Metabolism Of 5-fluorouracil Research Articles

Associations of various gene polymorphisms with toxicity in colorectal cancer patients receiving oral uracil and tegafur plus leucovorin: a prospective study

BackgroundTo assess the predictive value of polymorphism in nine genes, primarily thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT), which relates to 5-fluorouracil (5-FU) metabolism, for toxicity in patients treated with oral uracil/tegafur (UFT) plus leucovorin (LV). Patients and methodsWe treated 99 patients with stage II or III colorectal carcinoma with oral UFT + LV. Germline DNA from patients was genotyped for 5-FU and folate metabolism-relating genes. CYP2A6, tegafur-activating enzyme, and uridine diphosphate-glucuronosyltransferase 1A1 genetic variation were also assessed. Toxicity was graded by the National Cancer Institute Common Toxicity Criteria, version 2.0. ResultsThe multivariate logistic regression revealed that OPRT 638G>C polymorphism was associated with grade 3 diarrhea [odds ratio (OR) 19.84 for patients with the C/C homozygous type compared with patients with wild type, P = 0.014] and polymorphisms of UGT1A1 were associated with hyperbilirubinemia (OR 38.76 for homozygotes and double heterozygotes of *6 or *28 compared with wild type, P = 0.0008). No relationships were observed between TS polymorphisms and any toxicity. ConclusionsOPRT polymorphism predicts toxicity, especially grade 3 or greater diarrhea to oral UFT + LV adjuvant chemotherapy, whereas TS does not, in our study cohort. UGT1A1 polymorphism seems to be a risk factor for hyperbilirubinemia due to UFT+LV.

Role of dihydropyrimidine dehydrogenase and thymidylate synthase expression in immunohistochemistry of intrahepatic cholangiocarcinoma

Dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are key enzymes in the metabolism of 5-fluorouracil and have been implicated as possible prognostic markers for cancer patients. However, the clinical roles of DPD and TS in intrahepatic cholangiocarcinoma (IHCC) have not been investigated. The aim of this study was to clarify the clinicopathological role of DPD and TS expressions in IHCC. Twenty-nine patients who had undergone hepatic resection for IHCC were enrolled in this study. Expressions of DPD and TS in the resected IHCC specimens were examined using anti-DPD or anti-TS antibody. The patients were divided into positive and negative groups according to DPD/TS expressions: DPD-positive group (n = 18) and DPD-negative group (n = 11)/TS-positive group (n = 14) and TS-negative group (n = 15). Clinicopathological factors were compared between the two groups. The overall survival rate was significantly lower in the DPD-negative group than in the DPD-positive group (1-year 36.4% vs. 77.4%, 3-year 18.2% vs. 43.0%; P < 0.05). The disease-free survival rate in the DPD-negative group tended to be lower than that in the DPD-positive group. The overall survival rate or disease-free survival rate did not appear to be associated with the TS-expression status. The Ki-67 labeling index in the DPD-negative group was significantly higher than that in the DPD-positive group (16.9 ± 3.2% vs.13.2 ± 3.3%; P < 0.05). The negative DPD expression was significantly associated with the enhanced tumor cell proliferation and poorer prognosis in patients with IHCC. DPD expression is a potential prognostic indicator for IHCC.

Uracil pharmacokinetics in a DPD-deficient patient with a novel DPYD mutation compared to volunteers with normal DPD activity.

e13134 Background: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the metabolism of 5-fluorouracil (5-FU). Patients with a partial or complete DPD deficiency are at risk to develop severe toxicity after 5-FU administration. Uracil (U) is degraded in dihydrouracil (DHU) in a similar way as 5-FU. An oral uracil test dose might be useful to determine the systemic DPD activity by measuring uracil and its metabolite dihydrouracil in plasma. DPD deficiency is hypothesized to result in higher uracil levels and a reduced turnover of uracil into dihydrouracil. Methods: Uracil (500 mg/m2) was administered to 11 healthy volunteers with normal DPD activity (≥ 6 nmol/mg/hour) and 1 patient with colorectal carcinoma with a novel DPYD mutation and decreased DPD activity (4.6 nmol/mg/hour). DPD activity was measured in PBMC, as determined as described earlier. Blood samples were taken at t= 15, 30, 45, 60, 80, 100, 120, 150, 180, and 220 or 240 min after oral uracil intake. U and DHU pla...

DNA mismatch repair (MMR)‐dependent 5‐fluorouracil cytotoxicity and the potential for new therapeutic targets

The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.

HPLC with UV or Mass Spectrometric Detection for Quantifying Endogenous Uracil and Dihydrouracil in Human Plasma

We developed and compared 2 different methods for quantifying uracil (U) and dihydrouracil (UH(2)) in BSA and human plasma. Special attention was paid to the selectivity/specificity and the absence of a matrix effect. The UH(2)/U ratio is intended as a biomarker to identify patients with deficiency in 5-fluorouracil metabolism. We quantified U and UH(2) with 2 liquid chromatography methods after solid-phase extraction, one with UV detection (LC-UV) and the other with mass spectrometric detection (LC-MS). We selected 2 internal standards to prevent the risk of interferences. Separation was achieved with a Waters Atlantis dC18 column (LC-MS) or a Waters SymmetryShield RP18 column connected with an Atlantis dC18 (LC-UV). Mass spectrometric data were acquired in single-ion monitoring mode. Assay imprecision in BSA solution was <15% (LC-UV) and <12% (LC-MS); in plasma, assay imprecision was <9.5% and <9.0%, respectively. Recoveries were 88.2%-110% (LC-UV) and 94.8%-107% (LC-MS). Extraction efficiencies were >or=89.0%. In BSA, the lower limits of quantification for U and UH(2) were 2.5 microg/L and 6.25 microg/L, respectively, for the LC-UV method and 2.5 microg/L and 3.1 microg/L for LC-MS. The corresponding values in plasma were 11.6 microg/L and 21.5 microg/L, and 4.1 microg/L and 12.1 microg/L. To estimate endogenous U and UH(2) concentrations and their ratio, we recommend the use of a drug-free human plasma pool in which baseline U and UH(2) concentrations have previously been measured with the standard-addition method. Our LC-MS method, which has the better test performance and is useful for measuring UH(2)/U ratios in cancer patients, is preferred when this equipment is available.

Altered Dihydropyrimidine Dehydrogenase Activity Associated with Mild Toxicity in Patients Treated with 5-Fluorouracil Containing Chemotherapy

Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5-fluorouracil (5FU). In patients treated with capecitabine or 5FU combined with other chemotherapeutic drugs, DPD activity in peripheral blood mononuclear cells was increased in patients experiencing grade I/II neutropenia. In contrast, decreased DPD activity proved to be associated with grade I/II dermatological toxicity, including hand-foot syndrome. Thus, patients with a low-normal or high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-based chemotherapy, demonstrating the important role of DPD in the etiology of toxicity associated with 5FU and the catabolites of 5FU.

Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy

BckgroundA number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU) metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU.MethodsThis study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT) chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT) activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors.ResultsWhile there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity.ConclusionTS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.

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

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. 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. 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. 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.

Levels and expressions of orotate phosphoribosyltransferase in gastric carcinoma and normal gastric mucosa tissues

Orotate phosphoribosyltransferase (OPRT; EC 2.4.2.10), a key enzyme that catalyzes one of the primary steps in the phosphorylation of fluoropyrimidine, was recently recognized as an important enzyme that determines the anticancer effects of the dihydropyrimidine dehydrogenase-inhibitory fluoropyrimidine, S-1. Levels of OPRT were examined in 97 gastric carcinoma tissues and 65 normal gastric mucosa tissues obtained from patients with gastric carcinoma. The relation between OPRT levels and clinicopathological variables was evaluated, and correlations of OPRT with thymidylate synthase and dihydropyrimidine dehydrogenase levels in gastric carcinoma tissues were evaluated. Although OPRT levels were high in well-differentiated and localized carcinomas, they were not correlated with other clinicopathological variables or with the pathological stage of gastric carcinoma. Levels of OPRT were significantly higher in gastric carcinoma tissue than in normal gastric mucosa. OPRT levels were not correlated with levels of either thymidylate synthase or dihydropyrimidine dehydrogenase. In samples of gastric carcinoma tissues and normal gastric mucosa tissues obtained simultaneously from 24 patients, no correlation was found between OPRT levels in gastric carcinoma and levels in normal gastric mucosa. These results suggest that the OPRT level is significantly higher in gastric carcinoma tissue than in normal gastric mucosa and that the OPRT level in gastric carcinoma is a novel variable that is independent of the levels of other previously known enzymes related to 5-fluorouracil (FU) metabolism.

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

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. 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. 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). The TP/DPD ratio might be useful as a prognostic factor in patients with pancreatic cancer.

Thymidylate Synthase (TYMS) and Dihydropyrimidine Dehydrogenase (DPYD) Polymorphisms in the Korean Population for Prediction of 5-Fluorouracil-Associated Toxicity

The important cellular proteins for 5-fluorouracil (5-FU) metabolism are the major target enzymes, thymidylate synthase, and the rate-limiting enzyme in the degradation pathway, dihydropyrimidine dehydrogenase. Adverse drug reactions to 5-FU-based chemotherapy have been reported to be in part the result of polymorphisms in the thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) genes. Therefore, we examined the type and frequency of polymorphisms in the TYMS and DPYD genes in 100 healthy Korean individuals and compared these findings with 21 patients with colorectal cancer who had a grade 3 or greater toxic response to 5-FU treatment. Genotyping analysis of the promotor enhancer region (TSER) and the 3'-untranslated region (3'-UTR) of the TYMS gene as well as haplotype analysis were conducted in all 121 study participants. For the TSER and the 3'-UTR of the TYMS gene, similar genotypes and allele frequencies were observed in control subjects and patients. For the haplotype analysis of the single nucleotide polymorphism G > C at the 12th nucleotide of the second repeat of the 3R allele of the TSER, different haplotype frequencies were noted in comparisons between the two groups; in addition, we found that the 3RC-del 6bp was significantly associated with severe toxicity with 5-FU treatment. Extensive polymorphisms in the DPYD gene were observed; in addition, four polymorphisms were related to the known DPYD allelic variants or to allelic variants that alter protein structure, among which the most common polymorphism was 1627A > G, observed in 20.5% of all alleles. The 496A > G allele and a novel 1774C > T allele were identified in two patients. The DPYD*2A allele, causing exon 14 skipping, was not identified in the study group. The findings, from Korean patients with colon cancer, suggest that polymorphisms of the DPYD gene are not associated with an increased risk for toxic response to 5-FU. These findings suggest that there may be an important relationship between the TYMS haplotypes examined and 5-FU toxicity. The novel variant in the DPYD gene, identified in this study, should be further investigated to confirm its functional significance. A large sample is required before DPYD or TYMS genotyping could be used as markers for individualized treatment of patients with colorectal cancer.

The significance of the expression of dihydropyrimidine dehydrogenase in prostate cancer

To measure dihydropyrimidine dehydrogenase (DPD), an enzyme involved in the metabolism of 5-fluorouracil (5-FU), expression in prostate cancer and determine whether 5-chloro-2,4-dihydroxypyridine (CDHP), a potent inhibitor of DPD, enhances the antitumoral activity of 5-FU against prostate cancer. In all, 44 prostate tissue specimens were obtained from men who had a radical prostatectomy alone for prostate cancer, and 38 specimens from men who had had neoadjuvant hormonal therapy. We analysed the cancerous tissue and normal prostate tissue for DPD expression using immunohistochemistry, and determined its prognostic significance. In cultured human prostate cancer lines (DU145 and LNCaP), we compared the cytotoxicity of 5-FU/CDHP with that of 5-FU alone. Finally, in experiments on immunodeficient mice, we studied the effect of oral administration of tegafur, a pro-drug for 5-FU, with or without CDHP on the growth of tumours introduced by injection of DU145 cells. The expression of DPD was significantly higher in cancerous than normal prostate tissue; 36 of 44 (82%) specimens of prostate cancer expressed DPD, whereas only 25 of 44 (57%) specimens of normal prostate tissue expressed DPD. For men with prostate cancer who had radical prostatectomy alone, men with negative DPD expression tended to have a longer recurrence-free survival than those with positive expression; there were no recurrences in men with prostate cancer and negative DPD expression in the 5-year follow-up. DPD expression was significantly lower in men with prostate cancer who received neoadjuvant hormonal therapy. In vitro treatment of human prostate cancer cell lines with 5-FU/CDHP showed more cytotoxicity than with 5-FU treatment alone. Finally, DU145 tumours in mice treated with tegafur and CDHP were significantly smaller than in mice given tegafur alone. The present study showed that DPD expression is elevated in prostate cancer, and indicate that DPD inhibitors might enhance the antitumour activity of 5-FU against prostate cancer.

Pharmacokinetics of 5‐fluorouracil in mutant Nagase analbuminemic rats: faster metabolism of 5‐fluorouracil via CYP1A

It has been reported that plasma albumin concentrations were significantly lower in cancer patients than those in the healthy volunteers, and the expression and mRNA level of hepatic microsomal cytochrome P450 (CYP) 1A2 increased in mutant Nagase analbuminemic rats (NARs). After intravenous administration of 5-fluorouracil at a dose of 30 mg/kg to NARs, the time-averaged nonrenal clearance (Clnr) of the drug was significantly faster than the controls (51.3 versus 28.8 ml/min/kg), possibly due to an increase in the expression and mRNA level of CYP1A2 in NARs. In order to determine whether 5-fluorouracil is metabolized via CYP1A2 in male Sprague-Dawley rats, the rats were pretreated with 3-methylcholanthrene (a main inducer of CYP1A1/2 in rats). The Clnr of 5-fluorouracil was significantly faster (34.3 versus 27.3 ml/min/kg) in rats pretreated with 3-methylcholanthrene. The aforementioned data indicate that CYP1A is involved in the metabolism of 5-fluorouracil in rats.

Upregulation of Enzymes Metabolizing 5-Fluorouracil in Colorectal Cancer

Background: The purpose of this study was to compare the activities of various enzymes, participating in the metabolism of 5-fluorouracil, between colorectal cancer and nontumor tissues and to investigate the association of the enzyme activities with clinicopathological backgrounds. Methods: Activities of seven enzymes involved in nucleic acid metabolism – orotate phosphoribosyltransferase (OPRT), ribonucleotide reductase (RNR), thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, uridine phosphorylase and thymidine kinase (TK) – were measured in tumor and nontumor tissues from 28 patients who were operated on for colorectal cancers. Results: OPRT, thymidylate synthase, RNR, thymidine phosphorylase, uridine phosphorylase and thymidine kinase activities were significantly higher in tumor areas than in nontumor areas. OPRT showed the highest T/N ratio (the ratio of each enzyme activity in tumor areas to that in nontumor areas). The T/N ratio of RNR activity showed a tendency to be associated with lymph node metastasis and Dukes classification. Conclusion: The results suggest that OPRT is a main enzyme participating in the phosphorylation of 5-fluorouracil and has an important role in tumor growth. The T/N ratio of RNR may be predictive of tumor progression.

PREDICTIVE MARKERS OF TREATMENT RESPONSE TO 5-FU AND GEMCITABINE IN PANCREATIC CANCER

Introduction: The RTOG 9704 prospective multicenter randomized adjuvant treatment trial demonstrated that the addition of gemcitabine to postoperative adjuvant 5-FU chemoradiation therapy improves survival in patients with pancreatic adenocarcinoma. Thymidylate synthase (TS) thymidine phosphorylase (TP) and methylenetetrahydrofolate reductase (MTHFR) are key enzymes in the metabolism of folate and 5-fluorouracil. Human equilibrative nucleoside transporter (hENT1) is involved in gemcitabine transport into cells. Retrospective studies in pancreatic cancer suggest that these markers may have prognostic and predictive value. We aim to study protein expression and polymorphisms of these markers in resected tumors from patients participating in the RTOG 9704 trial. Methods: A Tissue Microarray was constructed using 3 separate cores from 225 resected pancreatic tumors. Immunohistochemistry was performed to define TS, TP and hENT1 protein expression. TS promoter genotype (2 or 3 tandem repeats) and MTHFR genotype (C667T) was assessed using tumor DNA. Correlations between protein expression or genotype and prognostic or predictive factors were sought, by unconditional logistic regression analysis. Results: High TS nuclear expression, TP cytoplasmic expression, TP nuclear expression and TP stromal expression is seen in 30%, 86%, 65% and 92% of cases. There are statistically significant associations between higher pathologic grade and high expression of TP nuclear (p = 0.05) and TP cytoplasmic (p = 0.02). There is a trend towards nodal status and high TP nuclear marker levels (p = 0.09). The frequencies of TS promoter tandem repeat 2R/2R, 2R/3R and 3R/3R are 27%, 33%, and 38%. There is a non-significant trend towards 3R/3R TS genotype and high TS protein expression. 22% of cases had no expression of hENT1, compared with 38% with high expression. The frequencies of the MTHFR 667CC, 667CT, and 667TT genotypes are 48.8%, 41.1%, and 10.1%, respectively. 27.6% of patients with either 667CC or 667CT vs. 8.3% of patients with 667TT are early T stage (p = 0.12). Correlation analysis with treatment outcome and toxicity for this trial will be presented. Conclusions: This is the first study to analyze prognostic and predictive markers in a prospective randomized pancreatic cancer clinical trial. There is limited association between TS or TP protein expression and tumor demographic factors. There is a trend towards correlation between TS genotype 3R/3R and high TS protein expression in pancreatic cancer. MTHFR TT genotype was more likely to be associated with advanced T stage and may be associated with disease progression. The majority of tumors expressed hENT1. These markers may serve as useful predictors markers of response and toxicity in pancreatic cancer treatment.

In-vitro differential metabolism and activity of 5-fluorouracil between short-term, high-dose and long-term, low-dose treatments in human squamous carcinoma cells

Although continuous infusion of 5-fluorouracil (5-FU) has been clinically demonstrated to be superior to bolus administration, the mechanistic difference between the treatments still remains unclear. Here, we investigated in vitro whether there were any differences in the metabolism and activity of 5-FU between these schedules. To simulate bolus and infusional treatments of 5-FU, HST-1 human squamous carcinoma cells were treated with short-term, high-doses and long-term, low-doses so that the area under the curve (AUC) of 5-FU became equivalent between both schedules, and compared the cytotoxicity, fluorinated RNA (F-RNA) levels, 5-fluorodeoxyuridine monophosphate (FdUMP) content and thymidylate synthase (TS) activity. F-RNA and FdUMP were measured by capillary gas chromatography-mass spectrometry and competitive ligand-binding assay, respectively. The [H]FdUMP binding site in TS was determined as an index of the amount of TS using the radio-binding assay. Long-term, low-dose treatment of 5-FU was found to be 1.3-1.7 times more cytotoxic than the short-term, high-dose treatment. F-RNA content increased as the AUC of 5-FU was increased and was 2-4 times significantly higher in the cells treated with the long-term, low-dose than those with the short-term, high-dose schedule, indicating that the levels of F-RNA are AUC and schedule dependent. In contrast, there were no significant differences in FdUMP levels, TS activity and TS inhibition rate between the schedules. These data suggest that the superior activity of 5-FU administered long-term, low-dose over short-term, high-dose could be explained by more 5-FU incorporated into RNA during a long-term, low-dose exposure, thus providing a strategic rationale for the clinical advantage of continuous infusion over bolus administration.

Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase Expression Levels in Tumor and Normal Tissue Specimens of T3 Human Colorectal Carcinoma

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are important enzymes related to the metabolism of 5-fluorouracil and its derivatives. We evaluated the association between the clinicopathological factors and these enzymes in patients with T3 colorectal carcinoma. The TP and DPD expression levels in 15 patients with T3 colorectal carcinomas were measured in tumor and adjacent normal tissue specimens by enzyme-linked immunosorbent assay. Correlations between each enzyme and clinicopathological factors were also statistically evaluated. The TP levels in tumor and normal tissue specimens were 77.9 +/- 33.6 and 24.7 +/- 10.3, respectively (P < 0.001). The DPD levels in tumor and normal tissue specimens were 44.1 +/- 18.2 and 53.1 +/- 24.1, respectively (P = 0.46). The TP/DPD ratios in tumor and normal tissue specimens were 1.84 +/- 0.52 and 0.53 +/- 0.26, respectively (P < 0.001). The tumor/normal ratios of TP level in patients with and without liver metastasis were 1.79 +/- 0.91 and 4.67 +/- 2.51, respectively (P = 0.024). The measurement of the enzyme expression levels of TP and DPD is considered to be useful for better understanding the conditions of tumor progression. The mechanisms of regulation of these enzymes thus require further evaluation.

Molecular factors of 5-fluorouracil metabolism in colorectal cancer: Analysis of primary tumor and lymph node metastasis

Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) are predictive markers for tumor response to 5-fluorouracil-based therapies. To determine whether gene expression values measured in primary cancer tissue would be useful for prediction of response of lymph node metastases, the expressions of these genes were quantitatively analyzed in 35 pairs of primary colorectal cancer (CRC) and corresponding lymph node metastases using real-time PCR. DPD and TP mRNA levels were significantly lower in the primary colorectal tumor and lymph node metastases compared with the normal adjacent stroma tissue (p<0.01), whereas TS mRNA levels were significantly higher in the primary tumor and lymph node metastases than in the normal adjacent tissue (p<0.001). Median gene expression levels of TP and TS did not differ significantly between primary colorectal tumor and corresponding lymph node metastasis but median DPD gene expression levels in the lymph node metastases were significantly higher compared to matched primary colorectal tumors (p=0.015). There was a significant correlation for DPD, TP and TS gene expression levels between primary colorectal tumor specimens and the matched lymph node metastasis. These results suggest that biopsies of the tumor of origin may be valid for determining predictive markers for chemotherapy response in patients with metastatic CRC.

Phase II study of tailored chemotherapy for advanced colorectal cancer with either 5-fluouracil and leucovorin or oxaliplatin and irinotecan based on the expression of thymidylate synthase and dihydropyrimidine dehydrogenase

Background: Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are essential enzymes for 5-fluorouracil (5-FU) metabolism. In patients with advanced colorectal cancer (ACRC), retrospective studies have shown that low expression levels of TS and DPD correlated with response to 5-FU. We performed a prospective study in which the choice of first-line chemotherapy with either 5-FU or a non-5-FU containing regimen was based on TS and DPD expression.Patients and methods: Fresh-frozen samples of metastases were obtained from 58 previously untreated patients with ACRC. mRNA expression of TS and DPD was quantified using an RT–PCR assay. Patients with low tumor expression of both TS and DPD received weekly bolus 5-FU/leucovorin (LV) 500mg/m2 (group A); patients with high TS and/or DPD received 3-weekly oxaliplatin 85mg/m2 and irinotecan 200mg/m2 (group B). After progression, cross-over to the alternative regimen was attempted.Results: Of 53 eligible patients, 31 had tumors with both low TS and low DPD, and were treated in group A. A response was observed in 11 patients [35%; 95% confidence interval (CI) 19% to 54%]. Cross-over to second-line oxaliplatin/irinotecan resulted in a partial response in two out of 16 patients (13%; 95% CI 1% to 38%). In group B, four out of 22 patients responded (18%; 95% CI 5% to 40%), while no responses were observed in 12 patients after cross-over to 5-FU/LV (0%; 95% CI 0% to 28%).Conclusions: Prospective selection of 5-FU/LV chemotherapy based on low TS and DPD expression in patients with ACRC did not confirm the high response rates reported in retrospective studies. The procedure of obtaining metastatic tissue and quantitation of enzymes appeared feasible but cumbersome. Before assessing the clinical utility of a predictive marker in a randomized trial, future studies should focus on prospective validation of the assay in a large and well defined population.

Polymorphism at the 3′-UTR of the thymidylate synthase gene: A potential predictor for outcomes in Caucasian patients with esophageal adenocarcinoma treated with preoperative chemoradiation

To test the hypothesis that TS3'UTR polymorphisms predict outcomes in 146 Caucasian patients with esophageal adenocarcinoma treated with preoperative 5-fluorouracil-based chemoradiation. DNA was extracted from hematoxylin-and-eosin stained histologic slides of normal esophageal or gastric mucosa sections from paraffin blocks of esophagectomy specimens. Genotypes of the TS3'UTR polymorphism were determined by polymerase chain reaction for a 6-bp insertion. The genotype groups (0bp/0bp, 6bp/0bp, and 6bp/6bp) were compared for clinical features and overall survival, recurrence-free-survival, locoregional control (LRC), and distant metastasis control. Multivariable Cox regression analyses were performed to find independent predictors for the stated outcomes. There was a trend of association between 6bp/6bp genotype and a decreased risk of local regional recurrence (hazards ratio = 0.211, 95% confidence interval = 0.041-1.095, p = 0.06) compared with other genotypes. There was a trend that patients with 6bp/6bp genotype had a higher 3-year probability of LRC compared with patients with the other two genotypes combined (p = 0.07); however, the difference was not statistically significant. The null hypotheses were not rejected in this study, probably owing to small sample size or the single gene examined. Prospective studies with adequate statistical power analyzing a family of genes involved in the 5-fluorouracil metabolism are needed to assess genetic determinants of treatment-related outcomes in esophageal adenocarcinoma.