Abstract
Background: The enzyme dihydropyrimidine dehydrogenase (DPD) is involved in the metabolism of 5-fluorouracil (5-FU). The aim of this study was to clarify the correlation between the expression of DPD and the efficacy of 5-FU therapy in patients with lung adenocarcinoma (AD). Patients and Methods: We examined surgically resected specimens from 90 stage I to IIIA patients with lung ADs to determine the level of intra-tumoral DPD mRNA. Results: Administration of 5-FU improved the prognosis of patients with low DPD-expressing tumors, whereas it did not do so for patients with high DPD expressing tumors. Patients with low DPD-expressing tumors administered with 5-FU had a significantly better prognosis than those who underwent surgery alone. A Cox proportional hazards regression model revealed that administration of 5-FU was an independent variable to predict prognosis in patients with low DPD-expressing tumors. Conclusion: Quantification of DPD mRNA levels is useful for determining the subgroup of lung AD patients who would benefit most from 5-FU after surgery.
- Lung adenocarcinoma
- 5-fluorouracil
- dihydropyrimidine dehydrogenase
- thymidylate synthase
- chemosensitivity
5-Fluorouracil (5-FU) and its derivatives are widely used for treatment of various types of cancer (1). A recent study showed that postoperative oral administration of tegafur-uracil (UFT) improves survival in patients following resection of stage I lung adenocarcinoma (AD) and its administration has become standard therapy after curative resection in early non-small cell lung cancer (NSCLC) cases (2, 3). Furthermore, a novel oral form of fluorouracil S-1 was shown to have promising effects against advanced NSCLC (4). These findings indicate that 5-FU is effective for NSCLC patients and highlight the importance of detection of biomarkers for prediction of its efficacy for treating NSCLC.
Thymidylate synthase (TYMS), the target enzyme for 5-FU, catalyzes an important process for DNA biosynthesis (5, 6) and we previously reported that the prognosis of NSCLC patients is related significantly to the intratumoral TYMS mRNA level (7). Dihydropyrimidine dehydrogenase (DPD) is one of the key enzymes involved in the catabolism of 5-FU and its expression was found useful in predicting the efficacy of 5-FU after surgery for NSCLC based on disease-free interval during short follow-up periods (8-10). In the present study, we examined the efficacy of 5-FU in association with DPD expression in regards to prognosis, including overall survival, in patients with lung ADs over a longer follow-up period.
Patients and Methods
Ninety specimens from lung AD patients determined to be p-stage I to IIIA were obtained during surgical procedures at Osaka University Hospital, Kinki Chuo Chest Medical Center, Toneyama Hospital, and Osaka Prefectural Medical Center for Respiratory and Allergic Disease between January 1999 and March 2003. Quantification of TYMS and DPD mRNA levels in AD tissues was performed as described previously (7, 10). The obtained copy numbers of TYMS and DPD were standardized with glyceralde-hydo-3-phosphate dehydrogenase (GAPDH) mRNA quantity, used as an endogenous control, with the following equation: ‘Result’=Log (TYMS or DPD RNA copy number in tumor)/(GAPDH RNA copy number in tumor)×(6.1×109; GAPDH RNA copy number in 1 μg of total RNA extracted from the peripheral blood of 30 healthy volunteers).
Patient background data.
Informed consent was obtained from all patients. Those administered UFT following surgery comprised the 5-FU group (n=30), while those who underwent surgery only, comprised the control group (n=60). UFT administration was started within 2 months after surgery and continued for more than 12 months. The dose of UFT was 300-400 mg/day and the mean duration of treatment was 21.5±7.3 months (mean±SD; range 12-26 months). The clinical backgrounds of the patients are summarized in Table I. There was no difference in clinical factors between the groups. The median follow-up period was 78±23 months (range 17-115 months) after surgery.
Chi-square, Mann-Whitney U, and Kruskal-Wallis tests were used to compare the results, while survival rates were estimated by the method of Kaplan and Meier and compared using log-rank test, using Statview version 5.0 for Windows (Abacus Concepts, Berkeley, CA, USA). A p-value of <0.05 was considered to be statistically significant.
Results
Quantification of TYMS and DPD mRNA levels in NSCLC tissues was successfully performed for all specimens. Intratumoral TYMS and DPD mRNA levels ranged from 6.28 to 8.04 (mean±SD; 6.98±0.34) and 5.36 to 8.28 (6.79±0.59), respectively. The results for TYMS and DPD mRNA levels are summarized in Table II. TYMS mRNA levels were associated with tumor status, while DPD mRNA levels were not associated with tumor or nodal status.
Thirty-five (39%) of the 90 patients developed distant metastasis after surgery. In regards to tumor stage, 12 (21%) out of 58 patients in stage I, 8 (53%) out of 15 patients in stage II, and 15 (88%) out of 17 patients in stage IIIA suffered from recurrent disease. Categorized by group, 27 (45%) out of 60 patients and 8 (27%) out of 30 in the control and 5-FU groups, respectively, had recurrence. There was no significant difference in overall survival rate between the groups (Figure 1A). Similar to a previous report (7), TYMS mRNA levels were significantly correlated to overall survival when dichotomized at the mean TYMS mRNA level (Figure 1B).
Thymidylate synthase (TYMS) and Dihydropyrimidine dehydrogenase (DPD) mRNA levels, and clinicopathologic factors.
Next, we evaluated the correlation between DPD expression and efficacy of 5-FU. DPD mRNA levels were significantly correlated to overall survival in the 5-FU group, but not in the control group when dichotomized at the mean DPD mRNA level (Figure 2). In the 5-FU-group, the 5-year survival rate was 92% for the low DPD-expressing subgroup and 68% for the high DPD-expressing subgroup. Patients with low DPD-expressing tumors, who were administered 5-FU had a significantly better prognosis than those who underwent surgery alone (Figure 3A); the 5-year survival rates were 92% for the 5-FU group and 53% for the control group. These findings suggest that the intratumoral DPD mRNA level may be a possible predictor for efficacy of 5-FU administration after surgery for NSCLC. On the other hand, patients with high DPD-expressing tumors administered 5-FU had a tendency for a worse prognosis as compared to those who underwent surgery alone (Figure 3B).
We analyzed 5 variables, namely tumor status, nodal metastasis, TYMS mRNA expression, DPD mRNA expression, and 5-FU administration, using a Cox proportional hazards regression model to determine their effects on overall survival in NSCLC patients (Table IIIA). Multivariate analysis revealed that p-N2 and TYMS mRNA expression were independent variables for predicting overall survival (Table IIIB). Furthermore, in patients with low DPD-expressing tumors, multivariate analysis showed that TYMS mRNA expression and administration of 5-FU, were each independent variables predicting prognosis (Table IIIC).
Discussion
We performed quantitative assays of intratumoral TYMS and DPD mRNA levels to assess their association with clinicopathological factors, as well as the feasibility of applying them to predict the efficacy of 5-FU therapy inpatients with NSCLC over a long term. TYMS activity is necessary for cell proliferation because it catalyses an essential step in DNA synthesis, while its overexpression is reported to be associated with tumor proliferation, as well as poor prognosis, in a variety of cancer types (11, 12). As shown in Table IIIB, multivariate analysis revealed that a high level of TYMS mRNA was independently correlated to overall survival with a high hazard ratio, indicating that this marker can precisely perform prognosis for patients with lung AD. Determination of gene expression by RT-PCR is a useful technique for small-sized specimens, thus quantification of TYMS mRNA levels is clinically sensitive and useful for determining the prognosis of AD patients (7).
As DPD is a rate-limiting enzyme in the catabolism of 5-FU, its high expression in tumors is reported to result in a low sensitivity to 5-FU therapy (13). In the present study, we evaluated the efficacy of 5-FU administration as adjuvant chemotherapy, in relation to intratumoral DPD mRNA levels in lung AD patients. Our results revealed that DPD expression was significantly inversely correlated to the overall survival of patients administered 5-FU following surgery, indicating that patients with low levels of DPD expression in cancer tissue are sensitive to 5-FU. Furthermore, for patients with low DPD-expressing tumors, those administered 5-FU had a significantly better prognosis than those who underwent surgery alone. These findings suggest that the intratumoral DPD mRNA level is a possible predictor for the efficacy of 5-FU administration after surgery in lung AD patients. Interestingly, in patients with high DPD-expressing tumors, those administered 5-FU had a tendency for worse prognosis than those who underwent surgery alone (Figure 3B), suggesting that 5-FU may not have benefits for patients with high DPD-expressing tumors. Multivariate analysis showed that administration of 5-FU was an independent variable predicting prognosis of patients with low DPD-expressing lung ADs. Based on these results, determination of DPD mRNA levels in lung AD tumors may provide important information for clinicians to decide whether or not to proceed with 5-FU-based chemotherapy for their patients.
Based on our findings for biomarkers associated with 5-FU therapy, it is considered important to evaluate the expressions of TYMS and DPD before establishing a protocol for made-to-order chemotherapy for NSCLC patients (14). In addition, investigation of the effects of more aggressive adjuvant therapy for patients with NSCLC who have elevated TYMS or DPD mRNA levels is also necessary. Takizawa et al. reported that in vitro sensitivity to platinum-derived drugs, such as cisplatin and carboplatin, was associated with the expression of TYMS and DPD in NSCLC specimens (15). They hypothesized that these may be novel markers of DNA repair capacity and may also be linked with chemosensitivity to drugs other than 5-FU. Furthermore, it is important to clarify the roles of TYMS and DPD in regards to chemosensitivity toward various chemotherapy regimens, as their inhibition is now receiving attention for new cancer treatment drugs development. Recently, S-1, a combination of tegafur, gimeracil, and oteracil potassium (Taiho Pharmaceutical), was developed for clinical use (4). Gimeracil is a stronger inhibitor of DPD than uracil when used with UFT. However, Takeda et al. reported that a high level of DPD expression predicted resistance to S-1-based chemotherapy in patients with advanced NSCLC (16). Therefore, additional investigations of the effects of new regimens with other anticancer drugs and molecular targeting therapies for NSCLC patients with high DPD-expressing tumors are necessary.
A: Overall survival curves for patients administered and those not administered 5-fluorouracil (5-FU) after surgery. B: Overall survival curves for patients with high and low thymidylate synthase (TYMS) mRNA levels in resected cancer tissues when dichotomized at the mean TYMS mRNA level.
A: Overall survival curves for patients with low and high dihydropyrimidine dehydrogenase (DPD)-expressing tumors who did not receive 5-fluorouracil (5-FU) when dichotomized at the mean DPD mRNA level. B: Overall survival curves for patients with low and high DPD-expressing tumors who received 5-FU.
A: Overall survival curves for patients with dihydropyrimidine dehydrogenase (DPD)-expressing tumors: comparison between those who underwent surgery alone and those who received 5-fluorouracil (5-FU) when dichotomized at the mean TYMS mRNA level. B: Overall survival curves for patients with high DPD-expressing tumors: comparison between those who underwent surgery alone and those who received 5-FU.
In conclusion, using real-time RT-PCR, assessment of TYMS and DPD expressions in tumors from patients with NSCLC can provide precise prognostic information and predict the efficacy of 5-FU therapy after resection.
Footnotes
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Conflicts of Interest Statement
The Authors have no conflicts of interest to declare.
- Received September 13, 2011.
- Revision received November 17, 2011.
- Accepted November 18, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
