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Originally published as JCO Early Release 10.1200/JCO.2005.00.992 on February 28 2005 © 2005 American Society of Clinical Oncology. 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
From the Departments of Thoracic Surgery, Thoracic Oncology, and Pathology and Molecular Diagnostics, Aichi Cancer Center Hospital; and Division of Molecular Oncology, Aichi Cancer Center Research Institute, Aichi Cancer Center, Nagoya, Japan Address reprint requests to Tetsuya Mitsudomi, MD, Department of Thoracic Surgery, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan; e-mail: mitsudom{at}aichi-cc.jp.
PURPOSE: To evaluate the relationship between mutations of the epidermal growth factor receptor (EGFR) gene and the effectiveness of gefitinib treatment in patients with recurrent lung cancer after pulmonary resection. PATIENTS AND METHODS: We sequenced exons 18-21 of the EGFR gene using total RNA extracted from 59 patients with lung cancer who were treated with gefitinib for recurrent lung cancer. Gefitinib effectiveness was evaluated by both imaging studies and change in serum carcinoembryonic antigen (CEA) levels. RESULTS: EGFR mutations were found in 33 patients (56%). Of these mutations, 17 were deletions around codons 746-750 and 15 were point mutations (12 at codon 858, three at other codons), and one was an insertion. EGFR mutations were significantly more prevalent in females, adenocarcinoma, and never-smokers. Gefitinib treatment resulted in tumor shrinkage and/or CEA decrease to less than half of the baseline level in 26 patients, tumor growth and/or CEA elevation in 24 patients, and gefitinib effect was not assessable in nine patients. Female, never-smoking patients with adenocarcinoma tended to respond better to gefitinib treatment. Gefitinib was effective in 24 of 29 patients with EGFR mutations, compared with two of 21 patients without mutations (P < .0001). Of note, del746-750 might be superior to L858R mutations for prediction of gefitinib response. Patients with EGFR mutations survived for a longer period than those without the mutations after initiation of gefitinib treatment (P = .0053). CONCLUSION: EGFR mutations were a good predictor of clinical benefit of gefitinib in this setting.
Lung cancer has long been the leading cause of cancer death in North America. In 1998, it became the leading cause of cancer death in Japan, and now claims more than 55,000 lives annually.1 Lung cancer is divided into two morphologic types: small-cell lung cancer and non–small-cell lung cancer (NSCLC). NSCLCs are further subdivided into adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma. Adenocarcinoma is the predominant histologic subtype, and is increasing among patients with lung cancer who are candidates for surgical treatment in Japan. In our institution, adenocarcinoma accounted for 76% of 407 patients who were operated on from 2001 through 2003. Adenocarcinomas are characterized by a high degree of morphologic heterogeneity. Analyses of various cancer-associated genes, including K-ras,2 p53,3,4 cyclin D1,5 p27Kip1,6 and cyclooxygenase-2,7 suggests a different molecular pathway for carcinogenesis in lung adenocarcinomas at least partly accounts for this heterogeneity. In addition, the NSCLC frequently overexpresses receptors of the ErbB family, including the epidermal growth factor receptor (EGFR) encoded by ErbB1 (HER-1).8,9 EGFR is a 170 kd receptor tyrosine kinases (TK) that dimerizes and phosphorylates several tyrosine residues upon binding of several specific ligands including epidermal growth factor and transforming growth factor alpha.8 These phosphorylated tyrosines serve as the binding sites for several signal transducers that initiate multiple signaling pathways resulting in cell proliferation, migration and metastasis, evasion from apoptosis, or angiogenesis, all of which are associated with cancer phenotypes.8 Downstream pathways include ras-raf-MEK-ERK, phosphatidylinositol-3 kinase-Akt, and PAK-JNKK-JNK.8 Gefitinib is an orally bioavailable small molecule that specifically inhibits EGFR tyrosine phosphorylation.10 Clinical trials revealed that there is significant variability in response to gefitinib. Good clinical responses have been observed most frequently in women, in nonsmokers, in patients with adenocarcinomas, and in Japanese patients.11,12 However, it was not possible to predict gefitinib sensitivity by levels of EGFR overexpression as determined by immunohistochemistry13 or immunoblotting.14 The factors that determine gefitinib sensitivity have long been an enigma. Recently, it has been reported that activating mutations of EGFR are present in a subset of pulmonary adenocarcinomas and that tumors with EGFR mutations are highly sensitive to gefitinib15-17 or erlotinib, another EGFR TK inhibitor. Furthermore, the incidence of EGFR mutations is significantly higher in female, never-smoking, Japanese patients with adenocarcinoma.15 These features coincide with those of good responders to gefitinib. In this study, we studied patients who had recurrent disease after pulmonary resection for NSCLC and who were subsequently treated with gefitinib. We searched for mutations of the EGFR gene in tumor specimens taken at the time of surgery and we correlated EGFR mutations with gefitinib effectiveness, including tumor response and patient survival.
Patients Seventy-five patients were treated with gefitinib for their recurrent diseases after they had undergone surgery between 1999 and 2003. We studied 59 patients whose tumors were available for RNA extraction, which was a sole determinant of inclusion into the present study. There were 32 men and 27 women with ages ranging from 48 to 79 years. Fifty patients had adenocarcinomas, five had squamous cell carcinomas, three had large-cell carcinomas, and one had adenosquamous carcinoma. Eight patients had stage IA disease; seven stage IB; three stage IIA; five stage IIB; 24 stage IIIA; eight stage IIIB; and three stage IV at the time of surgery. Lobectomy had been performed in 57, and pneumonectomy and partial resection in one patient each. Four patients received postoperative adjuvant chemotherapy (two with oral uracil/tegafur and two with gemcitabine monotherapy). Forty patients had had chemotherapy before gefitinib treatment (23 patients, platinum doublet; 16 patients, monotherapy with vinorelbine or gemcitabine, one patient, oral uracil/tegafur). Gefitinib treatment with a daily dose of 250 mg was initiated between July 2002 and May 2004, with the median interval between operation and gefitinib treatment being 778 days (range, 107 to 1,931 days). Fifty patients had distant metastatic tumors, eight patients had pleural dissemination and malignant effusion, and one patient had hilar lymph-node metastasis at initiation of gefitinib treatment.
Molecular Analysis of Lung Cancer Specimens The first four exons (exons 18-21) of the seven exons (exons 18-24) that code for TK domain of the EGFR gene (which includes all the mutations reported so far15-17) was amplified with primers F1 (5'-AGCTTGTGGAGCCTCTTACACC-3') and R1 (5'-TAAAATTGATTCCAATGCCATCC-3') in a one-step reverse transcription polymerase chain reaction (RT-PCR) using the QIAGEN OneStep RT-PCR Kit (Qiagen). The cDNA sequence of the EGFR gene was obtained from GenBank (accession number NM 005228). The RT-PCR conditions were: one cycle of 50°C for 30 minutes, 95°C for 15 minutes, 40 cycles of 94°C for 50 seconds, 62°C for 50 seconds, and 72°C for 60 seconds, followed by one cycle of 72°C for 10 minutes. RT-PCR products were diluted and cycle-sequenced using the Big Dye Terminator v3.1/1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Sequencing products were electrophoresed on an ABI PRISM 3100 (Applied Biosystems). Both the forward and reverse sequences obtained were analyzed by BLAST (basic local alignment search tool) and chromatograms by manual review. High-quality sequence variations found in both directions were scored as candidate mutations.
Definition of Effectiveness of Gefitinib As patients with recurrent lung cancer often do not have measurable disease, we also included change in serum carcinoembryonic antigen (CEA) level (cut off, 5 ng/mL) as an evaluation criterion to avoid underestimating gefitinib effectiveness. CEA has been reported as a useful clinical therapeutic marker.19 When the elevated CEA level decreased to a level less than half of the baseline level, gefitinib treatment was judged as effective. On the other hand, gefitinib treatment was judged as ineffective when the tumors showed any growth or a new lesion appeared in the imaging studies, or when the serum CEA level increased. Any patient who did not fit either of these criteria was classified as not assessable. All these evaluations were done before the EGFR gene analysis, without knowledge of mutational status of the EGFR gene.
Statistical Analysis
EGFR Mutations Mutations of the EGFR gene were detected in 33 (56%) of 59 patients. Seventeen were deletions, 15 were point mutations, and one was an insertion. Details of these mutations are shown in Figure 1. As previously reported,15-17 EGFR mutations were significantly associated with adenocarcinoma histology, female sex, and never-smoking status (Table 1). However, the mutations were not associated with the age or stage of the patients. Furthermore, median time from the original surgery to recurrence was almost identical in patients with EGFR mutations (362 days) and in those without EGFR mutations (363 days; P = .8265).
Clinical Improvement After Gefitinib Treatment Forty-one of 59 patients had measurable disease at recurrence with imaging studies. Of these, 20 showed appreciable tumor shrinkage after gefitinib treatment, whereas 17 tumors increased in size, and there was no change in tumor size in four patients. All of these 20 tumors (pulmonary metastases in 11, pleural disseminated nodules in two, hepatic metastases in two, mediastinal lymph node swelling in two, brain metastases in two, and chest wall tumor in one) showed at least a 30% decrease in diameter. Figure 2 shows representative imaging studies. A computed tomography scan of the chest in patient L703 (73-year-old woman, adenocarcinoma) showed masses in the right-lower lobe and marked improvement 8 weeks after gefitinib initiation. A computed tomography scan of the liver in patient L1492 (52-year-old woman, adenocarcinoma) showed masses in the right lobe of the liver and dramatic improvement 10 days after gefitinib initiation. A large chest-wall mass in the left back of patient L1362 (62-year-old man, adenosquamous carcinoma) before gefitinib treatment almost disappeared 13 weeks after gefitinib initiation. A left-lung tumor in patient L1171 (70-year-old woman, adenocarcinoma) was smaller 6 weeks after gefitinib initiation.
CEA was above the upper normal limit (5 ng/mL) at baseline in 32 patients. Serum CEA level decreased to < 10%, < 50%, and to > 50% of the baseline level in three, 12, and five patients, respectively, whereas CEA level increased in 12 patients. When we combined the results of imaging studies with CEA and judged according to our criteria, gefitinib treatment was effective in 26 (52%), not effective in 24 (48%), and not assessable in nine patients (Table 2). There was a good correlation between these two examinations. The imaging studies and change in CEA levels did not conflict in any patients. In 17 patients with measurable diseases and whose baseline CEA level was elevated, the CEA level decreased in all 11 patients showing tumor shrinkage and increased in all five patients showing tumor growth, except for one patient whose tumors showed no change in size (P < .001, Fisher's exact test), supporting the validity of our criteria.
We searched for a relation between gefitinib effectiveness and various clinical and pathologic features (Table 2). Never-smokers and patients with adenocarcinoma had a significantly higher incidence of gefitinib effect. However, we could not detect significant difference in gefitinib sensitivity by sex or presence of prior chemotherapy, probably because of the small sample size, although there was a trend that female and chemotherapy-naïve patients were more responsive.
Relationship Between Clinical Response to Gefitinib Treatment and EGFR Mutations
When we used our criteria combining the results of imaging studies with CEA, gefitinib was effective in 24 (83%) of 29 patients with EGFR mutations, whereas it was effective only in two (10%) of 21 patients without EGFR mutations (P < .0001; Table 2). There were three patients with EGFR mutations (two with L858R and one with G719A) whose CEA level increased after gefitinib treatment but did not have measurable diseases. There were also two patients with EGFR mutations, one with L858R+E709H and one with I744-K745 ins KIPVAI whose tumor progressed. Logistic regression analysis (Table 4) showed that EGFR mutation was the only significant factor contributing to gefitinib sensitivity.
On the other hand, patient L1171, who showed a decrease in size of multiple pulmonary metastatic nodules (Figs 2G and H) and a decrease in CEA level from 16.8 to 4.3 ng/mL, did not have EGFR mutations. In this patient, we extended our search for mutations to exons 22 and 23 of the EGFR gene, and still found none. Another patient without EGFR mutation in whom gefitinib was effective was a 59-year-old man who showed a decrease in serum CEA level from 10.6 to 1.5 ng/mL after 2 weeks of gefitinib treatment; this low level of CEA was maintained at least for 7 months. When we further analyzed gefitinib response by classes of EGFR mutation, we found that there was a difference of response between patients with deletion mutations and those with the other types of mutations. Gefitinib was effective in all 16 patients with deletions, and effective in eight of 13 with other types of mutation (P = .0108).
Effect of EGFR Mutation on Patient Survival After Gefitinib Treatment
Recurrence after complete resection of NSCLC often presents as a form of distant metastases.20 In clinical practice, chemotherapy is given to these patients except for a small number in whom re-resection of the tumor is indicated. Many studies have shown that chemotherapy prolongs survival and improves quality of life in unresectable stage IV tumors.21 However, patients with unresectable tumors and patients with recurrent diseases may not be the same. There have been no large-scale randomized clinical trials addressing whether chemotherapy improves survival of patients with recurrence. Yoshino et al22 found that chemotherapy for recurrence only tended to prolong survival in 118 of 468 consecutive patients who had recurrence after pulmonary resections. After introduction of gefitinib to clinical practice in 2002 in Japan, some patients with recurrent disease showed dramatic responses to gefitinib treatment, but many others did not respond. It has been unclear which patients respond to gefitinib and also whether gefitinib treatment prolongs survival in these patients. Recent studies have showed striking correlation between gefitinib sensitivity and EGFR mutations both in vitro and in clinical studies.15-17 Because this study was a retrospective analysis of response to gefitinib prescribed as routine care, judgment of gefitinib effectiveness tended to be less strict than that in a prospective clinical trial. Yet, changes in serum CEA level never conflicted with imaging studies. We were able to confirm a relation between EGFR mutations and gefitinib sensitivity in a slightly different clinical setting. We correlated EGFR mutations found in specimens taken at the time of surgery with response to gefitinib, often after several courses of cytotoxic chemotherapy for recurrent disease. Multivariate analysis revealed that EGFR mutation was the only independent predictor for gefitinib response among several allegedly contributing factors. As in previous studies, EGFR mutation was not a perfect predictor of gefitinib effectiveness.15-17 Two patients without EGFR mutations showed response to gefitinib. It is not clear at this time whether EGFR mutations are present in other parts of the gene or whether mechanisms other than EGFR mutations govern sensitivity in these patients. We found a significant difference in gefitinib sensitivity according to classes of EGFR mutations. All 16 patients with deletion mutants responded to gefitinib, compared with eight of 12 patients with other mutations (P = .0108). It is not clear whether this difference is based on differences in biologic activity of these mutant proteins. Gefitinib sensitivity was essentially the same in COS cells transfected with L858R and in cells transfected with del L747-P753insS.16 A more recent study showed that the tyrosine residue at codon 845 is highly phosphorylated in L858R mutants, but not in deletion mutants after epidermal growth factor binding.23 This might explain the difference in gefitinib response between tumors with L858R and those with deletions. Although our criteria for tumor response are soft, these are merely a surrogate marker for the effect on survival. We were able to show, for the first time, that EGFR mutation was the only significant and independent predictor for a prolonged survival after gefitinib treatment. In a previous study, we showed that EGFR mutation itself is not a predictor for better postoperative survival in 236 unselected patients with adenocarcinoma,24 and in the present study, median disease-free interval was almost identical in patients with or without EGFR mutations. A recent placebo-controlled clinical trial showed that treatment with erlotinib, another oral EGFR TK inhibitor, significantly prolongs survival after first and second chemotherapy for NSCLC,25 although EGFR mutation frequency is reported to be around 10% in Western countries.15-17 This result is interpreted to mean that a subset of patients without mutations have also benefited from erlotinib therapy. The present study suggests that if patients were selected by presence of EGFR mutations, it would be possible to concentrate patients with benefits from gefitinib treatment, avoiding unnecessary adverse reactions such as fatal interstitial lung disease, which is relatively common in Japanese patients.26 Furthermore, our results provide a basis for postoperative adjuvant gefitinib treatment in NSCLC patients with EGFR mutations, as adjuvant treatment is considered the earliest treatment of metastatic disease. These possibilities should be tested in future clinical trials. It is common for patients to show progressive disease soon after presenting an initial striking response to gefitinib. However, we could not detect any evidence that differences in classes of EGFR mutations are associated with duration of response (data not shown). In conclusion, tumors with EGFR mutations showed good, but not perfect, correlation with clinical response in patients with postoperative recurrence of NSCLC. Furthermore, patients with EGFR mutations survived for a significantly longer period than those without EGFR mutations. Future clinical trials using gefitinib should examine EGFR mutations for effective selection of patients who are most likely to benefit from this molecular-targeted drug.
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Honoraria: Tetsuya Mitsudomi, AstraZeneca Japan, Bristol-Myers Squibb Japan, TAIHO Pharmaceutical. For a detailed description of this category, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
We thank Kaori Hayashi-Hirano for excellent technical assistance in molecular analysis of tumors, and Ryuzo Ohno, President of Aichi Cancer Center, for special encouragement and support.
Supported in part by Grant-in-Aid (16591424) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
2 test or Fisher's exact test was used. The Kaplan-Meier method was used to estimate the probability of survival as a function of time, and survival differences were analyzed by the log-rank test. The two-sided significance level was set at P < .05. To identify which independent factors had a joint significant influence on gefitinib effectiveness, the logistic regression modeling technique was used, and for multivariate analysis of the overall survival, the Cox proportional hazards modeling technique was applied. All analyses were performed using StatView version 5 (SAS institute Inc, Cary, NC) software on a Macintosh computer. 
