Dihydropyrimidine Dehydrogenase Levels Research Articles

Phase I Study of Capecitabine With Concomitant Radiotherapy for Patients With Locally Advanced Pancreatic Cancer: Expression Analysis of Genes Related to Outcome

To establish the feasibility of capecitabine with concurrent radiotherapy (XRT) in patients with locally advanced (LA) pancreatic cancer and evaluate the effect of XRT on thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and tumor necrosis factor-alpha (TNF-alpha). Fifteen patients with LA pancreatic cancer received three-dimensional conformal XRT to a dose of 50.4 Gy with capecitabine at escalating doses from 600 to 1,250 mg/m2 bid (Monday through Friday). Following chemo-XRT, stable and responding patients were treated with capecitabine 2,000 mg/m2 orally bid for 14 days every 21 days. Tumor specimens were procured with endoscopic ultrasound-guided fine-needle aspiration 1 week before and 2 weeks after chemo-XRT to evaluate TP, DPD, and TNF-alpha mRNA levels. Dose-limiting grade 3 diarrhea was observed in two of six patients treated at a capecitabine dose of 1,000 mg/m2 with XRT. Three patients (20%) achieved partial response. Mean percent difference in TP pre- and post-XRT was 119.2% (P = .1934). There was no significant differences in mean TNF-alpha, or DPD levels pre- and post-XRT (P = .1934 and .4922, respectively). TP and TNF-alpha levels were not significantly correlated both at pre- and post-XRT (P = .670 and P < .154, respectively). Median value of TP:DPD ratios at baseline was 2.65 (range, 0.36 to 11.08). No association between TP:DPD ratio and efficacy of capecitabine or severity of toxicities was identified. The recommended dose for phase II evaluation is capecitabine 800 mg/m2 bid (Monday through Friday) with concurrent XRT. This approach offers an easy alternative to intravenous fluorouracil as a radiosensitizer in these patients. Role of TP and TP:DPD ratio warrants further investigation in a larger clinical trial.

Recurrence and 5-FU sensitivity of stage III/Dukes' C colorectal cancer with occult neoplastic cells in lymph node sinuses

In 103 patients without occult neoplastic cells (ONCs) in the lymph node sinuses, the 5-year relapse-free survival (RFS) rate and overall survival (OS) rate were 90.2% and 91.8%, respectively. In 21 patients with ONCs, the 5-year RFS and OS rates were 34.9% and 62.3%, respectively. There were marked differences of survival between the two groups (p=0.0000 and p=0.0003). In the primary tumors of the 21 ONC-positive patients, high and low TS levels were found in 46.2% (6/13) and 53.8% (7/13) of the recurrence group (n=13), respectively. High and low DPD levels were found in 23.1% (3/13) and 76.9% (10/13), respectively. In the non-recurrence group (n=8), high and low TS or DPD levels were found in 75.0% (6/8) and 25.0% (2/8) versus 12.5% (1/8) and 87.5% (7/8), respectively. The percentage of patients with high TS and low DPD levels was 23.1% (3/13) in the recurrence group and 62.5% (5/8) in the non-recurrence group (p=0.07). These results suggest that the presence of ONCs had a clear association with the 5-year RFS and OS rates. The recurrence group of ONC-positive patients with stage II/Dukes' B colorectal cancer was unlikely to be highly responsive to 5-FU-based treatment, thus requiring multi-combination chemotherapy using CPT-11 and/or L-OHP.

The activities of thymidine phosphorylase and dihydropyrimidine dehydrogenase in colorectal cancer depend on where the sample is taken

The significance of tumor tissue thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) levels, as well as the TP/DPD ratio have recently been reported as prognostic factors and for custom-made chemotherapy. However, there have been no distinct studies on actual tumor sampling methods. For 16 patients who had undergone resection of advanced colorectal cancer, we: i) measured TP and DPD levels in different portions of the tumor using enzyme-linked immunosorbent assay (ELISA); ii) categorized the tumor into an edge, center, and base area, and histo-pathologically calculated the ratio of cancer cell/cancer cell + stromal cell; and iii) examined the correlation between cancer and stromal cell TP expression and TP value. Variation within the same tumor was seen in each activity level and TP/DPD ratio. The ratio of cancer cell in the edge area was high, with the ratio of stromal cell in the center and base areas increasing in that order. A correlation was seen between TP expression and TP levels, and TP expression was evident in the stromal cells. It is therefore recommended to sample the edge area for tumor TP levels.

5-fluorouracil related gene expression levels in primary colorectal cancer and corresponding liver metastases

3568 Background: The most commonly used chemotherapeutic drug for colorectal cancer (CRC) is 5-fluorouracil (5-FU). Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), orotate phosphoribosyl transpherase (OPRT) are involved in the metabolic pathway of 5-FU and have been shown in several studies to be associated with response to 5-FU-based therapies. Since liver metastases are the main cause of death for most of CRC patients, it is reasonable to expect that measurement of these gene expression levels in liver metastases would provide the best prediction of therapy benefit, but in most cases, only biopsies of the patient’s primary tumor are readily available for analysis. Our aim was to determine how TS, DPD, TP, and OPRT gene expression levels in primary CRC were related to those in liver metastases. Methods: Thirty-one pairs of primary CRC and corresponding liver metastases were analyzed. (18 males and 13 females: Median age 66 (range 45–85)). Formalin-fixed, paraffin-embedded tumor specimens were dissected by using laser-captured microdissection. RNA was extracted and cDNA was prepared by reverse-transcription. Quantitation of target gene and internal reference gene was performed using real-time PCR (Taqman PCR). Results: No significant difference was seen between median mRNA expression levels of TS, DPD, TP, and OPRT in primary cancer and those in corresponding liver metastases (Median value: TS 1.48 vs. 1.43; p=0.92, DPD 0.19 vs.0.12; p=0.10, TP 1.20 vs. 0.98; p=0.39, OPRT 1.17 vs. 0.95; p=0.10, Wilcoxon signed rank test). When matched tissue sets were compared on an individual basis, there was a significant correlation for TS mRNA expression between primary cancer and corresponding liver metastases (rs=0.52, p=0.0026, Spearman rank correlation coefficient). However, no correlation was seen between matched sets for DPD, TP, or OPRT. Significant correlation was seen between DPD and TP expression levels in both primary CRC (rs=0.38, p=0.03) and liver metastases (rs=0.72, p<0.0001). Conclusions: A good prediction of TS mRNA levels in liver metastases can be obtained by measuring those of primary CRC, although no correlation was seen for DPD, TP, and OPRT. No significant financial relationships to disclose.

Predictive significance of thymidylate synthase and dihydropyrimidine dehydrogenase levels in patients with completely resected colorectal cancer receiving adjuvant 5-fluorouracil therapy

3629 Background: Cytotoxic effect of 5-FU is mediated through competitive binding to thymidylate synthetase (TS), leading to thymine depletion. 5-FU is catabolized by dihydropyrimidine dehydrogenase (DPD). It is speculated that tumorcells with high levels of TS or DPD will be less sensitive to 5-FU therapy. Aim. The primary objective was to evaluate the predictive significance of TS and DPD levels in colorectal tumors on overall survival following complete resection and adjuvant 5-FU therapy. Method: This is a retrospective study among consecutive patients completely resected for colorectal cancer stage III and treated with adjuvant 5-FU according to the Mayo-regimen. The intensity of TS and DPD were scored (0–3) using immunohistochemical staining Proportion of TS positive tumorcells were counted at the luminal surface of the tumor and at the border of tumor invasion of normal tissue, and analysed for the predictive significance to overall survival using Cox proportional hazard linear regression analysis. Results: A total of 190 patients were included. Sixty patients have deceased during 9 years of follow up. In the multivariate analysis T-stage (p=0.001), N-stage (p=0.003) and performance status (p=0.02) were significant prognostic factors, while tumor site (p=0.28), age (p=0.36), intestinal perforation (p=0.23), perineural tumorcells (p=0.26), intravascular tumorcells (p=0.50), differentiation grade (p=0.35) and histologic type (p=0.44) were non significant. The intensity of TS (p=0.18) and proportion of TS positive tumorcells (p=0.25) at the site of tumor invasion of normal tissue were better predictors for survival than the intensity of TS (p=0.64) and proportion of TS positive tumorcells (p=0.96) at the luminal surface of the tumor. The DPD intensity of tumorcells turned out not to predict overall survival (p=0.68). Conclusions: Levels of TS and DPD enzymes in colorectal tumors were not predictive factors of overall survival of adjuvant 5-FU therapy in patients at high risk of recurrence of resected colon carcinoma. No significant financial relationships to disclose.

Changes over time in thymidine phosphorylase and dihydropyrimidine dehydrogenase when colon cancer samples are left at room temperature

The changes over time in thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) were studied when colon cancer samples were left at room temperature. The TP levels and the TP/DPD ratio showed no changes over time in either the tumor or normal tissues. DPD levels increased over time in the normal tissues ( P=0.015), but showed no changes in the tumor tissues. When the DPD level in normal tissues is to be examined, the samples should be frozen as soon as possible. In all other cases, the ELISA results will not be affected if the samples are frozen within 6 h after collection.

Important Role of the Dihydrouracil/Uracil Ratio in Marked Interpatient Variations of Fluoropyrimidine Pharmacokinetics and Pharmacodynamics

Dihydropyrimidine dehydrogenase (DPD) deficiency in patients causes severe toxicities in 5-fluorouracil/floxuridine (5-FU/FUDR) treatments. To determine the plasma dihydrouracil/uracil ratio (DUUR) as a potential index for setting 5-FU/FUDR doses, the authors conducted a prospective study on the relationships of the DUUR with 5-FU/FUDR pharmacokinetic and pharmacodynamic parameters. Forty gestational trophoblastic tumor (GTT) patients were treated with 30 mg/kg of 5-FU or prodrug FUDR during a 10-day cycle. The pretreatment DUURs of the patients were determined prior to the treatments, and plasma 5-FU and FUDR concentrations on day 1 of the test cycle were measured to calculate the corresponding pharmacokinetic parameters. The absolute neutrophil count (ANC) and human chorionic gonadotrophins (HCG/beta-HCG) were recorded as the efficacy indexes. The correlation of the DUUR with pharmacokinetic parameters and efficacy indexes was analyzed to look for a relationship between individual doses (in milligrams) and the varied DUUR. Pretreatment DUUR was significantly correlated with the corresponding plasma AUC (r > 0.80, P < .01), the plasma drug clearance (r > 0.78, P < .01), the ANC (r > 0.76, P < 0.01), and the decrease of HCG/beta-HCG levels (r > 0.5, P < 0.01). In addition, the charts for setting 5-FU/FUDR doses were designed for further validation in clinical trials. These findings indicate the important roles of the DUUR in remarkable interpatient variations of fluoropyrimidine pharmacokinetics and pharmacodynamics and propose a better index for setting individual 5-FU/FUDR doses based on interpatient variations in DPD levels.

Thymidine phosphorylase and dihydropyrimidine dehydrogenase expression in hepatocellular carcinoma and metastatic liver cancer

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are considered to be key enzymes affecting the prognosis for patients with various cancers. We tried to prove the correlation of TP and DPD expression in hepatocellular carcinoma (HCC) and liver metastasis. We quantified TP and DPD levels by an enzyme-linked immunosorbent assay (ELISA) in the tumor (T) and adjacent normal tissue (N) obtained from 8 HCC patients, and 11 liver metastasis patients together with 9 of their primary cancers. TP levels were higher in the primary cancer, liver metastasis, and HCC compared with each adjacent tissue. TP levels were higher in HCC than in liver metastasis, and TP levels in the adjacent tissues of HCC were also higher than those in adjacent tissues of liver metastasis. TP levels were higher in liver metastasis than in primary cancer, and TP levels in adjacent tissues of liver metastasis were also higher than those in adjacent tissues of primary cancer. However, there were no differences in TP T/N ratio between HCC and liver metastasis, and between primary cancer and liver metastasis. DPD levels were lower in the liver metastasis compared with the adjacent liver tissues, and DPD levels in liver metastasis or its adjacent liver tissues were higher than those in primary cancer or its adjacent tissues. There were no differences in DPD T/N ratio between HCC and liver metastasis, and between primary cancer and liver metastasis. Thus, we demonstrated that TP was highly expressed in liver malignancy. We may be able to increase the success of anticancer chemotherapy for liver malignancy while decreasing the side effects by analysis of T/N ratios in TP, DPD, and TP/DPD in addition to TP expression.

Chemopredictive diagnostic project in patients (pts) receiving capecitabine (X) or 5-FU/leucovorin (LV) as first-line chemotherapy for metastatic colorectal cancer (MCRC)

9652 Background X (Xeloda®) is a tumor-activated oral fluoropyrimidine, with superior response rate and improved safety compared to bolus 5-FU/LV in 1st-line MCRC. Variation in tumor expression levels of thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) may impact efficacy or toxicity of X-containing regimen. We are retrospectively evaluating the relationship of the expression of these enzymes to outcomes in a 1st-line MCRC phase III trial of X vs 5-FU/LV (Hoff, JCO 2001). 9 Canadian sites participated in the trial (N=164). Methods Pts were eligible if they received at least one dose of chemotherapy. We obtained informed consent through next-of-kin or Ethics Committee waiver. We obtained formalin-fixed, paraffin-embedded tissue from primary tumors +/- synchronous metastases. We analyzed TP, DPD and TS expression levels using immunohistochemistry (IHC) and quantitative reverse transcriptase - polymerase chain reaction (RT-PCR) kits, both from Roche Diagnostics. Overall IHC score was calculated using tumor and stromal staining intensity and pattern. IHC intensity and percentage of staining were scored blinded to pt treatment and clinical outcome.Results Tumor blocks from 37/164 pts have been analyzed to date, 34 primaries (see table), 4 metastases (not shown). Data on correlation of IHC and RT-PCR results to clinical outcomes will be presented at the meeting. Conclusions Retrospective sample collection was feasible in a large multicenter trial. Sophisticated molecular analysis of archival samples was performed with a success rate of 97%. Results of this biomarker analysis could generate a hypothesis to be tested in further prospective research with X, eventually allowing tailored therapy for the individual patient. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Hoffmann-La Roche Hoffmann-La Roche Hoffmann-La Roche

Chemopredictive diagnostic project in patients (pts) receiving capecitabine (X) or 5-FU/leucovorin (LV) as first-line chemotherapy for metastatic colorectal cancer (MCRC)

9652 Background X (Xeloda®) is a tumor-activated oral fluoropyrimidine, with superior response rate and improved safety compared to bolus 5-FU/LV in 1st-line MCRC. Variation in tumor expression levels of thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) may impact efficacy or toxicity of X-containing regimen. We are retrospectively evaluating the relationship of the expression of these enzymes to outcomes in a 1st-line MCRC phase III trial of X vs 5-FU/LV (Hoff, JCO 2001). 9 Canadian sites participated in the trial (N=164). Methods Pts were eligible if they received at least one dose of chemotherapy. We obtained informed consent through next-of-kin or Ethics Committee waiver. We obtained formalin-fixed, paraffin-embedded tissue from primary tumors +/- synchronous metastases. We analyzed TP, DPD and TS expression levels using immunohistochemistry (IHC) and quantitative reverse transcriptase - polymerase chain reaction (RT-PCR) kits, both from Roche Diagnostics. Overall IHC score was calculated using tumor and stromal staining intensity and pattern. IHC intensity and percentage of staining were scored blinded to pt treatment and clinical outcome.Results Tumor blocks from 37/164 pts have been analyzed to date, 34 primaries (see table), 4 metastases (not shown). Data on correlation of IHC and RT-PCR results to clinical outcomes will be presented at the meeting. Conclusions Retrospective sample collection was feasible in a large multicenter trial. Sophisticated molecular analysis of archival samples was performed with a success rate of 97%. Results of this biomarker analysis could generate a hypothesis to be tested in further prospective research with X, eventually allowing tailored therapy for the individual patient. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Hoffmann-La Roche Hoffmann-La Roche Hoffmann-La Roche

Preoperative endoscopic analysis of thymidine phosphorylase and dihydropyrimidine dehydrogenase in gastrointestinal cancer

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are considered to be key enzymes affecting the prognosis for patients with advanced gastrointestinal cancer. Preoperative examination of TP and DPD expression levels and assessment of these enzymes in inoperable cancer patients may contribute to successful treatment. We tried to prove the correlation of TP and DPD expression in preoperative specimens by endoscopy and in surgical specimens. The present study was designed to quantify TP and DPD levels by enzyme-linked immunosorbent assay (ELISA) in tumor tissue obtained from 30 gastrointestinal cancer patients by preoperative endoscopy and surgery, including 15 gastric and 15 colorectal cancers. Successful cases as those in which cancer cells were demonstrated histologically in preoperative specimens by endoscopy were 12 (success rate: 80%) in gastric cancer patients, and 15 (success rate: 100%) in colorectal cancer patients. In successful cases, there were almost significant correlations in all cases, gastric cancer, and colorectal cancer among the expression of TP, DPD, and TP/DPD ratio in each preoperative specimen by endoscopy and surgical specimen, respectively. On the other hand, in the gastric cancer group, 3 unsuccessful cases resulted in a significant departure from ideal equation compared with 12 successful cases. In actual clinical care, physicians should pay attention to and evaluate carefully the data from endoscopical biopsy specimens in which cancer cells may not be demonstrated histologically. Thus, endoscopic analysis of TP and DPD expression in preoperative or inoperable cancer patients may contribute to successful treatment.

The ratio of thymidine phosphorylase to dihydropyrimidine dehydrogenase in tumour tissues of patients with metastatic gastric cancer is predictive of the clinical response to 5 ′-deoxy-5-fluorouridine

The aim of this work was to determine whether intratumour contents of thymidine phosphorylase (TP), which converts 5 ′-deoxy-5-fluorouridine (5 ′-DFUR) to 5-fluorouracil, and dihydropyrimidine dehydrogenase (DPD), which degrades 5-fluorouracil to inactive molecules, could be useful in predicting the response of patients with metastatic gastric cancer to chemotherapy using 5 ′-DFUR. Endoscopic biopsy specimens for the measurement of TP and DPD were obtained from the primary lesions before the start of combination chemotherapy, in which 5 ′-DFUR, cisplatin and mitomycin C were administered. TP and DPD were measured by enzyme-linked immunosorbent assays after the objective responses to chemotherapy had been confirmed. Twenty five patients were enrolled in this study and data for 22 patients in whom responses were confirmed were analysed. The median levels (ranges) of TP and DPD were 80 (4.9–360) and 44 (15–82) U/mg protein, respectively. The median value (range) of TP to DPD ratios was 1.9 (0.25–5.1). Eight patients with a complete or partial response to chemotherapy had significantly higher TP to DPD ratios than did the remaining patients with stable or progressive disease ( P=0.014). When a cut-off level of TP to DPD ratio was defined as the median value, the high-ratio group ( n=11) showed a significantly higher response rate (64% vs. 9.1%, P=0.024) than the low-ratio group ( n=11). Overall survival of the high-ratio group was significantly longer than that of the low-ratio group (the median survival time; 300 days vs. 183 days, P=0.047). The efficacy of 5 ′-DFUR could be optimised by preselecting patients with high TP/ DPD ratios in their tumour tissues, and this would be applicable to the treatment with capecitabine.

Establishment of enzyme-linked immunosorbent assays for thymidylate synthase and dihydropyriminide dehydrogenase in cancer tissues

Thymidilate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. Intratumoral activity and expression levels of both enzymes are suggested to be predictive markers for response to 5-FU-based chemotherapy in cancer patients. Several different methods are used to measure intratumoral levels of TS and DPD. We developed new enzyme-linked immunosorbent assays (ELISAs) for these enzymes. New ELISAs were produced using anti-TS and anti-DPD polyclonal antibodies obtained using recombinant human TS and purified DPD from pig liver, respectively. Intra-assay coefficients of variations (CVs) were less than 5% in both ELISAs. Inter-assay CVs tested using the extract from 20 breast cancer specimens were 1.5-24.2% for TS-ELISA and 0.4-7.2% for DPD. Importantly, TS and DPD levels measured by the respective ELISAs closely related to TS activity (r(2)=0.8492) and DPD activity (r(2)=0.7666), respectively, measured by the respective substrate assays. To investigate the correlations between clinicopathological characteristics and intratumoral TS and DPD levels, data on 52 breast cancer patients were analyzed. Estrogen receptor (ER)-negative tumors had significantly higher TS and DPD levels than ER-positive tumors. Progesterone receptor (PgR)-negative tumors showed a significantly higher DPD level than PgR-positive tumors. There was no significant correlation of the TS or DPD levels with other clinicopathological factors in this preliminary study. Further studies are warranted to investigate predictive and prognostic values of intratumoral TS and DPD levels in various malignancies using these ELISAs.

Comparative analysis of thymidine phosphorylase and dihydropyrimidine dehydrogenase expression in gastric and colorectal cancers

Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are considered to be key enzymes affecting the prognosis for patients with gastric and colorectal cancers. We tried to prove the correlation of TP and DPD expressions in gastric and colorectal cancers. The present study was designed to quantify TP and DPD levels by an enzyme-linked immunosorbent assay (ELISA) in tumors and normal tissues obtained from 16 gastric and 20 colorectal cancer patients. TP and TP/DPD ratio in the tumor specimens were almost all higher than those in each normal tissue, especially for tumors in the progressive state. In the early stage of the colorectal cancer group, DPD in the normal tissues were higher than those in the tumor specimens. There were no significant differences between TP levels in the tumor specimens of the two groups, whereas in stages III and IV, those of the gastric cancer group tended to be higher than those of colorectal cancer group. In stages I and II, DPD levels in the tumor specimens tended to be higher in the gastric cancer group than in the colorectal cancer group. DPD T/N was higher in the gastric cancer group than in the colorectal cancer group. There were no significant differences between TP/DPD ratios in the tumor specimens of the two groups, whereas those in normal tissue were higher in the gastric cancer group than in the colorectal cancer group. We may be able to achieve the successful effects or reduction of side effects of anticancer chemotherapy for gastric and colorectal cancer using the results of this study.

Gene expression of 5-fluorouracil metabolic enzymes in primary colorectal cancer and corresponding liver metastasis.

Expression of thymidylate synthase (TS) and the 5-fluorouracil (5-FU) metabolic enzymes, including dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), thymidine phosphorylase (TP), and uridine phosphorylase (UP), has been reported to be associated with the sensitivity to 5-FU-based chemotherapy in colorectal cancer. We evaluated the correlation of the expression of these genes between primary tumors and corresponding liver metastases. The mRNA levels of TS, DPD, OPRT, TP, and UP were measured by real-time quantitative RT-PCR in samples from 23 consecutive patients with both primary colorectal adenocarcinoma and liver metastasis. The DPD, OPRT, TP, and UP mRNA levels were significantly higher in liver metastases than in primary tumor (expression in relation to that of beta-actin mRNA: 0.42 vs 0.16, P=0.00053; 1.4 vs 0.92, P=0.016; 23 vs 11, P=0.00014; 0.36 vs 0.25, P=0.0026; respectively). However, the TS mRNA level did not differ significantly between liver metastases than primary tumor (0.20 vs 0.16, P=0.28). No correlation was observed for any gene between primary tumor and liver metastases. In both primary tumor and liver metastasis, the TS mRNA levels correlated significantly with the OPRT mRNA level (primary rS=0.83, P=0.00000081; liver metastasis rS=0.49, P=0.017), while the DPD mRNA level correlated significantly with the TP mRNA level rS=0.81, P=0.0000024; rS=0.63, P=0.0014; respectively). The differential gene expression of 5-FU metabolic enzymes between primary colorectal cancer and corresponding liver metastases should be taken into consideration when estimating the sensitivity to 5-FU-based chemotherapy in colorectal cancer. The gene expression of TS and OPRT, which are involved in de novo pyrimidine synthesis, and that of DPD and TP, may be coregulated.

Dihydropyrimidine dehydrogenase pharmacogenetics in the Taiwanese population.

5-Fluorouracil (5-FU) remains the most frequently used chemotherapy agent in various human cancers. Over 80% of the 5-FU administered is metabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. However, mutations in the DPD gene have been found to be associated with low DPD activity causing severe complications. The aim of this study was to determine the frequency of 11 known mutations in Taiwanese subjects and the relationship between mutation and DPD level. Samples from a total of 300 subjects were investigated in this study. The PCR-RFLP method was used to identify 11 mutations of the DPYD gene, including 62G>A, 74A>G, 85T>C (DPYD*9A), 812delT, 1003G>T, 1156G>T, 1627A>G (DPYD*5), 1714C>G, 1897delC (DPYD*3), 2194G>A (DPYD*6), and IVS14+1G>A (DPYD*2A). DPD protein levels were determined using a DPD ELISA kit. Four mutations, including 74A>G, 85T>C (DPYD*9A), 1627A>G (DPYD*5), and 2194G>A (DPYD*6), were found in our 300 samples. The following mutations were not detected: 62G>A, 812delT, 1003G>T, 1156G>T, 1714C>G, 1897delC (DPYD*3), and IVS14+1G>A (DPYD*2A). The phenotype analysis by DPD protein level indicated that the 1627A>G (DPYD*5) mutation was not associated with the DPD protein level and might be a polymorphism in the DPD gene. The DPD level was also not correlated with gender. No significant correlations between these 11 mutations and DPD protein level were found indicating that examination of these mutations is insufficient to provide a high-value prediction of the 5-FU pharmacogenetic syndrome in Taiwanese. Genotype and phenotype analysis indicated the 1627A>G (DPYD*5) mutation to be a polymorphism.

Correlation of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2'-deoxyuridine.

To determine the expression levels of three metabolic enzymes of fluoropyrimidines: thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines, and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd). TS, TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR, TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50), representing the sensitivity to drugs, were determined by MTT assay. IC50 values ranged from 1.28 to 12.26 microM for 5-FU, and from 5.02 to 24.21 microM for FdUrd, respectively. Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P<0.05), but neither TS nor TP correlated with 5-FU IC50 (P>0.05). Only TS mRNA level was sharply related with FdUrd sensitivity (P<0.05), but TP and DPD were not (P>0.05). A correlation was found between mRNA and protein levels of DPD (P<0.05), but not TP (P<0.05). DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd, respectively.

Dihydropyrimidine dehydrogenase and thymidylate synthase activities in hepatocellular carcinomas and in diseased livers.

Dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are key enzymes for predicting the efficacy of 5-FU in the treatment of malignant tumors. However, 5-FU is not commonly commonly chosen for chemotherapeutic treatment of hepatocellular carcinoma (HCC) in practice. The aim of this study was to determine the activities of both DPD and TS in HCCs and corresponding liver parenchyma and to assess the correlation between the activities of these enzymes and clinicopathological features. The possibility of using 5-FU as a first-choice chemotherapeutic agent for HCC was also evaluated. The study material comprised 33 pairs of hepatocellular carcinoma and noncancerous liver samples. The DPD and TS activities were quantified by a radiometric enzymatic assay and a 5-fluoro-2'-deoxyuridine-5'- monophosphate (FdUMP) ligand-binding assay, respectively. Pathologically invasive HCCs tended to show higher DPD activity and lower TS activity with some exceptions. DPD activity was lower in the HCCs regardless of their clinical features than in the noncancerous liver parenchyma, whereas TS activity was generally lower in HCCs except for those with certain clinical features. HCCs with multiple nodules showed lower DPD activity and those with a diameter of more than 5 cm showed lower TS activity. In the noncancerous liver parenchyma, a gradual decrease in DPD activity and an increase in TS activity were associated with the age of the patient, liver damage and z-factor. Of 30 HCC samples, 10 exhibited comparatively low DPD and TS activity, and these could be considered 5-FU-sensitive HCC. DPD and TS activity may be affected by the clinicopathological status in both the HCC and the corresponding liver parenchyma. However, further investigation is necessary. Some HCC patients may be good candidates for 5-FU-based chemotherapy based on measurements of tumor levels of DPD and TS.

1211 Evidence based symptom management for patients with breast cancer: clinical pathways teach process, implementation and evaluation

Objective: To measure the gene expression of the metabolic enzymes of 5 fluorouracil (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase) to assess the efficacy of 5 fluorouracil inpatients with head and neck cancerPatients and Methods: Tissue specimens of 64 patients with head and neck cancer (41 men and 23 women with a mean age of 62.6 years) who had been surgically treated from April 1997 to March 2004 were included in this study. The mRNA expressions of thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase genes were examined from the surgically resected tissues using the Danenberg tumour profile method. The mRNA levels of the 4 enzymes were compared between tumour tissues and adjacent healthy tissues.Results: All specimens showed that thymidylate synthase, thymidine phosphorylase, and orotate phosphoribosyltransferase levels were significantly higher in the primary tumours than in the adjacent healthy tissues, while the levels of dihydropyrimidine dehydrogenase were significantly lower in the primary tumours than in the adjacent healthy tissues.Conclusion: Prediction of the efficacy of 5 fluorouracil therapy in the treatment of head and neck cancer from the measurement of mRNA levels of its metabolic enzymes appears to be feasible and possibly beneficial. Key words: Head and neck neoplasms, Orotate phosphoribosyltransferase, Thymidine phosphorylase, Thymidylate synthase

Quantitative Analysis of Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase in Renal Cell Carcinoma

Objective: The purpose of the present study was to clarify the clinicopathological significance of both thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in renal cell carcinoma (RCC) based on a quantitative analysis of RCC patients. Methods: Levels of TP and DPD in RCC and/or uninvolved renal tissues from 65 RCC patients were measured by enzyme-linked immunosorbent assay. Results: The TP level and TP/DPD ratio were significantly higher in RCC than in adjacent uninvolved renal tissues (p < 0.0001). There was no significant difference in DPD levels between RCC and uninvolved renal tissues. The ratio of the highest to the lowest level was 623 in TP level, 28.9 in DPD level, and 985 in TP/DPD ratio. In the univariate analysis, patient’s age (p = 0.04), tumor stage (p < 0.0001), tumor size (p = 0.007), TP expression (p = 0.03), and DPD expression (p = 0.04) were significantly associated with increased risk of death. Multivariate analysis showed that patient’s age, tumor stage, and TP expression were independent prognostic factors. Conclusions: TP and DPD in RCC provide prognostic information although DPD was not an independent prognostic factor. The present finding of a wide range in these enzyme expressions in RCC suggests that a certain subpopulation with a high TP/DPD ratio has potential responsiveness to fluoropyrimidines, especially 5’-deoxy-5-fluorouridine and capecitabine.