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 unclearwhether this occurs because such tumours have better prognosisor they are more sensitive to 5-FU treatment. Patients and methods: Associations between TS, DPD and TP levels,determined by tissue microarrays and immunohistochemistry, andsurvival was evaluated in 945 CRC patients according to treatmentstatus. Results: Low TS and DPD expression associated with worse prognosisin 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 surgeryalone [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 associatedwith trends for better outcome in stage III patients treatedwith 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 worseoutcome in CRC patients treated by surgery alone, whereas lowTS, DPD and TP expression are prognostic for better outcomein patients treated with 5-FU chemotherapy. These results provideindirect evidence that low TS, DPD and TP protein expressionare predictive of good response to 5-FU chemotherapy. Key words: colorectal cancer, fluorouracil, predictive, prognostic, thymidylate synthase Received for publication July 12, 2007. Revision received December 10, 2007. Accepted for publication December 17, 2007.     

Expression of mRNA levels of thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase and orotate phosphoribosyltransferase in diffusely infiltrating colorectal cancer

Little is known about the expression levels of thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) in diffusely infiltrating colorectal cancer. We semiquantified the mRNA levels of these enzymes in colorectal cancer specimens from 10 patients with diffusely infiltrating type and 20 patients with other types (control) matched by the maximal diameter and pathological stage, using the Dannenberg tumor profiling method. The relative expression of these enzymes did not significantly differ between diffusely infiltrating and other types of tumors. There were no significant relationships among the relative expression of the four enzymes in the diffusely infiltrating tumors, while the following three combinations were found to be correlated with each other in the control tumors: TS versus TP,TS versus DPD, and TP versus DPD. In 5 patients who received 5-fluorouracil-based chemotherapy for evaluable lesions, there were no specific relationships between the expression of these enzymes and therapeutic response. In conclusion, diffusely infiltrating colorectal cancer does not appear to have any characteristics in terms of the expression of these enzymes, which may not alter the effect of 5-fluorouracil-based chemotherapy.     

乳腺癌组织中TP和TS及DPD mRNA表达与预后的关系

目的　探讨乳腺癌组织中胸苷酸化酶 (TP)、胸苷酸合成酶 (TS)和二氢嘧啶脱氢酶(DPD)mRNA表达水平及其与预后的关系。方法　采用real time定量PCR技术检测经过微选的 9例正常乳腺组织和 86例乳腺癌组织TP、TS和DPD的mRNA表达水平。结果　肿瘤组织中TP、TS和DPDmRNA表达水平中位数分别为 16 .5 4 ,0 .38和 2 .74 ,正常乳腺组织分别为 11.75 ,0 .2 5和 8.33,差异均无显著性 (P >0 .0 5 )。除年龄与DPD表达呈负相关外 ,TP、TS和DPDmRNA表达与肿瘤体积、淋巴结转移、组织学分级、临床分期均无相关性。TP、DPD高表达组和低表达组之间 ,无瘤生存期和总生存期差异均无显著性。TS高表达组和低表达组无瘤生存期差异无显著性 (P =0 .0 6 9) ;平均总生存期分别为 5 9.0 0和 70 .30个月 ,差异有显著性 (P =0 .0 4 96 )。结论　仅检测TSmRNA对判断乳腺癌预后有参考价值 ,同时检测TP、TS和DPD具有更好的预测价值。     

The prognostic significance of thymidylate synthase and dihydropyrimidine dehydrogenase in colorectal cancer of 303 patients adjuvantly treated with 5-fluorouracil

Cytotoxic effect of 5-fluorouracil 5-FU is mediated through inhibition of thymidylate synthase (TS), and 5-FU is catabolised by dihydropyrimidine dehydrogenase (DPD). Efficacy of 5-FU may therefore depend on the TS and DPD activity of colorectal cancer. Archival tumour specimens from 303 consecutive patients were analysed for the expression of TS and DPD using immunohistochemistry. All patients were completely resected for colorectal cancer stages II-III and have subsequently received adjuvant treatment with 5-FU. In a multivariate analysis adjusting for the impact of bowel obstruction and vascular tumour invasion, diffuse TS pattern was significantly associated with increased risk of recurrence (hazard ratio (HR) = 1.9; 95% confidence interval (CI): 1.1-3.2; p = 0.02), but without significant association to death (HR = 1.6; 95% CI: 0.9-2.8; p = 0.08). High TS intensity was not significantly associated with lower risk of recurrence (HR = 0.6; 95% CI: 0.3-1.1; p = 0.07) or death (HR = 0.6; 95% CI: 0.3-1.2; p = 0.2). High DPD intensity was significantly associated with increased risk of recurrence (HR = 1.5; 95% CI: 1.1-2.3; p = 0.03) and death (HR = 1.6; 95% CI: 1.1-2.5; p = 0.02). Patients with a combination of low TS and high DPD intensity were at significantly increased risk of both recurrence (HR = 2.1; 95% CI: 1.0-4.2; p = 0.04) and death (HR = 2.0; 95% CI: 1.0-4.0; p = 0.05). No relationship between tolerability and toxicity of 5-FU and TS and DPD expression was found. It is concluded that characterizing colorectal carcinomas by TS and DPD expression may disclose subsets of patients with significantly greater risk of disease recurrence and early death. This may be utilized in the selection of patients for treatment approaches and for decision on follow-up programs.     

Activities of thymidylate synthase and dihydropyrimidine dehydrogenase in patients with colorectal cancer]

Between 1998 and 2001, 82 colorectal cancers were resected in our hospital. The activities of TS and DPD were evaluated. TS activities in tumor tissues were significantly higher than in normal tissue, but the DPD activities had no significant difference between them. TS and DPD showed a correlation between normal and tumor tissues in stage III or IV patients. The TS value of patients with recurrence tended to be higher than that of patients with no recurrence. Especially in stage I or II patients with recurrence, who were administered 5-FU before recurrence, the TS value was significantly higher than in non-treated patients. In stage III or IV patients, it was considered that DPD prevention was important for 5-FU to effectively prevent TS. The TS value might be a new prospective risk factor for recurrence. Moreover, TS and DPD would be the index of biological malignancy.     

Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression after administration of 5-fluorouracil to patients with colorectal cancer

This study explores the effect of 5-fluorouracil (5FU) exposure on mRNA levels of its target enzyme thymidylate synthase (TS) and the rate-limiting catabolic enzyme dihydropyrimidine dehydrogenase (DPD) in tumors of colorectal cancer patients. TS and DPD mRNA levels were determined in primary tumor and liver metastasis samples from patients who were either not pretreated (n = 29) or given one presurgery bolus of 5FU (n = 67). In both groups a wide variation in TS mRNA levels was observed. Median TS mRNA expression in 17 primary tumors of exposed patients was 3.0-fold higher than in 19 primary tumors of unexposed patients (p = 0.015). TS mRNA expression in liver metastasis samples of exposed patients (n = 16) was also higher (5.2-fold) than that of unexposed patients (n = 48; p < 0.001). Also DPD mRNA expression displayed a large degree of interpatient variation. No difference in DPD expression in liver metastasis samples was observed between exposed and unexposed patients. However, median DPD mRNA expression in 15 primary tumors of exposed patients was 3.2-fold lower than in 18 primary tumors of unexposed patients (p = 0.027). In conclusion, administration of 5FU in vivo influences the gene expression of TS and DPD.     

The role of thymidylate synthase and dihydropyrimidine dehydrogenase in resistance to 5-fluorouracil in human lung cancer cells

The expressions of thymidylate synthase (TS) and intracellular metabolic enzymes have been reported to be associated with the sensitivity and/or resistance to 5-fluorouracil (5-FU). However, since the role of these enzymes in the mechanism of resistance to 5-FU has not been fully examined in lung cancer, in the present study we measured the expression levels of TS, dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyltransferase (OPRT) genes in lung cancer cell lines by real-time PCR, and the sensitivity to 5-FU using the MTS assay. The expression of DPD was significantly correlated with the concentration of 5-FU for 50% cell survival in 15 non-small-cell lung cancer (NSCLC) cell lines (p<0.05), but the expressions of TS, TP, and OPRT were not. Treatment with 5-chloro-2,4-dihydroxypyridine, an inhibitor of DPD, altered the sensitivity to 5-FU in DPD-expressing RERF-LC-MT cells, indicating that modulation of DPD activity could increase the 5-FU sensitivity in lung cancer. In contrast, TS expression was dramatically higher in a 5-FU-resistant small-cell lung cancer cell line than in the parent cell line, whereas the expressions of DPD, TP, and OPRT genes were not markedly different. In order to examine the effect of other cytotoxic agents on TS and DPD expression, we compared the expressions of both genes between cisplatin-, paclitaxel-, gemcitabine-, or 7-ethyl-10-hydroxycamptothecin-resistant lung cancer cells and their respective parent cells, but found no differences between any pair of resistant subline and the corresponding parent cell line. Our results indicate that degradation of 5-FU due to DPD is an important determinant in 5-FU sensitivity, while induction of TS contributes to acquired resistance against 5-FU in lung cancer. Therefore, the expression levels of TS and DPD genes may be useful indicators of 5-FU activity in lung cancer.     

Microsatellite instability in colorectal cancer and association with thymidylate synthase and dihydropyrimidine dehydrogenase expression

Background  

Microsatellite instability (MSI) refers to mutations in short motifs of tandemly repeated nucleotides resulting from replication errors and deficient mismatch repair (MMR). Colorectal cancer with MSI has characteristic biology and chemosensitivity, however the molecular basis remains unclarified. The association of MSI and MMR status with outcome and with thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression in colorectal cancer were evaluated.     

Tumoral levels of thymidine phosphorylase and dihydropyrimidine dehydrogenase in elderly colorectal cancer patients]

Fluoropyrimidine therapy for elderly colorectal cancer patients remains controversial. Tumoral levels of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and the ratio of TP to DPD determined by an enzyme-linked immunosorbent assay were compared between colorectal cancer patients aged 75 or over (elderly group, n = 25) and those 74 years or less (control group, n = 87), in order to examine the characteristics of colorectal cancers in the elderly from the viewpoint of metabolic and anabolic pathways of fluoropyrimidines. The level of TP was 78.4 +/- 47.0 unit/mg protein in the elderly group and 82.4 +/- 70.9 unit/mg protein in the control group (p = 0.86). The level of DPD was 53.7 +/- 43.1 unit/mg protein in the elderly group and 52.6 +/- 37.7 unit/mg protein in the control group (p = 0.73). The ratio of TP to DPD was 2.0 +/- 1.2 in the elderly group and 1.8 +/- 0.9 in the control group (p = 0.44). These three parameters did not differ between the groups when divided according to Dukes' stage (Dukes' A.B versus Dukes' C.D). These results suggest that there are no age-specific characteristics in relation to conversion of fluoropyrimidines such as capecitabine and doxifluridine to 5-fluorouracil (FU) and degradation of 5-FU in colorectal cancers.     

Estimation of level of enzyme induction of thymidine phosphorylase, dihydropyrimidine dehydrogenase, and thymidylate synthase in primary and recurrent breast cancer

Several enzymes are closely related with the mechanism of action of fluoropyrimidine (FP). Dihydropyrimidine dehydrogenase (DPD) which catalyzes 5-fluorouracil (5-FU), thymidine phosphorylase (TP), responsible for catalyzing doxifluridine to 5-FU, and thymidylate synthase (TS) were estimated for breast cancer. TP level determined by enzyme-linked immunoabsorbant assay (ELISA), DPD level by ELISA and catalytic assay and TS level by fluorodeoxyuridine monophosphate (FdUMP) binding assay were estimated for 210 specimens from 125 consenting patients with primary or metastatic breast cancer who gave consent. TS level of T1 was higher than that of T2-4 (p < 0.05). A high positive correlation was observed between TP and DPD in the same specimen. The ratio of simultaneously resected metastasis/primary and postoperative recurrent/primary never exceeded two. Enzyme level, generally decreased with or without interventing FP therapy. Enzyme level increase was frequently observed in a series of no-drug, non-FP, and FP therapy regimens. For selection of chemotherapy or estimation of chemosensitivity, TS can be used, with either TP or DPD at first surgery, and also with intervening chemotherapy after recurrence.     

Thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression in soft-tissue sarcoma versus capecitabine potential

Expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) can predict for clinical outcome of fluoropyrimidine-based therapy and there is every likelihood that relevant tumors will respond. High TP expression was observed in 35 (42%) patients with soft-tissue sarcoma. Seven out of 26 (27%) such patients revealed molecular phenotype prognostically favorable for capecitabine therapy. More clinical research is required to assess the efficacy of capecitabine-based therapy.     

Clinical significance of dihydropyrimidine dehydrogenase and thymidylate synthase expression in patients with pancreatic cancer

Background

Little is known about the clinical significance of TS and DPD in pancreatic cancer. We aimed to evaluate TS and DPD expression levels in not only pancreatic cancer but also surrounding normal pancreatic tissues to assess the clinical implications of the expression of TS and DPD in this study.

Patients and methods

Pancreatic cancer and normal pancreatic tissues were obtained from 18 patients with pancreatic cancer who underwent pancreatic resection to measure TS and DPD activities. The TS and DPD activities were determined by enzyme-linked immunosorbent assay using non-fixed fresh-frozen specimens.

Results

Pancreatic cancer tissues had significantly higher DPD and TS enzyme activities than surrounding normal tissue. Anaplastic ductal carcinoma had higher DPD and TS activities than the other histological types. Patients with high DPD in this study demonstrated poorer prognosis than those with low DPD. On the other hand, there was no statistically significant difference in survival between the high and the low TS groups.

Conclusions

The efficacy of 5-FU may be lower in pancreatic cancer tissue than in normal tissue because DPD activity is upregulated in pancreatic cancer tissue compared to normal pancreatic tissue. It is necessary to develop an effective 5-FU delivery system and/or 5-FU combined with an inhibitor for DPD that can be used when 5-FU must be administered to patients with pancreatic cancer. High DPD activity may be a prognostic factor in patients with pancreatic cancer.     

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.     

Relationships between the expression of thymidylate synthase,dihydropyrimidine dehydrogenase,and orotate phosphoribosyltransferase and cell proliferative activity and 5-fluorouracil sensitivity in colorectal carcinoma

Background: The site of action of the 5-fluorouracil (5-FU) antitumor effect has been explicated in recent years. Many studies have investigated enzymes involved in 5-FU metabolism in attempts to predict this effect, and a correlation of enzyme activity with the 5-FU drug sensitivity test has been reported. The aim of this study was to identify the biochemical response determinants of 5-FU. Additionally, we aimed to clarify the association between cell proliferative activity and the response to 5-FU of colorectal cancer. Methods: Our research subjects were 54 patients with colorectal carcinoma who had undergone operations between August 1999 and July 2001 in our department. Assays of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) activities in colorectal carcinoma tissue and assays of 5-FU sensitivity by the collagen gel droplet embedded culture drug sensitivity test (CD-DST) were conducted to investigate the relationships between each enzyme activity and 5-FU sensitivity. In addition, the proliferative activity of cancer cells was evaluated with Ki-67 antibody, and the relationship of this activity to each enzyme activity and 5-FU sensitivity were investigated. Results: 5-FU sensitivity was high in the low-TS-activity group and in the high-OPRT-activity group. Cancers with high cell proliferative activity showed good sensitivity to 5-FU, and TS and OPRT activities were high in such cancers. Conclusion: The results suggest that OPRT activity can predict sensitivity to 5-FU, and high OPRT activity may cause good 5-FU sensitivity in cancers with high cell proliferative activity. Received: April 23, 2002 / Accepted: January 20, 2003 Correspondence to:R. Fujii     

Thymidine phosphorylase and dihydropyrimidine dehydrogenase protein expression in colorectal cancer.

It is essential for actively proliferating cells to increase their rate of DNA synthesis to progress through the cell cycle. This is reflected in the increased uracil usage that is a common feature in solid tumours. Thymidine phosphorylase (TP) anabolises formation of pyrimidine nucleosides available for DNA synthesis, whereas dihydropyrimidine dehydrogenase (DPD) catabolises the degradation of pyrimidine bases, thereby reducing levels of uracil and thymine available for DNA synthesis. In addition, tissue levels of TP or DPD have been associated with the clinical efficacy of pyrimidine anti-metabolites commonly used in the treatment of colorectal cancer. There is little information, however, on the relative expression or degree of co-ordinated regulation of either protein in primary or metastatic colorectal cancer. DPD and TP protein levels were measured in 15 primary colorectal carcinomas, 10 colorectal liver metastases and 25 adjacent uninvolved tissues. DPD was reduced in 67% (10/15) of colorectal tumours (mean tumour/normal = 0.52) and in all liver metastases (mean tumour/normal = 0.41) compared with the corresponding normal tissue. In contrast, TP was increased in 80% (12/15) of colorectal tumours (mean tumour/normal = 18.91) and in all metastases (mean tumour/normal = 3.70). TP and DPD protein expression were highly variable in uninvolved and tumour tissues. The ratio of TP:DPD was higher in 87% of colorectal tumours and in all liver metastases compared with the adjacent uninvolved tissues. This suggests the presence of co-ordinated regulation of these pyrimidine metabolic enzymes and offers a strategy for optimising the use of pyrimidine-based chemotherapy.     

Expression of thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase, E2F-1, Bak, Bcl-X, and Bcl-2, and clinical outcomes for gastric cancer patients treated with bolus 5-fluorouracil

Few studies have investigated the biological factors associated with sensitivity to bolus infusions of 5-fluorouracil (5FU), including sequential methotrexate (MTX)/5FU therapy. We investigated the relationship between the expression of thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), E2F-1, Bcl-2, Bak, and Bcl-X, and the chemotherapeutic effects of sequential MTX/5FU. We studied 38 patients with unresectable or recurrent gastric cancer, treated weekly with sequential MTX/5FU (MTX 100 mg/m2, 5FU 600 mg/m2, by bolus infusions, with a three-hour interval). Expression of the above proteins was examined in initial biopsy samples with immunohistochemical methods. Immunohistochemical reactivity was defined as positive when over 25% of cancer cells showed strong staining in the cytoplasm for TS, TP, DPD, Bak, Bcl-2, and Bcl-X, and in the nucleus for E2F-1. The overall response rate was 28% in the 29 patients who had measurable lesions. Bak-negative patients showed a higher response rate than Bak-positive patients (39% versus 9%, respectively; p=0.1096), although expression of the other proteins was not associated with chemosensitivity. The median survival time (MST) of all patients was 256 days. Bak-negative patients survived significantly longer than Bak-positive patients (MST, 302 days versus 134 days, respectively; p=0.0044). Bcl-X-negative patients survived significantly longer than Bcl-X-positive patients (MST, 302 days versus 215 days, respectively; p=0.0080). Furthermore, patients negative for both Bak and Bcl-X had significantly better prognoses than other patients (MST, 373 days; p<0.0001). Within the limits of the small patient population, multivariate analysis using the Cox proportional hazards model showed that Bak, Bcl-X, and histological type were independent variables predicting survival (p=0.0008, 0.0081, and 0.0082, respectively). Although previously described predictive markers for protracted infusion of 5FU, including TS, TP, and DPD, might not be associated with clinical outcome in patients treated with sequential MTX/5FU, Bak may be a useful marker for chemoresponse and survival. Furthermore, both Bcl-X expression and the coupled expression of Bak and Bcl-X, as well as histological type, may be useful predictive markers for survival.     

Clinical significance of thymidine phosphorylase and dihydropyrimidine dehydrogenase expression in transitional cell cancer

PURPOSE: Thymidine phosphorylase (TdR-Pase) and dihydropyrimidine dehydrogenase (DPD) are thought to be key enzymes in the metabolic pathways of 5-fluorouracil (5-FU). Theoretically, tumors which have low DPD and/or high TdR-Pase expression should be 5-FU-sensitive. TdR-Pase also has angiogenic expression which aids tumor progression and metastasis. However, little is known concerning the relationship between DPD expression and clinical malignant potential, especially in urological cancer. MATERIALS AND METHODS: Transitional cell cancer (TCC) tissues were obtained from 50 patients, and TdR-Pase and DPD expression was measured by ELISA and radioenzyme assay, respectively. The sensitivity of 23 of the 50 specimens to 5-FU was assessed in a histoculture drug response assay (HDRA), an in vitro chemosensitivity test. RESULTS: TdR-Pase and DPD expression in TCC tissues was higher than in normal urothelial tissues. The expression of both TdR-Pase and DPD in TCC increased with histological grade and stage. Superficial bladder cancer patients who had undergone transurethral resection were divided into two groups, a recurrent and a nonrecurrent group. The expression of TdR-Pase and DPD was higher in the recurrent group than in the nonrecurrent group, but the differences were not significant. There was a significant inverse correlation between DPD expression and 5-FU sensitivity. However, TdR-Pase exhibited no correlation with 5-FU sensitivity. CONCLUSIONS: The expressions of both enzymes may be a good indicator of the malignant potential of TCC. Although DPD may be a good indicator of sensitivity of TCC to 5-FU, TdR-Pase appeared not to regulate the sensitivity of TCC to 5-FU.