Japanese Journal of Clinical Oncology Advance Access originally published online on November 17, 2008
Japanese Journal of Clinical Oncology 2009 39(1):33-36; doi:10.1093/jjco/hyn121
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© The Author (2008). Published by Oxford University Press. All rights reserved
Comparison of 5-Fluorouracil-related Gene Expression Levels Between Adenocarcinomas and Squamous Cell Carcinomas of the Lung
1 Department of Cardiothoracic Surgery
2 Department of Anatomic and Diagnostic Pathology, Dokkyo Medical University, Shimotsuga, Tochigi, Japan
For reprints and all correspondence: Shinichiro Miyoshi, Dokkyo Medical University, 880 Kitakobayashi, Mibu-machi, Shimotsuga, Tochigi 321-0293, Japan. E-mail: miyoshi{at}dokkyomed.ac.jp
Received August 12, 2008; accepted October 1, 2008
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Abstract |
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Background: A recent meta-analysis study showed that post-operative adjuvant chemotherapy with UFT, an oral combination drug composed of tegafur [prodrug of 5-fluorouracil (5-FU)] and uracil [inhibitor of dihydropyrimidine dehydrogenase (DPD)] was associated with improved survival in patients with lung adenocarcinomas, but not in those with lung squamous cell carcinomas.
Methods: We investigated the 5-FU-related gene expression levels of thymidylate synthase (TS), DPD, thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) in resected tumor specimens from 51 patients with adenocarcinomas and 47 with squamous cell carcinomas using quantitative reverse transcription–PCR, and compared those levels between the two histological types.
Results: The relative gene expression values of TS, TP and OPRT were significantly lower in adenocarcinomas compared with squamous cell carcinomas, 1.60 ± 0.86 versus 4.33 ± 3.40 (P < 0.001), 0.84 ± 0.52 versus 2.27 ± 1.16 (P = 0.006) and 9.59 ± 6.30 versus 16.94 ± 12.04 (P < 0.001), respectively. The relative gene expression value of DPD was significantly greater in adenocarcinomas than those in squamous cell carcinomas, 2.33 ± 1.22 versus 1.50 ± 1.20 (P = 0.01). Lower expressions of TS and TP were observed more in adenocarcinomas (89.8%) than in squamous cell carcinomas (48.9%) (P < 0.001).
Conclusion: These data may explain that post-operative adjuvant chemotherapy with UFT was associated with improved survival in stage I patients with adenocarcinoma, but less with squamous cell carcinoma.
Key Words: lung cancer • gene expression • UFT • thymidylate synthase • orotate phosphoribosyl transferase
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INTRODUCTION |
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The expressions of thymidylate synthase (TS) and intracellular metabolic enzymes, such as dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT), have been reported to be associated with sensitivity and/or resistance to 5-fluorouracil (5-FU) in patients with colorectal and lung cancer (1–4). Recently, a meta-analysis study (5) showed that post-operative adjuvant chemotherapy with UFT, an oral combination drug composed of tegafur (prodrug of 5-FU) and uracil (competitive inhibitor of DPD), was associated with improved survival in a Japanese population composed primarily of stage I lung adenocarcinoma patients. However, that study failed to show the efficacy of UFT in patients with squamous cell carcinomas of the lung. Those findings suggest that the gene expression levels of the four 5-FU related enzymes differ between adenocarcinomas and squamous cell carcinomas in lung cancer patients. In the present study, we conducted the first known investigation of messenger RNA (mRNA) expressions of these four enzymes in resected lung cancer specimens and compared them between the two histological types.
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SUBJECTS |
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Resected specimens from 51 patients with adenocarcinomas and 47 with squamous cell carcinomas who underwent surgery from April 2001 to June 2005 at our university hospital were studied. Patients who received preoperative induction therapy including chemotherapy or chemoradiotherapy were excluded. The pathological stage of the disease was based on the TNM classification of the Union Internationale Contre Cancer (UICC) (6). Informed consent was obtained from all patients and the study protocol was approved by the Ethics Committee of our hospital.
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METHODS |
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Surgically resected tumor samples were fixed in 10% buffered formalin for 48 h and embedded in paraffin. Histological diagnosis was made following hematoxylin/eosin staining of 5-µm sections. Danenberg’s tumor profile method3 was performed by Response Genetic Inc. (New York, USA). In brief, a laser captured micro-dissection technique (7) was used on 10-µm sections with a magnification of x100 to obtain only cancer cells. Next, 1500–2000 round spots 80 µm in diameter were dissected for each case, from which total RNA was extracted. Real-time quantitative reverse transcription–PCR assays of the four genes, TS, DPD, TP and OPRT, were performed on those samples using an ABI 7700 and TaqMan Probes. Sequences of the primers used have been described previously (3). The level of β-actin mRNA was used as a reference gene. Relative gene expression values are expressed as a ratio of PCR products of the gene of interest to that of the internal reference gene β-actin.
We used cutoff values that were estimated for responsiveness to 5-FU (3), for TS (
4.0), TP (18) and DPD (2.5).
Fisher’s exact test or Pearson’s chi-square test was used to compare categorical data for gender and pathological TNM staging classification. Comparisons of mean ages of the patients and gene expression values for TS, DPD, TP and OPRT were done with an unpaired t-test. The StatView statistical software package (version 5) was used for these statistical analyses. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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Patient characteristics are shown in Table 1. The mean age and frequency values for males were significantly higher in patients with squamous cell carcinomas. The pathological stage was not different between the two histological types.
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The relative gene expression levels of the four enzymes studied are shown in Fig. 1. No expression of TS was detectable in two cases with adenocarcinomas. Relative gene expression values of TS were 1.60 ± 0.86 in adenocarcinomas and 4.33 ± 3.40 in squamous cell carcinomas. The gene expressions of TS were significantly lower in the adenocarcinoma specimens as compared with the squamous cell carcinoma specimens (P < 0.001). In contrast, relative gene expression values of DPD were 2.33 ± 1.22 in adenocarcinomas and 1.50 ± 1.20 in squamous cell carcinomas. Those of DPD were significantly greater in adenocarcinomas than those in squamous cell carcinomas (P = 0.01). Those of OPRT and TP in adenocarcinomas were 0.84 ± 0.52 and 9.59 ± 6.30, and in squamous cell carcinomas 2.27 ± 1.16 and 16.94 ± 12.04, respectively. Those for TP and OPRT were significantly lower in the adenocarcinoma specimens as compared with the squamous cell carcinoma specimens (P = 0.006 and <0.001, respectively).
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Relative DPD gene expression in the specimens with TS
4.0 and TP 18 were replotted in Fig. 2. Forty-four of 49 (89.8%) adenocarcinomas and 23 of 47 (48.9%) squamous cell carcinomas were categorized as TS 4.0 and TP 18. The percentage of selected adenocarcinomas was significantly greater than that of squamous cell carcinomas (P < 0.001). Relative gene expression values from only a few specimens in squamous cell carcinomas were >2.5.
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DISCUSSION |
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This is the first known investigation of the gene expressions of TS, DPD, TP and OPRT as a set in resected lung cancer specimens. Our results showed significant differences for the expressions of all four enzymes between adenocarcinoma and squamous cell carcinoma of the lung.
Shintani et al. (8) reported that there were no significant differences in expression levels of TS mRNA between adenocarcinomas and squamous cell carcinomas of the lung. However, the RT–PCR technique used in that study was based on a homogeneous solution methodology, thus the TS gene expression value was presented as an average that included contributions from tumor cells as well as from any non-tumor tissues that may have infiltrated the specimens. In contrast, the present laser micro-dissection technique allowed us to analyze only cancer cells and provided more accurate results.
5-FU and its derivatives are anti-metabolic drugs widely used in cancer chemotherapy. The effects of 5-FU have been attributed to inhibition of TS, and incorporation of its metabolites into RNA and DNA. The drug enters tumor cells rapidly and is converted by metabolic enzymes, with DPD, a rate-limiting enzyme that converts 5-FU into the inactive metabolite dihydrofluoro-uracil, one of the first. In contrast, the other first metabolic enzymes, namely TP and OPRT, catalyze the synthesis of active metabolites, resulting in disruption of the activities of TS and DNA, or RNA synthesis (4,9).
The gene expression values of TS, DPD and OPRT in other organ carcinomas, such as head and neck, gastric, colorectal, breast and pancreatic cancers, were previously extensively investigated using the same method (10), and the median gene expression values of those were compared between the studied lung cancers and other organ carcinomas. The median values for TS and OPRT in lung squamous cell carcinomas were greater than those in other organ carcinomas, while that for DPD was slightly greater in lung adenocarcinomas as compared with the other organ carcinomas (Table 2). The gene expression levels of these three enzymes in other organ carcinomas, where there were mostly adenocarcinomas, were quite similar to those in lung adenocarcinomas, as compared with the lung squamous cell carcinomas.
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Solonga et al. (3) reported that all tested-colorectal tumors that responded to 5-FU therapy and had longer survival had low expression values of TS, TP and DPD. And they indicated cutoff values for TS (
4.0), TP (18) and DPD (2.5). UFT includes uracil, a DPD competitive inhibitor, at the molar ratio of 1:4. When UFT is administered to patients with non-small-cell lung cancer, the functional DPD levels in most will likely decrease to below the non-response cutoff value of DPD (2.5). Thus, in our study, a larger percentage of patients with adenocarcinoma was categorized into this responder group (TS 4.0 and TP 18) compared with those having squamous cell carcinomas. These data may explain the findings that post-operative adjuvant chemotherapy with UFT was associated with improved survival in Stage I lung adenocarcinoma patients, though that study failed to show the efficacy of UFT in patients with lung squamous cell carcinomas (5). However, some population of squamous cell carcinomas (23 of 47) in this study was also categorized into the responder group. UFT might be effective for patients with squamous cell carcinoma, if enough number of patients were enrolled. More recently, TS-1, an oral combination drug of tegafur, gimeracil and oteracil potassium, was developed (11). Gimeracil directly inhibits the function of DPD and is about 200-fold more potent than uracil used in UFT. Therefore, TS-1 is expected to be more effective for treatment of lung adenocarcinoma patients, which results in disruption of the activities of TS and DNA or RNA synthesis (4,7). |
CONCLUSIONS |
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TOPAbstractINTRODUCTIONSUBJECTSMETHODSRESULTSDISCUSSIONCONCLUSIONSFundingReferences |
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We investigated mRNA expressions of TS, DPD, TP and OPRT as a set in resected lung cancer specimens, and compared the results between adenocarcinomas and squamous cell carcinomas. There were significant differences in all four gene expressions between the two histological types. These findings provide some insight into the mechanism involved with the higher sensitivity to UFT or TS-1 of adenocarcinomas as compared with squamous cell carcinomas.
Conflict of interest statement
None declared.
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Funding |
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This work was partially supported by grants from Taiho Pharmaceutical Co. Ltd (Tokyo) and Dokkyo Medical University Fund (Shimotsuga, Japan).
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References |
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