+-enriched cells with this construct and incubation with 5-FU (10(-6) M) for 14 days also resulted in an increased number of 5-FU-resistant colonies. Retroviral transduction of human hematopoietic progenitor cells with a cDNA-expressing human DPD conferred resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and human CD34+-enriched cells. These results encourage the use of this gene as a method to protect patients from 5-FU myelotoxicity.

Oral DPD-inhibitory fluoropyrimidine drugs.

Over the past decade, increasing data have emphasized both the importance of dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting enzyme in the catabolism of fluorouracil (5-FU), and its role as a control step in 5-FU metabolism, regulating the availability of 5-FU for anabolism. It is now clear that DPD also accounts for much of the variability observed with therapeutic use of 5-FU, including variabilities in 5-FU levels over a 24-hour infusion, interindividual pharmacokinetics, bioavailability, toxicity, and drug response (resistance). This variability makes effective dosing of 5-FU and related drugs difficult. In order to lessen this variability, and potentially improve 5-FU pharmacology, the pharmaceutical industry has made an effort to develop DPD inhibitors to modulate 5-FU metabolism, which has resulted in the creation of a new subclass of orally administered fluoropyrimidines, known as DPD-inhibiting fluoropyrimidines (DIF). Four drugs--uracil and tegafur (UFT) or the combination of UFT and leucovorin, ethynyluracil (eniluracil), S-1, and BOF-A2--have recently undergone clinical evaluation in the United States. The biochemical basis for using these drugs is reviewed.

Molecular cloning and characterization of the human dihydropyrimidine dehydrogenase promoter.

Several studies have demonstrated that dihydropyrimidine dehydrogenase (EC 1.3.1.2) has a critical role in the pharmacokinetics of the anticancer agent 5-fluorouracil. We previously reported the structural organization of the human DPYD gene. In this article, we describe the molecular cloning and functional characterization of 1.2 kb of the 5' flanking region of the DPYD gene. Sequence analysis demonstrated that this region of the DPYD gene lacks the typical TATA or CCAAT boxes with several GC-rich regions containing potential cis-regulatory elements. Progressive 5' deletions of the 5' flanking region were fused to the luciferase reporter gene and transient expression measured following transfection into HeLa and 293 cells. Comparative analysis of luciferase activity revealed that a 208 bp region of the DPYD gene (-121/+86) contained equivalent transcriptional activity to the complete 1.2 kb 5' flanking region of the DPYD gene. Site-directed mutagenesis of the luciferase reporter constructs demonstrated that the -72/-23 sequence contained two regulatory regions (designated elements I and II) essential for promoter activity. Gel shift experiments demonstrated that both regulatory elements specifically bind with protein(s) from nuclear extracts of 293 cells. Competitive binding experiments with 293 nuclear extracts and radiolabeled oligonucleotides (corresponding to elements I and II) suggest that the same protein(s) bind to both regulatory elements. We conclude that constitutive expression of the DPYD gene involves a limited GC-rich region of the 5' flanking sequence of the DPYD gene which contains two regulatory elements.

The role of pharmacogenetics and pharmacogenomics in cancer chemotherapy with 5-fluorouracil.

There is increasing evidence supporting the important role of genetics in determining the effect (response and toxicity) to cancer chemotherapy. This has included both pharmacogenetics, where the alteration of a gene coding for an important drug metabolizing enzyme results in increased toxicity (and occasionally altered efficacy), and pharmacogenomics, where knowledge of the expression of genes critical to the action of the cancer chemotherapy drug can be used to individualize therapy. This manuscript focuses on the widely used cancer chemotherapy drug 5-fluorouracil (5-FU) to illustrate the following concepts: (1) The effect of the pharmacogenetic syndrome known as dihydropyrimidine dehydrogenase (DPD) deficiency on 5-FU pharmacology; (2) the role of pharmacogenomics in individualizing 5-FU therapy, and (3) the potential value of pharmacogenomics in designing new drugs.

Activation of protein kinase C induces nuclear translocation of RFX1 and down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60 cells.

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inhibition of Myc synthesis accounted for the drop in Myc protein, because PMA treatment had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein complexes. Increased MIE1 binding was independent of protein synthesis and abolished by a selective PKC inhibitor, which also prevented the effect of PMA on myc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol without affecting total RFX1. Transfection of HL-60 cells with myc reporter gene constructs showed that the RFX1-binding X box was required for the down-regulation of reporter gene expression by PMA. These findings suggest that nuclear translocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 cells.

Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase.

We had previously shown that high gene expressions (mRNA levels) of thymidylate synthase (TS; Leichman et al., J. Clin. Oncol., 15: 3223-3229, 1997) and thymidine phosphorylase (TP; Metzger et al., Clin. Cancer Res., 4: 2371-2376, 1998) in pretreatment tumor biopsies could identify tumors that would be nonresponsive to 5-fluorouracil (5-FU)-based therapy. In this study, we investigated the association between intratumoral gene expression of the pyrimidine catabolism enzyme dihydropyrimidine dehydrogenase (DPD) and the response of colorectal tumors to the same 5-FU-based protocol. DPD expressions were measured by quantitative reverse transcription-PCR in 33 pretreatment biopsies of colorectal tumors from patients who went on to receive treatment with 5-FU and leucovorin (LV). The range of DPD gene expression in those tumors that were nonresponsive to 5-FU was much broader than that of the responding tumors. None of the tumors with basal-level DPD expressions above a DPD:beta-actin ratio of 2.5 x 10(-3) (14 of 33) were responders to 5-FU/LV therapy, whereas those tumors with DPD gene expressions below DPD: beta-actin ratio of 2.5 x 10(-3) had a response rate of 50%. There was no correlation among DPD, TS, and TP expression values in this set of colorectal tumors, which indicated that these gene expressions are independent variables. All of the tumors that responded to 5-FU therapy (11 of 33) had expression values of all three of the genes, TS, TP, and DPD, below their respective nonresponse cutoff values, whereas, in each of the nonresponding tumors, at least one of these gene expressions was high. The patients with low expression of all three of the genes had significantly longer survival than patients with a high value of any one of the gene expressions. The results of this study show that intratumoral gene expression level of DPD is associated with tumor response to 5-FU and that the use of more than one independent determinant of response permits the identification of a high percentage of responding patients.

Phase I and pharmacologic study of oral fluorouracil on a chronic daily schedule in combination with the dihydropyrimidine dehydrogenase inactivator eniluracil.

PURPOSE: To determine the maximum-tolerated dose (MTD), toxicities, and pharmacokinetics of oral fluorouracil (5-FU) administered twice daily in combination with oral eniluracil, an inactivator of dihydropyrimidine dehydrogenase, administered for 28 days every 35 days. PATIENTS AND METHODS: Oral 5-FU 1.35 mg/m(2) twice daily was administered with oral eniluracil 10 mg daily for 14 to 28 days, followed by a 1-week rest period. Eniluracil was started 1 day before 5-FU. Patients then received escalated doses of oral 5-FU 1. 35 to 1.8 mg/m(2) twice daily with an increased dose of eniluracil 10 mg twice daily for 28 days. A reduced dose of 5-FU 1.0 mg/m(2) with eniluracil 20 mg twice daily was evaluated. RESULTS: Thirty-six patients with solid malignancies were enrolled onto the study. Diarrhea was the principal dose-limiting toxicity of oral 5-FU and eniluracil given on this chronic schedule. The recommended phase II dose is 5-FU 1.0 mg/m(2) twice daily with eniluracil 20 mg twice daily. Mean (SD) values for terminal half-life, apparent volume of distribution, and systemic clearance of 4.5 hours (0.83 hours), 19 L/m(2) (3.0 L/m(2)), and 51 mL/min/m(2) (13 mL/min/m(2)), respectively. An average of 77% of 5-FU was excreted unchanged in urine after 28 days of treatment. The mean (range) 5-FU C(SS,min) values achieved at the 1.0 mg/m(2) dose level were 22 ng/mL (8 to 38 ng/mL). CONCLUSION: Chronic oral administration of 5-FU with oral eniluracil is tolerable and produces 5-FU steady-state concentrations similar to those achieved with protracted intravenous administration of 5-FU on clinically relevant dose schedules. Eniluracil provides an attractive means of administering 5-FU on protracted schedules.

Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction.

Several recent studies have reported a correlation between intratumor dihydropyrimidine dehydrogenase (DPD) messenger RNA (mRNA) levels and sensitivity to 5-fluorouracil (5-FU). However, significant tissue requirements and labor-intensive methodology have limited the large-scale studies necessary for statistical validation. In addition, the semiquantitative results obtained by these methods further limit their application. We have developed a real-time reverse transcription-PCR (RT-PCR) assay, based on TaqMan fluorescence methodology, capable of rapid and accurate quantitation of DPD mRNA levels in biopsy-sized tissue samples. Results obtained with this approach indicate a linear dynamic range of 10(8)-10(3) DPD mRNA copies, with an intra-assay variation of <5%. We evaluated the data using three different methods (absolute standard curve, relative standard curve, and comparative C(T)) and show them to be equivalent. This RT-PCR assay was validated by quantitative comparison to Northern blot analysis in five tissues. In addition, analysis of 18 colorectal tumor and liver tissue specimens demonstrated a significant correlation (r(2) = 0.90) between DPD enzyme activity and mRNA levels. This method provides the first high-throughput, reproducible, and sensitive technique capable of determining DPD mRNA expression levels in nanogram amounts of total RNA.