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Journal of Clinical Oncology, Vol 25, No 25 (September 1), 2007: pp. 3952-3957 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.11.8646 Epidermal Growth Factor Receptor Mutation Status and Adjuvant Chemotherapy With Uracil-Tegafur for Adenocarcinoma of the Lung
From the Departments of Cancer and Thoracic Surgery; Hematology, Oncology, and Respiratory Medicine; and Molecular Genetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama; Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima; and the Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan Address reprint requests to Shinichi Toyooka, MD, Department of Cancer and Thoracic Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan; e-mail: toyooka{at}md.okayama-u.ac.jp
Purpose Adjuvant chemotherapy with uracil-tegafur has been demonstrated to prolong survival among patients with resected lung adenocarcinomas. Epidermal growth factor receptor (EGFR) mutations have been reported to be present in lung adenocarcinomas. The present study evaluated whether the EGFR status could be used as a biologic predictor of the outcome of adjuvant chemotherapy with uracil-tegafur. Patients and Methods The EGFR mutational status of 187 patients with resected lung adenocarcinomas was determined using a polymerase chain reaction–based assay for EGFR exons 19 and 21; the results were then correlated with the effect of adjuvant uracil-tegafur chemotherapy on survival. The antiproliferative effect of fluorouracil (FU) on adenocarcinoma cell lines with EGFR wild-type or mutant type status was examined by measuring the inhibitory concentrations at 50% (IC50s). Results Among the 187 patients, 68 received uracil-tegafur as adjuvant chemotherapy, and 119 were not treated with any chemotherapeutic agents. EGFR mutations were present in 79 patients (43%). Overall, the adjuvant chemotherapy with uracil-tegafur significantly prolonged survival compared with the control group (hazard ratio = 0.38; P = .005). The survival benefit of adjuvant chemotherapy with uracil-tegafur was also examined after stratifying the patients according to EGFR mutation status. Adjuvant chemotherapy significantly prolonged survival among patients with EGFR wild-type tumors (hazard ratio = 0.34; P = .013) but not among patients with EGFR mutant tumors. In an in vitro experiment, the IC50s of EGFR mutant cells to FU were higher than those of wild-type cells, indicating that EGFR wild-type cells are more sensitive to FU than mutant cells. Conclusion EGFR status influenced the effect of adjuvant chemotherapy with uracil-tegafur. Adjuvant chemotherapy could be customized based on EGFR status.
Lung cancer is the most common cause of death from cancer in industrialized countries.1 Human lung cancers are divided into two major types, small-cell lung cancer and non–small-cell lung cancer (NSCLC), the latter of which consists of several subtypes.2 Despite great effort to improve the survival of patients with NSCLC, satisfactory outcomes have not been achieved. Even in early-stage NSCLC patients who undergo surgical resections, recurrent disease often impairs the clinical outcome. Recent studies have demonstrated that adjuvant chemotherapy provides a survival benefit in patients with resected NSCLC.3-5 Uracil-tegafur, an antimetabolite that combines tegafur (a fluorouracil [FU] prodrug) and uracil in a 1:4 molar ratio, has been approved for the treatment of patients with resected NSCLC and extensively examined for use in an adjuvant setting in Japan, which had shown that adjuvant chemotherapy with uracil-tegafur provides a survival advantage compared with surgery alone.6-9 Even among patients with pathologic stage IA disease in whom a good prognosis should be expected, patients with tumors more than 2 cm in diameter benefited from adjuvant uracil-tegafur therapy in a large randomized study.8 Accumulating knowledge of molecular oncology is enabling us not only to understand the pathogenesis of NSCLC but also to predict the sensitivity of tumors to anticancer drugs. In adjuvant settings, patients with excision of repair cross-complementation group 1 (ERCC1) –negative tumors showed prolonged survival compared with patients with ERCC1-positive tumors.10 In addition, epidermal growth factor receptor (EGFR) gene mutations have been identified in NSCLC and are significantly related to a favorable clinical outcome among patients treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs).11-13 These findings have prompted the investigation of molecular predictor–based therapeutic strategies for patients with lung cancers. Previous studies have indicated that an EGFR mutation status was significantly associated with adenocarcinoma histology, never-smoking status, and East Asian ethnicity.14 Regarding the relationship between the EGFR pathway and sensitivity to FU, Ueda et al15 reported that epidermal growth factor decreased the dihydropyrimidine dehydrogenase activity, which is an enzyme involved in the catabolism of FU, thereby increasing the sensitivity of the cells to FU. By contrast, Magne et al16 reported that gefitinib downregulates the thymidylate synthase activity that is inversely related to the antiproliferative effect of FU, thereby increasing sensitivity to FU. Because the EGFR mutation is an oncogenic alteration mainly causing the lung adenocarcinoma17 and is frequently present in Japanese patients with adenocarcinoma,14 it would be interesting to examine the effect of the EGFR mutation on the clinical outcome of patients with lung adenocarcinoma treated with uracil-tegafur. This study retrospectively examined the value of the EGFR status for predicting the clinical outcome of uracil-tegafur adjuvant chemotherapy in patients with resected lung adenocarcinoma. We also examined the effect of FU, which is a major component of uracil-tegafur, on lung adenocarcinoma cell lines with EGFR wild type or mutation.
Patients and Treatment We reviewed surgically resected patients with lung adenocarcinoma who were treated at Okayama University Hospital between January 1994 and December 2003 and found that a total of 551 consecutive patients with pathologic stage I to IIIA disease underwent complete tumor resection. Among them, patients who received any preoperative therapy or postoperative adjuvant therapy other than uracil-tegafur were excluded. In addition, patients with stage IA disease whose tumor sizes were less than 2 cm were also excluded.8 As a result, 187 patients with lung adenocarcinoma were eligible for this study. Sixty-eight patients (36%) received adjuvant chemotherapy with uracil-tegafur (uracil-tegafur group), whereas 119 patients (64%) underwent surgery alone (control group). The administration of uracil-tegafur to patients was left to the physician's discretion. In the uracil-tegafur group, the administration dose was 300 mg per body per day (body weight < 50 kg) or 400 mg per body per day (body weight  50 kg), and treatment was initiated within 1 month postoperatively.18 Uracil-tegafur was administered for at least 1 year if adverse reactions did not occur or if the patient did not refuse the treatment; 67% of the patients completed the full 1-year course, and 79% of patients continued the treatment for more than 6 months. Clinicopathologic staging was basically determined according to the International Union Against Cancer's TNM classification for malignant tumors.19 The extent of pulmonary resection and mediastinal node dissection was left to the physician's discretion, although a lobectomy and ipsilateral mediastinal lymph node dissection were regarded as the standard procedures.
DNA Extraction and EGFR Mutation Screening
FU Effect on Cell Lines Cell proliferation was determined by a modified MTS assay with CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI). Cells were incubated with FU that was kindly provided by Kyowa Hakko Kogyo Co, Ltd (Tokyo, Japan) for 72 hours; then, optical densities of samples were measured using Immuno Mini NJ-2300 (Nalge Nunc International KK, Rochester, NY). The antiproliferative effects of FU were shown in terms of inhibitory concentration at 50% (IC50), which was calculated using a Web-based program (http://chiryo.phar.nagoya-cu.ac.jp/javastat/Graded50-j.htm).
Statistical Analyses
Patient Characteristics and EGFR Mutations The patient characteristics were as follows. The median age was 66 years (range, 24 to 84 years), and 98 patients were male, and 89 were female. Smoking categories were defined as follows: never-smokers were those with a lifetime exposure of 100 cigarettes or less, smokers were those with a lifetime exposure of more than 100 cigarettes, current smokers were those who smoked within 12 months at the time of diagnosis, and former smokers were those who had quit smoking more than 12 months before diagnosis.22 A total of 103 patients were smokers (44 former smokers and 59 current smokers), and 84 patients were never-smokers. One hundred forty-nine patients had pathologic stage I disease (86 patients with stage IA disease with tumor size 2 cm and 63 patients with stage IB), 23 patients had stage II disease (seven patients with stage IIA and 16 patients with stage IIB), and 15 patients had stage IIIA disease.
EGFR mutations were detected in 79 (42.2%) of 187 patients; 49 patients had exon 19 deletions, and 30 patients had an exon 21 mutation (L858R). The relationships between the patient characteristics and the EGFR mutation status are listed in Table 1. The
Survival, EGFR Mutation Status, and Adjuvant Chemotherapy With Uracil-Tegafur Of the 187 patients, 68 (36.4%) received adjuvant chemotherapy with uracil-tegafur, whereas 119 (63.6%) underwent surgery alone. By March 2006, 55 (39%) of 187 patients (15 with adjuvant chemotherapy and 40 with surgery alone) had died, with a median follow-up time among the surviving patients of 65.7 months for the adjuvant chemotherapy group and 56.2 months for the control group. The follow-up records were documented in 178 (95.2%) of 187 patients. The remaining nine patients were censored for the overall survival analysis at the time of last follow-up. The effects of adjuvant chemotherapy with uracil-tegafur and the EGFR mutation status on survival were examined. On the basis of a univariate analysis, patients with adjuvant chemotherapy (P = .073) and patients with the EGFR mutation (P = .092) tended to be correlated with a prolonged survival status compared with patients in the control group and patients with the EGFR wild-type status, respectively (5-year survival rate, 80.4% for the adjuvant group and 70.8% for the control group, and 77.9% for the EGFR mutant type group and 71.8% for the EGFR wild-type group; Fig 1). There was no significant difference in survival time between exon 19 deletion and exon 21 mutation (Appendix Fig A1A, online only). In a multivariate analysis, uracil-tegafur administration was one of the independent factors for prolonged overall survival in our cohort (hazard ratio = 0.38; 95% CI, 0.19 to 0.75; P = .005; Table 2).
Next, we examined the effect of uracil-tegafur on survival according to the EGFR mutation status. Among the EGFR wild-type patients, the univariate analysis showed that adjuvant chemotherapy was significantly related to prolonged survival (5-year survival rate, 81% for the adjuvant group and 65.4% for the control group; P = .039; Fig 2A). In the multivariate analysis, adjuvant chemotherapy with uracil-tegafur was identified as one of the independent factors for prolonged survival (hazard ratio = 0.34; 95% CI, 0.14 to 0.80; P = .013; Table 2). By contrast, among EGFR mutant patients, adjuvant chemotherapy was not associated with prolonged survival both in the univariate and multivariate analyses (5-year survival rates, 79.5% for the adjuvant group and 77.2% for the control group; Fig 2B; Table 2). In addition, a difference in survival between patients with exon 19 deletion and exon 21 mutation was not observed in the adjuvant or control groups (Appendix Figs A1B and A1C).
Treatment of Wild-Type or Mutant EGFR Transfectants With FU The expression levels of EGFR stable transfectants were examined to select transfectants with the same levels of EGFR protein expression. The types of mRNA expression in the transfectants were then confirmed. In particular, the expression of EGFR-L858R protein was confirmed in EGFR-L858R transfectants by Western blotting (data not shown). FU inhibited cell proliferation in a concentration-dependent manner in all ABC1 transfectants. The IC50 values were 9.3 ± 1.8 µmol/L for the vector control, 5.2 ± 0.6 µmol/L for the EGFR wild-type, 21.8 ± 8.6 µmol/L for the E746-A750del mutant, and 17.8 ± 4.6 µmol/L for the L858R mutant transfectants (Table 3). These results indicate that the EGFR mutant transfectant had a lower sensitivity to FU than the wild-type transfectant. The IC50 values were determined for seven nontransfected lung adenocarcinoma cell lines. In EGFR wild-type cell lines, the IC50 values were 13.6 ± 2.2 µmol/L for A549, 11.3 ± 2.4 µmol/L for H1299, and 24.5 ± 3.3 µmol/L for H1437 cell lines. In EGFR mutant cell lines, the IC50 values were 44.2 ± 4.0 µmol/L for PC-3, 24.5 ± 4.0 µmol/L for PC-9, 35.0 ± 5.6 µmol/L for H1975, and more than 1,500 µmol/L for H3255 cell lines. These data also suggest that sensitivity to FU tends to be lower in EGFR mutant than in EGFR wild-type cell lines, although a remarkable difference was not seen in all examined cell lines.
In this study, we showed that adjuvant chemotherapy with uracil-tegafur significantly prolonged survival among patients with EGFR wild-type tumors but not among patients with EGFR mutant tumors. In addition, we examined the effect of FU on lung adenocarcinoma cell lines with EGFR wild-type or mutation status to find that the sensitivity to FU was higher in EGFR wild-type versus mutant type tumors. Although the mechanism of how uracil-tegafur works differently in EGFR mutant or wild-type tumors was unknown, previous reports suggest some clues. Mutant EGFRs, such as exon 19 deletions and L858R, activate Akt and STAT signaling pathways, which protect cells against apoptosis and promote cell survival.23 NSCLC cells expressing mutant EGFRs were relatively resistant to apoptosis induced by conventional chemotherapeutic drugs, such as cisplatin and doxorubicin.23 Of note, uracil-tegafur also acts as a cytotoxic drug on cancer cells through the induction of apoptosis.24 Indeed, Tanaka et al18 reported that postoperative adjuvant therapy for NSCLC had a larger effect on the prolonged survival of patients with tumors that had a high apoptotic index compared with patients with tumors with a low apoptotic index. A similar relationship between apoptosis of tumor cells and uracil-tegafur has been reported in GI tumor xenografts.24 Taken together, these results suggest that EGFR mutant tumors have a low sensitivity to uracil-tegafur and FU because of the highly activated status of their antiapoptosis pathway. Although a survival advantage of adjuvant chemotherapy using several reagents has been demonstrated, the next goal is to select patients who will benefit from individual chemotherapeutic reagents. For this purpose, the identification of biologic predictors for each reagent is essential. The usefulness of EGFR-TKI as an adjuvant chemotherapeutic reagent for patients with EGFR mutations is being investigated.25 Our results suggest that uracil-tegafur or FU-based chemotherapy may be one option for adjuvant therapy among patients with EGFR wild-type NSCLCs. Because there is still a possibility that other chemotherapeutic reagents are also beneficial, even in patients with EGFR wild-type status, further studies, including biologic work and prospective trials, are crucial to examine the usefulness of other reagents based on EGFR status. There are two possible major limitations for this study. First, all of the clinical analyses were performed retrospectively, and the treatment plan was not clearly defined in each patient. Interestingly, our results confirm an improved survival among patients with uracil-tegafur adjuvant chemotherapy,8 suggesting that our cohort may be appropriate for the present study. Second, the difference in FU sensitivity seems to be not remarkable in some cells between EGFR wild-type and mutant cell lines or transfectants. Because the effect of uracil-tegafur is assumed not to be strong, we consider that our in vitro results would be compatible with clinical findings.8,26 Regarding the impact of uracil-tegafur on patients with EGFR mutation, the Kaplan-Meier plot suggested a small benefit of adjuvant therapy among patients with EGFR mutation. There is a possibility that our sample size may not be large enough to detect such a small difference. Further investigation is necessary to confirm our findings. In conclusion, we showed that the EGFR mutation status influences the clinical benefit of adjuvant chemotherapy with uracil-tegafur in patients with resected lung adenocarcinomas. Patients with EGFR wild-type status could be encouraged to receive adjuvant chemotherapy using FU-associated reagents. By contrast, other adjuvant treatments, such as EGFR-TKIs, should be investigated for patients with EGFR mutant tumors.
The author(s) indicated no potential conflicts of interest.
Conception and design: Shinichi Toyooka, Katsuyuki Hotta Financial support: Shinichi Toyooka Administrative support: Hiroshi Date Provision of study materials or patients: Akiko Uchida, Minoru Takata, Katsuyuki Kiura, Hiroshi Date Collection and assembly of data: Hiroshi Suehisa, Shinichi Toyooka, Junichi Soh Data analysis and interpretation: Hiroshi Suehisa, Shinichi Toyooka, Katsuyuki Hotta, Yoshiro Fujiwara, Keitaro Matsuo, Mamoru Ouchida Manuscript writing: Hiroshi Suehisa, Shinichi Toyooka, Katsuyuki Hotta Final approval of manuscript: Hiroshi Date
We thank Yukinari Isomoto, Central Research Laboratory, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, for excellent technical support.
Supported by Grant-in-Aid No. 18790993 for scientific research from the Ministry of Education, Science, Sports, Culture and Technology of Japan. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-2139, 2004 12. Paez JG, Janne PA, Lee JC, et al: EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497-1500, 2004 13. Mitsudomi T, Kosaka T, Endoh H, et al: Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J Clin Oncol 23:2513-2520, 2005 14. Shigematsu H, Lin L, Takahashi T, et al: Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 97:339-346, 2005 15. 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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test. Overall survival for resected patients was defined as the time from surgery to the time of death from any cause or to the date the patient was last known to be alive. Univariate and multivariate analyses of overall survival were carried out with the Kaplan-Meier method using the log-rank test and the Cox proportional hazards model, respectively. All data were analyzed using StatView 5.0 Program for Windows (SAS Institute Inc, Cary, NC). All statistical tests were two sided, and P < .05 was considered statistically significant. 
