Confirmatory DPYD Testing in Patients Receiving Fluoropyrimidines Who are Suspected DPYD Variant Carriers Based on a Genetic Data Repository.

Using pharmacogenetics (PGx) to inform clinical decision making can benefit patients but clinical use of PGx testing has been limited. Existing genetics data obtained in the course of research could be used to identify patients who are suspected, but have not yet been confirmed, to carry clinically actionable genotypes, in whom confirmatory genetic testing could be conducted for highly efficient PGx implementation. Herein, we demonstrate that it is regulatorily and technically feasible to implement PGx by identifying suspected carriers of actionable genotypes within an institutional genetics data repository and conduct confirmatory PGx testing immediately prior to that patient receiving the PGx-relevant drug, using a case study of DPYD testing prior to fluoropyrimidine chemotherapy. In 2 years since launching this program, ~ 3,000 suspected DPYD carriers have been passively monitored and one confirmed DPYD carrier was prevented from receiving unacceptably toxic fluoropyrimidine treatment, for minimal cost and effort. Now that we have demonstrated the feasibility of this strategy, we plan to transition to PGx panel testing and expand implementation to other genes and drugs for which the evidence of clinical benefit of PGx-informed treatment is high but PGx testing is not generally conducted. This highly efficient implementation process will maximize the clinical benefits of testing and could be explored at other institutions that have research-only genetic data repositories to expand the number of patients who benefit from PGx-informed treatment while we continue to work toward wide-scale adoption of PGx testing and implementation.

Effects of a bolus injection of 5-fluorouracil on dihydropyrimidine dehydrogenase activity in rats receiving continuous infusion of 5-fluorouracil.

PURPOSE: The options for improving the chemotherapeutic regimen consisting of bolus plus infusion of 5-fluorouracil (5-FU) include omitting the 5-FU bolus injection. We examined the effects of a 5-FU bolus injection on the activity of dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme of 5-FU catabolism, in rats. METHODS: The rats were divided into three groups, and then continuous infusion (50 mg/m(2)/h) for 4 h was started with a bolus injection of saline, 20 mg/kg 5-FU, or 60 mg/kg 5-FU. Plasma 5-FU, uracil (Ura), dihydrouracil (UH2) levels, and hepatic DPD activity were determined after administration of 5-FU. RESULTS: The half-life after the end of the infusion (t 1/2, 4-8 h) of 5-FU in the rats given the bolus injection was significantly longer than in those that had been given saline, and it increased with increasing 5-FU bolus injection dosage (r = 0.801, p < 0.01). The plasma UH2/Ura ratio, an indirect biomarker of hepatic DPD activity, tended to be lower in the rats that had received a 5-FU bolus injection than in those that had not, and it remained low after infusion ended. The hepatic DPD activity in rats that had received a 5-FU bolus injection was significantly lower than in those that had not. Negative correlation was observed between DPD activity and bolus injection dosage (r = -0.691, p < 0.05). CONCLUSIONS: A bolus injection suppresses hepatic DPD activity and its effects are dependent on dosage, resulting in slower elimination of 5-FU from the blood and contributing to long-term systemic exposure to 5-FU.

A DPYD variant (Y186C) specific to individuals of African descent in a patient with life-threatening 5-FU toxic effects: potential for an individualized medicine approach.

5-Fluorouracil (5-FU) is commonly administered as a therapeutic agent for the treatment of various aggressive cancers. Severe toxic reactions to 5-FU have been associated with decreased levels of dihydropyrimidine dehydrogenase (DPD) enzyme activity. Manifestations of 5-FU toxicity typically include cytopenia, diarrhea, stomatitis, mucositis, neurotoxicity, and, in extreme cases, death. A variety of genetic variations in DPYD, the gene encoding DPD, are known to result in decreased DPD enzyme activity and to contribute to 5-FU toxic effects. Recently, it was reported that healthy African American individuals carrying the Y186C DPYD variant (rs115232898) had significantly reduced DPD enzyme activity compared with noncarriers of Y186C. Herein, we describe for the first time, to our knowledge, an African American patient with cancer with the Y186C variant who had severe toxic effects after administration of the standard dose of 5-FU chemotherapy. The patient lacked any additional toxic effect-associated variations in the DPYD gene or the thymidylate synthase (TYMS) promoter. This case suggests that Y186C may have contributed to 5-FU toxicity in this patient and supports the use of Y186C as a predictive marker for 5-FU toxic effects in individuals of African ancestry.

[Dihydropirymidine dehydrogenase (DPD)--a toxicity marker for 5-fluorouracil?]. / Dehydrogenaza dihydopirymidynowa (DPD)--marker toksycznosci 5-fluorouracylu?

In proceedings relating to patients suffering from cancer, an important step is predicting response and toxicity to treatment. Depending on the type of cancer, physicians use the generally accepted schema of treatment, for example pharmacotherapy. 5-fluorouracil (5-FU) is the most widely used anticancer drug in chemotherapy for colon, breast, and head and neck cancer. Patients with dihydropyrimidine dehydrogenase (DPD) deficiency, which is responsible for the metabolism of 5-FU, may experience severe side effects during treatment, and even death. In many publications the need for determining the activity of DPD is discussed, which would protect the patient from the numerous side effects of treatment. However, in practice these assays are not done routinely, despite the high demand. In most cases, a genetic test is used to detect changes in the gene encoding DPD (such as in the USA), but because of the large number of mutations the genetic test cannot be used as a screening test. Dihydropyrimidine dehydrogenase activity has been shown to have high variability among the general population, with an estimated proportion of at least 3-5% of individuals showing low or deficient DPD activity. In this publication we presents data about average dihydropirymidine dehydrogenase activity in various populations of the world (e.g. Japan, Ghana, Great Britain) including gender differences and collected information about the possibility of determination of DPD activity in different countries. Detection of reduced DPD activity in patients with planned chemotherapy will allow a lower dosage of 5-FU or alternative treatment without exposing them to adverse reactions.

The effect of chemotherapy or radiotherapy on thymidine phosphorylase and dihydropyrimidine dehydrogenase expression in cancer of the uterine cervix.

OBJECTIVE: Levels of 5-FU metabolic or related enzymes, particularly thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD), have been investigated in various cancer types, including uterine cervical cancer. Intratumoral TP levels have been reported to increase in response to several chemotherapeutic agents or irradiation in both xenografts and clinical studies. In cervical cancer, however, only a few studies about changes in TP and DPD expression associated with cancer treatment have been published. We evaluated the effect of chemotherapy and/or irradiation on TP and DPD expression in cervical squamous cell carcinoma. STUDY DESIGN: Of 27 patients in this study, 12 patients underwent neoadjuvant chemotherapy consisting of nedaplatin, ifosfamide, and/or peplomycin followed by radical surgery, and 15 patients underwent radiotherapy (n=8) or chemoradiotherapy with nedaplatin (n=7) as initial treatment. Tumor specimens were obtained from biopsies acquired before treatment and after administration of chemotherapy (2 weeks after the first and second cycles), and after irradiation with 10 Gy, 20 Gy, and 30 Gy. These specimens were used to measure TP and DPD levels by ELISA. RESULTS: In the 12 patients who received neoadjuvant chemotherapy, intratumoral TP and DPD levels did not change. In contrast, in the 15 patients who underwent radiotherapy or chemoradiotherapy with nedaplatin, TP or DPD expression appeared to be slightly increased or decreased, respectively, after irradiation with 20 Gy, and consequently the TP/DPD ratio was significantly higher after irradiation with 20 Gy than before irradiation. CONCLUSIONS: These results suggest a clinical advantage of chemoradiotherapy with capecitabine or doxyfluridine over radiotherapy alone via the elevation of the TP/DPD ratio in cervical squamous cell carcinoma. However, no advantage of combination chemotherapy with these 5-FU derivatives was demonstrated. Therefore, further evaluation with a larger number of patients or with other chemotherapeutic agents is required to confirm these observations.

[Review of methods for determination of dihydropyrimidine dehydrogenase and possible application in screening previous chemotheraphy with 5-fluorouracil]. / Przeglad metod oznaczania dehydrogenazy dihydropirymidynowej oraz mozliwosci ich zastosowania w badaniach przesiewowych poprzedzajacych chemoterapie 5-fluorouracylem.

5-fluorouracil (5-FU) and its prodrug capecitabine are one of the most commonly used chemotherapeutic drugs. DPD-deficient cancer patients may be at risk of severe and sometimes lethal toxicity after the administration of 5-FU. In 39-61% of the cases severe toxicity of 5-FU is caused by decreased DPD acivity. DPD is the initial and rate-limiting enzyme of the metabolism of pyrimidines. 80-90% of the administered 5-FU is catabolised by DPD. Mutation of the DPYD gene encoding DPD result in decreased enzyme activity--total (0.2% of population) or partial (3-5% of population). Determination of DPD activity can be used as a screening procedure to identify patients with a DPD deficiency, before the start of treatment with 5-FU. There are several methods for DPD activity determination: the detection of relevant DPYD gene single-nucleotide polymorphism (SNPs), measurement of the level of DPYDmRNA expression, the evaluation of DPD activity in PBMC, the measurement of uracil in plasma and urea, evaluation of the UH2/U (dihydrouracil/uracil) and THYH2/THY (dihydrothymine/tymine) ratio in plasma and urea, [2-C13]uracil breath test, the analysis of fluorouracil and dihydrofluorouracil in plasma after administered a test dose of fluorouracil and measurement of 2-fluoro-beta-alanine. So far more than 30 mutations of DPYD gene have been identified in patients with cancer. A large number of them limits introduction of simple genetic test, which could be used for detection of DPD deficiency. Therefore scientists in searching of the simplest, the cheapest and the most available technique for detection DPD deficiency, generally use methods associated with measurement of DPD activity.

Effects of combined administration of DPD-inhibitory oral fluoropyrimidine, S-1, plus paclitaxel on gene expressions of fluoropyrimidine metabolism-related enzymes in human gastric xenografts.

BACKGROUND: S-1 is the most effective oral fluoropyrimidine derivative widely used for patients with gastric carcinoma in Japan. Although S-1 plus taxane has been a promising candidate as an effective chemotherapeutic regimen, the mechanisms of its additive or synergistic anticancer effects and changes in gene expression after the administration of these agents have not yet been fully elucidated. METHODS: Experimental chemotherapy was performed using human gastric carcinoma xenografts, MKN-45 and TMK-1, to examine anticancer effects and gene expressions of fluoropyrimidine metabolism-related enzymes including thymidine phosphorylase (TP), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and uridine phosphorylase (UP). Nude mice were treated with S-1, paclitaxel, and their combination. After treatment, in vivo antitumor effects of S-1, paclitaxel alone, and their combination and the effects on gene expressions of enzymes involved in 5-fluorouracil metabolism were examined using the RT-PCR method. RESULTS: The combined use of S-1 and paclitaxel showed additive to synergistic antitumor effects on both gastric cancer xenografts. While consistent upregulation of dThPase and DPD gene expression was exhibited after administration of S-1, no further increase of dThPase gene expression after combined use of S-1 with paclitaxel was observed. There was no increase in TS gene expression after the administration of either S-1 alone or paclitaxel alone. CONCLUSION: These results provide some insight into the mechanism and/or rationale underlying the additive to synergistic effect of combined administration of S-1 and paclitaxel in gastric carcinoma.

Synergistic antitumor effect of combined 5-fluorouracil (5-FU) with 5-chloro-2,4-dihydroxypyridine on 5-FU-resistant gastric cancer cells: possible role of a dihydropyrimidine dehydrogenase-independent mechanism.

BACKGROUND: S-1 is an oral fluorouracil antitumor drug that combines three pharmacological agents: tegafur, a prodrug of 5-fluorouracil (5-FU); 5-chloro-2,4-dihydroxypyridine (CDHP), an inhibitor of dihydropyrimidine dehydrogenase (DPD); and potassium oxonate, an agent included to reduce gastrointestinal toxicity. S-1 has a potent antitumor effect on gastric cancer, even in 5-FU-refractory cases. However, there is a lack of basic information to account for this clinical outcome. This study was performed to determine the differences in antitumor effects of combined administration of 5-FU and CDHP between NUGC-3 cells and NUGC-3/5FU/L cells, which are resistant to 5-FU (established by repeated cultures of NUGC-3 with escalating concentrations of 5-FU), and to determine the mechanisms involved. METHODS: Both cell lines were incubated with various concentrations of 5-FU and/or CDHP. The antitumor effect was assessed using an MTS assay and cell counts. DPD levels were assayed by using enzyme-linked immunosorbent assay. Expression of DPD and thymidylate synthase (TS) mRNA was quantified using real-time quantitative polymerase chain reaction analysis. RESULTS: The combination of 5-FU (IC15) with CDHP exerted a synergistic antitumor effect on NUGC-3/5FU/L, but not on NUGC-3, while CDHP by itself did not affect cell growth in either cell line. Expression of DPD was not detected in NUGC-3/5FU/L. In NUGC-3/5FU/L, 5-FU-enhanced expression of TS mRNA was inhibited by the addition of CDHP. In contrast, in NUGC-3, administration of 5-FU with or without CDHP did not alter TS mRNA expression. CONCLUSIONS: The inhibitory mechanism of CDHP, which is independent of DPD, may in part contribute to the antitumor effect of S-1 even in 5-FU-resistant gastric cancer cases.

Role of thymidine phosphorylase and orotate phosphoribosyltransferase mRNA expression and its ratio to dihydropyrimidine dehydrogenase in the prognosis and clinicopathological features of patients with pancreatic cancer.

BACKGROUND: Thymidine phosphorylase (TP), orotate phosphoribosyltransferase (OPRT), and dihydropyrimidine dehydrogenase (DPD) are important enzymes related to the metabolism of 5-fluorouracil and its derivatives. In this study, we analyzed the expression of these enzymes and evaluated the association between the expression of these enzymes and clinicopathological features and prognosis in patients with pancreatic cancer. METHODS: TP, OPRT, and DPD mRNA expressions were detected using a real-time reverse transcriptional-polymerase chain reaction method or by immunohistochemistry, using surgical specimens obtained from 25 patients with pancreatic cancer. RESULTS: TP mRNA expression was lower in cases with an alpha infiltration growth pattern than in cases with other infiltration growth patterns (P < 0.05). OPRT mRNA expression was higher in poorly differentiated-type cases than in differentiated type cases (P < 0.05). TP-, OPRT-, and DPD-positive stainings were found in 15 of 24 cases (63%), 10 of 19 cases (53%), and 14 of 21 cases (67%), respectively. There were significant correlations or trends between the mRNA and protein expressions of TP, OPRT, and DPD. Patients with a low TP/DPD ratio survived significantly longer than those with a high ratio (P < 0.05). Multivariate analysis demonstrated a significantly poorer outcome in patients with a high TP/DPD ratio compared with in patients with a low ratio (P < 0.05). CONCLUSION: The TP/DPD ratio might be useful as a prognostic factor in patients with pancreatic cancer.

Clinical role of thymidine phosphorylase and dihydropyrimidine dehydrogenase in colorectal cancer treated with postoperative fluoropyrimidine.

BACKGROUND/AIMS: Thymidine phosphorylase (TP) is an essential enzyme for activation of 5-fluorouracil (5-FU) and its derivatives. Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme for degradation of 5-FU. In colorectal cancer (CRC), several studies have evaluated the relationship between TP, DPD, and clinicopathological features. However, the results may not be definitive since monoclonal antibody sensitive for human TP and DPD has not been established. Now, new monoclonal antibodies for human TP (1C6-203) and human DPD (2H9-1b) are available. METHODOLOGY: The study included 150 patients whose CRCs were classified into stage II to IV, and resected surgically. TP and DPD expression were evaluated using immunohistochemistry with new antibodies and relationships between their expressions and clinicopathological features. Survival curves were calculated using Kaplan-Meier method, and differences were evaluated with log-rank test. Cox proportional hazards model was also used. RESULTS: TP and DPD expression showed positive correlations with advances in lymphatic invasion (p=0.049 and 0.032 respectively), venous invasion (p=0.027 and 0.005), and cancer stage (p=0.0003 and 0.006). The patients survival rates were higher in those TP(-) than in those TP(+) (p=0.007), and higher in those DPD(-) than in those DPD(+) (p=0.008). The survival rate was best in the patients negative for TP and DPD, and worst in those positive for both TP and DPD. In regard to the combination of TP and DPD expression, the best survival curve was obtained for the TP(-) DPD(-) group followed by those for the TP(+) DPD(-), TP(-) DPD(+), and TP(+) DPD(+) groups in descending order. The estimated hazard ratio for patients' death with TP and DPD expression were 3.10 and 6.55 (p=0.01 and 0.01) respectively. CONCLUSIONS: Using new sensitive monoclonal antibodies, the present results indicated that TP and DPD expression were associated with CRC progression, and closely related with poor prognosis in postoperative CRC patients. CRC patients positive for both TP and DPD expression had a poorer prognosis than those negative for one of these expressions in treatment with fluoropyrimidine after surgery. The use of two determinants of response may identify a high percentage of responding patients.

Changes in thymidylate synthase and its inhibition rate and changes in dihydropyrimidine dehydrogenase after the administration of 5-fluorouracil with cisplatin to nude mice with gastric cancer xenograft SC-1-NU.

BACKGROUND: Although 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum (cisplatin) in combination have synergistic cytotoxicity against both murine and human neoplasms, the precise mechanism of the synergism, and the effects on thymidylate synthase and its percent inhibition, and the effects on dihydropyrimidine dehydrogenase (DPD) remained to be elucidated. METHODS: Experimental chemotherapy was performed using SC-1-NU, a human gastric carcinoma xenograft. SC-1-NU was maintained by serial transplantation in male BALB/c nude mice. The nude mice received various chemotherapeutic regimens consisting of 5-FU and/or cisplatin, with different dosages and periods of administration. After the treatment, we examined the in vivo effects of 5-FU and cisplatin in each regimen on thymidylate synthase and its percent inhibition, and the effects on DPD, in addition to the observation of tumor growth inhibition. RESULTS: The combined use of 5-FU (20 mg/kg per day) and cisplatin (either 1.5 or 7.5 mg/kg per day) showed a synergistic antitumor effect, regardless of the different doses of cisplatin. The long-term administration of 5-FU significantly increased both total thymidylate synthase and the percent thymidylate synthase inhibition rate. The short-term administration of 5-FU significantly decreased DPD. Nevertheless, these changes showed no relation to the combined use of cisplatin. CONCLUSION: Combined administration of cisplatin with 5-FU did not further increase thymidylate synthase inhibition over that occurring with 5-FU alone, which does not support the hypothesis that cisplatin combined with 5-FU modulates thymidylate synthase inhibition in enhancing the anticancer effect of 5-FU. Changes in DPD after the administration of 5-FU may provide an insight into tumor sensitivity and resistance to 5-FU.