Thymidylate synthetase and dihydropyrimidine dehydrogenase mRNA levels in esophageal cancer

This study investigated the mRNA levels of thymidylate synthetase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) in esophageal squamous cell carcinoma (ESCC). TYMS and DPYD gene expression was quantified using real-time RT-PCR in 56 patients with ESCC, coamplified with glyceraldehyde-3-phosphate dehydrogenase as an internal standard. The results were analyzed with reference to the clinicopathological characteristics and the prognosis of the ESCC patients. The TYMS and DPYD expression levels in patients positive with lymphatic invasion were significantly higher compared to those in patients who exhibited negative lymphatic invasion (TYMS P=0.0127, DPYD P=0.0127). Patients were classified into the groups high TYMS/DPYD, high TYMS but low DPYD, low TYMS but high DPYD and low TYMS/DPYD. The highest survival rate was found in the group with low TYMS/DPYD and the lowest survival rate in the group with high TYMS/DPYD (P=0.017). It was concluded that, on the basis of the multivariate analysis, TYMS mRNA expression is a candidate that serves as an independent prognostic factor for ESCC patients.


Introduction
The prognosis of patients with esophageal cancer remains poor, prompting the search for novel treatment strategies.Given the high malignant potential of this type of cancer, many patients developed local recurrence of the tumor or distant metastasis within a short period of time.Molecular biological studies have shown that esophageal squamous cell carcinoma (ESCC) is caused by the accumulation of multiple genetic changes in oncogenes and tumor suppressor genes (1,2).Thymidylate synthetase (TYMS) plays a role in catalyzing the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), a crucial synthetic step in nucleotide metabolism.Dihydropyrimidine dehydrogenase (DPYD) is also a key enzyme in the metabolic pathway involved in the degradation of the pyrimidine bases uracil and thymine.
TYMS and DPYD are significant enzymes in de novo DNA synthesis and the salvage pathway in cancer cells, respectively.This study investigated the TYMS and DPYD mRNA expression in ESCC by real-time RT-PCR using a LightCycler system.The results were analyzed with reference to the clinicopathological characteristics and prognosis of the ESCC patients.

Materials and methods
Tissue samples.Tissue samples were obtained from 56 patients with primary ESCC who underwent radical esophagectomy at the Department of Surgery, Nagoya City University Medical School, between 1996 and 2000.The study design was approved by the Institutional Review Board of the university hospital and written consent was obtained from each patient.The tumors were classified according to the Guidelines for the Clinical and Pathological Studies on Carcinoma of the Esophagus (3).The patient population comprised 44 males and 12 females (mean age 63.2±8.4 years; range 46-80).The samples were immediately frozen in liquid nitrogen and stored at -80˚C until use.None of the patients received chemotherapy or radiation therapy prior to or following surgery.
RT-PCR assays for thymidylate synthetase and dihydropyrimidine dehydrogenase.The RNA concentration was determined using a spectrophotometer and adjusted to a concentration of 200 ng/ml.RNA (1 µg) was reverse transcribed by the Superscript Ⅱ enzyme (Gibco BRL, Gaitherburg, MD, USA) with 0.5 mg oligo(dT) (Amersham Pharmacia Biotech, Piscataway, NJ, USA).The reaction mixture was incubated at 42˚C for 50 min followed by incubation at 72˚C for 15 min.To ensure the quality of mRNA extraction and reverse transcription, the samples were subjected to PCR amplification with oligonucleotide primers specific for the constitutively

Thymidylate synthetase and dihydropyrimidine dehydrogenase mRNA levels in esophageal cancer
expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and normalized to it.PCR was performed using LightCycler-Fast start DNA Master SYBR-Green Ⅰ kit (Roche Molecular Biochemicals, Mannheim, Germany).The primer sequences for the TYMS gene were: forward primer, 5'-TTACCTGAATCACATCGAGC-3' and reverse primer, 5'-ATATCCTTCGAGCTCCTTTG-3' .The cycling conditions were: initial denaturation at 95˚C for 10 min, followed by 60 cycles at 94˚C for 15 sec, 55˚C for 5 sec and 72˚C for 8 sec.The primer sequences for the DPYD gene were: forward primer, 5'-GTTCTGGCTACCAGGCTAT-3' and reverse primer, 5'-CATAAGGTGTTGTCCTGGAA-3' .The cycling conditions were: initial denaturation at 95˚C for 10 min, followed by 60 cycles at 94˚C for 15 sec, 56˚C for 5 sec and 72˚C for 6 sec.Amplified cDNAs were separated on 1% agarose gels and the bands were visualized by ethidium bromide.
Statistical methods.Data are expressed as the means ± standard deviation (SD).Statistical analysis was performed using the Stat-View software package (Abacus Concepts, Berkeley, CA, USA).The Mann-whitney U test was used to evaluate the significance of the expression in paired groups.The survival of patients with ESCC was examined using the Kaplan-Meier method, and the survival times were compared using the log-rank test.Survival was measured from the day of surgery.Multivariate analysis was performed using Cox's regression model and the logistic multivariate regression model.P<0.05 was considered to be statistically significant.

Analysis of thymidylate synthetase and dihydropyrimidine dehydrogenase mRNA levels by real-time RT-PCR assay using
LightCycler.TYMS/GAPDH mRNA levels of the 56 esophageal cancer tissue samples were 1.553±0.275.The relationship between TYMS/GAPDH mRNA and the patient clinicopathological characteristics were examined (Table Ⅰ).No significant differences were noted in TYMS/GAPDH mRNA with respect to age, gender, pathological differentiation, tumor status, lymph node status, stage or vessel invasion.The TYMS/GAPDH mRNA expression levels in patients positive with lymphatic invasion were significantly higher compared to those in patients who exhibited negative lymphatic invasion (P=0.0127).
The DPYD/GAPDH mRNA expression levels were 5.463±1.807.By contrast, the DPYD/GAPDH mRNA expression levels in patients positive with lymphatic invasion were significantly lower compared to those in patients who exhibited negative lymphatic invasion (P=0.0417).Moreover, no significant differences were noted with respect to other factors (Table Ⅱ).
No significant clinicopathological differences were noted in patients classified into groups with TYMS levels higher (n=27) and lower (n=29) than 0.855.however, a significantly higher risk of lymph node metastasis was noted in patients with higher levels of DPYD (n=28, DPYD/GAPDH mRNA levels >1.70).
Relationship between TYMS and DPYD and survival.The correlation between the TYMS and DPYD mRNA expression levels and the survival of ESCC patients following surgery (median follow-up 19.7 months) was investigated.Patients with high TYMS mRNA expression levels had a significantly shorter survival after surgery compared to patients with low TYMS mRNA expression levels (P=0.031) (Fig. 1).Patients with high DPYD mRNA expression levels had a shorter survival, but no significant difference was found (P=0.099) (Fig. 2).Of the four groups of patients (high TYMS/DPYD, high TYMS but low DPYD, low TYMS but high DPYD and low TYMS/DPYD), the best survival rate was found in the group with low TYMS/DPYD and the worst survival rate was observed in the group with high TYMS/DPYD (P=0.017) (Fig. 3).The univariate analysis showed that among the clinicopathological characteristics, local invasiveness (tumor status) (risk ratio 7.38; P=0.0003), lymph node metastasis (node status) (risk ratio 6.07; P=0.0032), lymphatic invasion (risk ratio 5.48; P=0.021), blood vessel invasion (risk ratio 3.56; P=0.0069) and TYMS mRNA expression (risk ratio 2.19; P=0.038) were statistically significant prognostic factors.The multivariate analysis showed that local invasiveness (P=0.045) and TYMS mRNA expression (P=0.041) were independent prognostic factors (Table Ⅲ).

Discussion
Esophageal cancer is a digestive cancer with poor prognosis and the mortality rate is steadily increasing.Three types of treatment are currently available, i.e., operation, chemotherapy and radiation therapy.Frequently, chemotherapy and radiation therapy are combined, both before and after surgery.In esophageal cancer, tumor growth is extremely rapid.Consequently, prompt and correct diagnosis and staging, including identification of remote metastases, and individual treatment are required.The prediction of sensitivity to chemotherapeutic agents prior to therapy is relevant.Chemotherapy for esophageal cancer relies heavily on 5-fluorouracil (5-FU) and cisplatin.However, individual variations in responsiveness to these chemotherapies exist.Therefore, the susceptibility testing of the anti-cancer drug treatment in esophageal cancer was reported.we also examined the relationship between the expression of TYMS, DPYD, thymidylate synthetase (TYMP) and orotate phosphoribosyl transferase (OPRT) and 5-FU sensitivity in 25 ESCC cell lines.our findings showed that the TYMS and DPYD mRNA expression levels may aid in predicting the anti-tumor activity of 5-FU in ESCC (4).In colorectal cancer, Salon et al and Nishimura et al reported a correlation between the clinical effect of 5-FU and the expression of those genes (5,6).oguri et al reported that the degradation of 5-FU via DPYD is a significant determination of 5-FU sensitivity, while the induction of TYMS contributes to acquired resistance against 5-FU in lung cancer (7).
On the other hand, certain authors have reported that TYMS and DPYD exhibit the malignant potential of gastric and colon cancers.Terashima et al reported that in a group of patients who did not receive adjuvant chemotherapy, survival was poor in patients with high TYMS activity (8).Shirota et al investigated the correlation between DPYD and malignant potential in colon cancer, reporting that higher DPYD levels were associated with higher pathological classification, micro-scopic lymph node metastasis and liver metastasis (9).Suda et al found that the expression of TYMS in gastric cancer correlated with recurrence and survival rate (10).
Therefore, TYMS and DPYD affect the clinical outcome of esophageal cancer in two ways.One possibility is that TYMS and DPYD affect the malignancy of cancer, the other is that they affect the outcome of the anti-cancer drug treatment.Therefore, in the present study cases in which anti-cancer drug treatments were used pre-and post-surgery were excluded.Additionally, Tanaka et al reported that the expression of TYMS and DPYD was altered by chemoradiation therapy (CRT) in residual tumor cells of esophageal cancer, when comparing mRNA levels in pre-CRT biopsies and post-CRT specimens (11).Brucher et al found no significant correlation between clinical or histological factors and the relative gene expression of TYMS, TYMP, DPYD or Her-2/neu.However, patients exhibiting these factors underwent pre-operative, combined radiochemotherapy (12).Therefore, not only were the cases with anti-cancer treatment excluded, but also those cases with radiation therapy.As a result, we examined the  correlation between the malignant potential of esophageal cancer and the expression of TYMS and DPYD.
In this study, TYMS mRNA expression was significantly correlated with lymphatic invasion.However, no other clinicopathological characteristics correlated with TYMS mRNA levels.with regard to post-surgical survival, a high expression of TYMS was associated with a poor prognosis.Only the parameter and tumor status were noted in the multivariate analysis.Comparable results were reported by Suda et al in gastric cancer.These authors reported that the survival curve for the TYMS-positive group was significantly lower compared to that of the TYMS-negative group in the immunohistochemical study (10).In addition to TYMS, DPYD mRNA expression was statistically correlated with lymphatic invasion.Nevertheless, no other factors, including prognosis, correlated with DPYD.Certain studies reported the usefulness of the combination analysis.Suda et al reported that the TYMS-positive TYMP-positive group was more inhibited than in other groups (8).Beck et al reported that in cultured cells from colorectal cancers, those with low DPYD and TYMS expression were experimentally more sensitive, while the patients were clinically more sensitive to 5-FU (13).Danenberg et al reported that in colorectal cancer, patients with low TYMS, DPYD and TYMP levels exhibited the best survival curves (14).Ichikawa et al reported that the combined expression of TYMS and DPYD predicted the efficacy of chemotherapy (15).In the present study, combination analysis was useful.The low TYMS/DPYD group showed the best survival curves statistically.A combined evaluation of the expression of other genes, such as TYMP, is required for a more accurate prediction of the response.
In conclusion, the present study showed that there was a significant correlation between TYMS and DPYD mRNA levels in esophageal cancer and the survival of patients presenting with type of cancer.Based on the present data and the relationship between gene expression and 5-FU sensitivity in esophageal carcinoma cell lines, more effective treatment should be established for individual patients.

Figure 1 .
Figure 1.The overall survival rate of esophageal squamous cell carcinoma patients and the expression levels of thymidylate synthetase (TYMS).The expression levels of TYMS were high/low.

Figure 2 .
Figure 2. The overall survival rate of esophageal squamous cell carcinoma patients and the expression levels of dihydropyrimidine dehydrogenase (DPYD).The expression levels of DPYD were high/low.

Figure 3 .
Figure 3.The overall survival rate of esophageal squamous cell carcinoma patients and the expression levels of thymidylate synthetase (TYMS) and dihydropyrimidine dehydrogenase (DPYD).The expression levels of TYMS and DPYD were divided into the groups: high TYMS/DPYD, high TYMS but low DPYD, low TYMS but high DPYD and low TYMS/DPYD.

Table Ⅰ .
Correlation of TYMS mRNA expression in esophageal cancer with clinicopathological characteristics.

Table IⅠ .
Correlation of DPYD mRNA expression in esophageal cancer with clinicopathological characteristics.

Table Ⅲ .
Univariate and multivariate analysis of the expression levels of thymidylate synthetase and dihydropyrimidine dehydrogenase and various clinical characteristics.