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Keywords:

  • orotate phosphoribosyl transferase;
  • thymidylate synthasel;
  • dihydropyrimidine dehydrogenase;
  • oral 5-fluorouracil;
  • adjuvant chemotherapy;
  • colorectal cancer

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in phosphoribosylation of 5-fluorouracil (5-FU), an essential step that leads to tumor growth inhibition. In our study, the prognostic relevance of OPRT, thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in resectable colorectal cancer (CRC) patients treated by oral 5-FU were compared to further clarify the prognostic value of OPRT. Tumor tissue was collected from 90 CRC patients and the patients were followed for 5.2 years (Median). TS, DPD and OPRT activities in the extract of tumor tissue were determined enzymatically. The cut-off value of OPRT (0.147 nmol/(min mg), TS (0.044 pmol/mg) and DPD (72.10 pmol/(min mg) were determined by maximal χ2 method. Among these 5-FU metabolic enzymes, only high OPRT group demonstrated significantly better disease-free survival (DFS) (p = 0.0152) and better overall survival (p = 0.0078). In Cox regression analysis, node status (p < 0.0005) and OPRT (p = 0.044) were significant factors for DFS. OPRT activity in tumor tissue was a predictor of prognosis in resectable CRC patients treated by oral 5-FU-based adjuvant chemotherapy, and was useful to pick-up high risk patients independent from known prognosis factors. © 2006 Wiley-Liss, Inc.

5-fluorouracil (5-FU) is widely used all over the world for the treatment of a variety of tumors, and it is especially a key drug in the treatment of colorectal cancer (CRC). In western countries, 5-FU injection has been evolved as a constituent of multidrug chemotherapy, as represented by biochemical modulation of leucovorin. In Japan, in contrast, oral form of the drug was developed for the convenient administration, and has been used widely in adjuvant chemotherapy. In this connection, the usefulness of chemotherapy with oral fluoropyrimidine has been recently reported in adjuvant chemotherapy of CRC.1, 2

The analysis of correlation between 5-FU metabolic enzymes and antitumor effect has been widely investigated to predict the effect of 5-FU. Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are such enzymes and the association to 5-FU efficacy has been frequently reported in the literature. TS plays a part in DNA synthesis from thymidine and known be inhibited by forming ternary complex together with 5-fluorodeoxyuridine monophosphate (FdUMP) and active folic acid when 5-FU is administered.3, 4 High TS activity in tumor cells often associate with low 5-FU sensitivity and low TS activity associate with high sensitivity.5 It has been reported that patients with high DPD activity in the tumor showed low 5-FU sensitivity, whereas those with low DPD activity showed high 5-FU sensitivity.6 Also, Salonga et al. have studied the impact of the enzymes on the 5-FU efficacy and then clarified that a combination of two enzyme activities, TS and DPD, is useful to predict the response to 5-FU and the accuracy is very high.7 TS, however, is a target for DNA synthesis inhibition by 5-FU, and another effect of 5-FU to impair the RNA function, could not be taken into consideration.

On the other hand, orotate phosphoribosyl transferase (OPRT) is an essential enzyme that catalyzes the conversion of orotic acid to orotidine monophosphate in the de novo biosynthesis pathway of pyrimidine nucleotide and also controls the salvage biosynthesis pathway. OPRT also involves in the conversion of 5-FU to the active nucleotide and is considered to be a key enzyme in the first step, leading to DNA synthesis inhibition and RNA dysfunction. Thus, OPRT activity could be an important indicator, which can simultaneously assess DNA synthesis inhibition and RNA dysfunction by 5-FU. Our study indicated that there was a strong positive correlation between OPRT activity and CRC cancer cell ex vivo 5-FU sensitivity8 and the association of tumor OPRT activity and prognosis in resectable CRC patients. Namely, low tumor OPRT activity associates with poor prognosis.9

In the present retrospective study, the association of OPRT, TS and DPD to prognosis was evaluated, respectively, and compared to further clarify the contribution of tumor OPRT activity in prediction of prognosis in CRC patients.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Patients

Ninety patients (49 males and 41 females with a mean age of 67.0 years) who underwent complete resection of CRC without preoperative chemotherapy at the institute between November 1998 and April 2003 were enrolled. Written informed consent for using the surgical specimen for research purpose and performing statistical analysis was obtained from all patients. The protocol was reviewed and approved by the institutional review board of Tobu Chiiki Hospital.

The study population included resectable CRC patients subjected to oral 5-FU adjuvant chemotherapy. Dukes A patients are not subjected to adjuvant chemotherapy in general, however, adjuvant therapy was applied to some Dukes A patients because of their poor prognosis histology,10, 11 and they were enrolled in the study.

Methods

OPRT activity

A tissue sample (300 mg) was obtained from the resected tumor. The sample was immediately frozen and stored at −80°C until determination of OPRT activity by radioassay.12 The tissue was homogenized and then centrifuged to take a supernatant. The supernatant was mixed in an equal volume of the substrate solution containing 100 mM Tris–HCl (pH 7.5), 100 mM MgCl2, 10 mM phosphoribosyl pyrophosphate, 30 mM 2-glycerophosphate, 1.6 mM α,β-methylene adnosine diphosphate, and 8 mM [3H]-5-FU, and the reaction was stopped at 0, 5, 10 and 15 min after the addition of the substrate solution. The solution was then centrifuged to remove unreacted [3H]-5-FU, and reaction rate per minute was calculated by measuring the production of 5-fluorouridine monophosphate (FUMP) over time, with a liquid scintillation counter.

DPD activity

A tissue sample (300 mg) was taken from the resected tumor. The sample was immediately frozen and stored until determination of DPD activity by radioassay.13, 14 DPD activity was calculated by measuring a total production of metabolites of [14C]-5-FU, which was added to the homogenized tissue samples, i.e., dihydrofluorouracil (DHFU), 2-fluoro-β-ureidopropionate (F-β-UPA) and α-fluoro-β-alanine (F-β-Ala). 5-FU and the metabolites were separated by the thin-layer chromatography.

TS activity

A tissue sample (300 mg) was taken from the resected tumor, and it was immediately frozen for storage. TS (TS-free) activity was measured by FdUMP ligand binding assay.15

Adjuvant chemotherapy

The patients received adjuvant chemotherapy with oral 5-FU tablet (150 mg/day) (Kyowa Hakko Kogyo, Tokyo, Japan) for a minimum of 1 year. The treatment was switched to non-5-FU based chemotherapy when cancer recurrence was confirmed.

Statistical analysis

Cut-off values of OPRT, TS and DPD activities against recurrence were calculated by the maximal χ2 statistics.16, 17 In this statistics, patients were classified into below or equal to that value, or above that value for each OPRT, DPD or TS value. The Pearson χ2 test statistics was used to compare the recurrence rate of the two resulting groups of patients. OPRT, DPD or TS values that yield the largest χ2 test statistic was selected as the optimum cut-off value. Disease-free survival (DFS) and overall survival (OS) rates by cut-off values of OPRT, TS and DPD activities were calculated by the Kaplan-Meier analysis. DFS and OS curves of two groups categorized by cut-off values were compared by the log-rank test. The Cox regression was used in the multivariate analysis of prognostic factors for DFS and OS.

p values <0.05 were regarded as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Patients

Patient characteristics are shown in Table I. Dukes A patients with either poorly differentiated adenocarcinoma or mucinous adenocarcinoma were also included in the study. The type of chemotherapy administered and Dukes stage were shown in Table II. Five-year DFS rates were 74.3%, 71.4% and 65.3% for 5-FU, 5′-DFUR and Tegafur/Uracil group, respectively. Five-year OS rates were 79.2%, 66.7% and 70.9%, respectively. No significant difference in DFS and OS between types of chemotherapy was observed by log-rank test.

Table I. Patient Characteristics
No. of patients90
Age, years, median (range)67 (30–81)
Sex (male/female)49/41
Histological type (Wel/Mod/Poor/Muc)24/52/4/10
pT classification (pT2/pT3/pT4)18/69/3
Lymph node metastasis (pN0/pN(+))57/33
Dukes (A/B/C)13/44/33
Table II. The Type of Chemotherapy Administered and Dukes Stage
 Number of patients
5-FU5′-DFURTegafur/uracil
Dukes A814
Dukes B26 18
Dukes C1968

Cut-off values

The cut-off values obtained by the maximal χ2 statistics were 0.147 nmol/(min mg) for OPRT activity, 72.10 pmol/(min mg) for DPD activity and 0.044 pmol/mg for TS activity.

Correlation between 5-FU metabolic enzyme activities and prognosis

The patients were divided into high OPRT activity group and low OPRT activity group with the cut-off value of 0.147 nmol/min/mg. DFS and OS rates for each group are shown in Figure 1. The patients in the high OPRT activity group had significantly better prognosis for both DFS and OS compared to those in the low OPRT activity group (DFS: p = 0.0152, OS: p = 0.0078).

thumbnail image

Figure 1. Disease free survival rate (a) and overall survival rate (b) for high OPRT group (solid line) and low OPRT group (dotted line).

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The patients were divided into high DPD activity group and low DPD activity group with the cut-off value of 72.10 pmol/(min mg). DFS and OS rates for each group are shown in Figure 2. The patients in the low DPD activity group had better prognosis compared to those in the high DPD activity group in terms of both DFS and OS without significant differences.

thumbnail image

Figure 2. Disease free survival rate (a) and overall survival rate (b) for low DPD group (solid line) and low high DPD group (dotted line).

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DFS and OS rates in the high TS activity group and the low TS activity group divided by the cut-off value of 0.044 pmol/mg are shown in Figure 3. There were no differences in prognosis between the two groups.

thumbnail image

Figure 3. Disease free survival rate (a) and overall survival rate (b) for high TS group (solid line) and low TS group (dotted line).

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Five-year DFS and OS for each group by OPRT, DPD and TS were summarized in Table III.

Table III. Five-Year Disease-Free Survival and 5-Year Overall Survival Rate For Each Groups
 n5-year DFS (%)p value5 year OS (%)P value
OPRT
 <0.1472050.00.015248.80.0078
 ≥0.1477076.683.8 
DPD
 <72.13079.00.156982.10.1801
 ≥72.16066.667.5 
TS
 <0.0443968.20.474769.30.3163
 ≥0.0445172.575.6 

The subgroup analysis, DFS and OS of high and low OPRT group by Dukes stage, was shown in Table IV. DFS and OS were significantly better for high OPRT group when Dukes band C patients were combined. There was a trend toward better DFS and OS for high OPRT group in Dukes B only and C only, but these differences were not significant.

Table IV. Disease-Free Survival and Overall Survival of High and Low OPRT Group by Dukes Stage
 n5-year DFS (%)P value5-year OS (%)p value
Dukes A
 <0.1471100.00.676100.00.773
 ≥0.1471283.391.7 
Dukes B
 <0.1471070.00.10168.60.074
 ≥0.1473490.293.7 
Dukes C
 <0.147922.20.09022.20.092
 ≥0.1472454.375.0 
Dukes B+C
 <0.1471950.00.01548.80.008
 ≥0.1475876.683.8 

Multivariate analysis of prognostic factors

Node negative (HR: 5.211, 95% CI: 2.159–12.727, p < 0.0005) and high OPRT activity (HR: 0.364, 95% CI: 0.136–0.974, p = 0.044) were identified as independent prognostic factors for better DFS (Table V). Node negative (HR: 5.642, 95% CI: 2.011–15.857, p = 0.001) was the only independent prognostic factor for better OS (Table VI).

Table V. Multivariate Analysis Against Disease-Free Survival
 Hazard ratio95% CIp value
Age0.9970.955–1.0400.890
Sex0.8440.369–1.9330.689
pT2.5690.689–9.5830.160
pN5.2412.159–12.727<0.0005
OPRT cut-off0.3640.136–0.9740.044
TS cut-off0.5380.208–1.3920.202
DPD cut-off2.5580.950–6.8880.063
Table VI. Multivariate Analysis Against Overall Survival
 Hazard ratio95% CIp value
Age0.9990.952–1.0480.969
Sex0.7320.283–1.8930.520
pT3.7700.746–19.0580.108
pN5.6422.011–15.8570.001
OPRT cut-off0.4530.165–1.2460.125
TS cut-off0.5700.213–1.5220.262
DPD cut-off2.5150.830–7.6240.103

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

OPRT, which is also an enzyme to convert 5-FU to active nucleotide, is anticipated to play a key role in the first step, leading to DNA synthesis inhibition and RNA dysfunction. Thus, OPRT, together with TS and DPD, is thought to largely associate with the induction of 5-FU antitumor effect.18, 19 In the present study, we retrospectively evaluated the correlation to prognosis among OPRT, DPD and TS, in CRC subjected to 5-FU-based adjuvant chemotherapy.

The high OPRT activity group in the present study had significantly better DFS and OS compared to the low activity group. These results were consistent with the findings that 5-FU could be more easily metabolized to FdUMP in the high OPRT group, thus resulting in the induction of higher antitumor effect of 5-FU. DPD and TS alone did not demonstrate significant difference for both DFS and OS on the contrary to OPRT, although trends toward better survival were observed for low DPD group and high TS group.

Nodal status and OPRT activity were identified as significant prognosis factors for DFS by multivariate analysis in the present study. The identification of nodal status as the variant was very reasonable. The identification of only OPRT among 5-FU metabolic enzymes reflected the very important role of OPRT activity among other metabolic enzymes associated with the effect of 5-FU.

In the analysis for OS, on the other hand, nodal status was the only factor identified. With this respect, it should be taken into consideration that postrecurrence treatment varies from patient to patient, including best supportive care. Second line non-FU chemotherapy treatment also varied between patients, for instance CPT-11 and others. This may be the main reason for the identification of nodal status alone in the analysis for OS.

For all cancers, the very purpose of adjuvant chemotherapy is the elimination of minimal residual disease, which likely exists in high risk patients. In resectable CRC, the risk of recurrence is mainly evaluated by histopathology-based TNM staging and nodal status is especially important. On the other hand, in the area of leukemia treatment, the treatment is stratified more precisely using not only histopathological feature but also incorporating many prognostic factors derived from immunology, cytogenetics and molecular genetics achievement.20 In this context, there might be a room for progress in resectable CRC treatment. This study clarified that tumor OPRT activity is a useful independent factor to select high risk patient. To further stratify resectable CRC treatment, tumor OPRT activity would be one of candidate prognosis factors.

In colorectal carcinoma, relevance of prognostic factors had been retrospectively evaluated. These prognostic factors included Allelic loss of chromosome 18q, TS, DCC protein, proliferative activity, DNA aneuploidy, p53 oncoprotein and microsatellite instability.21, 22, 23, 24, 25, 26 Further efforts to sort out the importance of these prognostic factors would be necessary.

This study suggests that tumor OPRT may be a prognosis factor among 5-FU metabolic enzymes and the patients with 0.147 nmol/(min mg) or more tumor OPRT activity had better prognosis. The findings suggested that the patients satisfying this criteria would well respond to 5-FU-based adjuvant chemotherapy. Studies discussing the usefulness of oral fluoropyrimidines have been recently reported.27 As described in national NCCN clinical practice guideline in oncology (v.3.2005), however, there are several options for adjuvant therapy for CRC patients at present, and adverse effects cannot be avoided even in the so-called standard cancer therapies. Under such circumstances, other therapy should be chosen for those patients who are unlikely to respond well to the standard therapy. OPRT, among others, may play a key role in planning an individualized treatment for CRC patient, and it is critical to establish the applicability of 5-FU-based adjuvant chemotherapy by further studying the expression of OPRT, TS and DPD, enzymes associated with the induction of the antitumor effect of 5-FU.

References

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  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References
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