Key Words. EGFR mutation Gefitinib Lung cancer EGFR TKI Complex mutation pattern

Transcription

1 The Oncologist Lung Cancer Good Response to Gefitinib in Lung Adenocarcinoma of Complex Epidermal Growth Factor Receptor (EGFR) Mutations with the Classical Mutation Pattern SHANG-GIN WU, a YIH-LEONG CHANG, b YA-CHIEH HSU, a JENN-YU WU, a CHIH-HSIN YANG, c CHONG-JEN YU, a MENG-FENG TSAI, d JIN-YUAN SHIH, a PAN-CHYR YANG a a Department of Internal Medicine, b Department of Pathology, and c Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan; d Department of Molecular Biotechnology, Da-Yeh University, Chang-Hua, Taiwan Key Words. EGFR mutation Gefitinib Lung cancer EGFR TKI Complex mutation pattern Disclosure: Employment/leadership position: None; Intellectual property rights/inventor/patent holder: None; Consultant/advisory role: Chih-Hsin Yang, AstraZeneca; Honoraria: None; Research funding/contracted research: None; Ownership interest: None; Expert testimony: None; Other: None. ABSTRACT Background. Epidermal growth factor receptor (EGFR) mutations are usually detected in lung adenocarcinoma and are associated with a response to EGFR tyrosine kinase inhibitors (TKIs). However, not all EGFR mutations have similarly high clinical response rates. This study aimed to investigate the clinical characteristics and response to gefitinib in lung adenocarcinoma patients with complex EGFR mutations. Materials and Methods. Three hundred thirty-nine specimens of lung adenocarcinoma from patients treated with gefitinib were collected for EGFR sequencing. Nineteen patients with complex EGFR mutations were enrolled for the study after excluding three patients with the EGFR T790M mutation, which confers resistance to gefitinib. Results. Among the 19 patients, 12 had complex mutations with the classical mutation pattern (L858R or deletion in exon 19). When compared with those without the classical mutation pattern, patients with this mutation pattern had a higher response rate (83% versus 29%), longer progression-free survival duration (median, 12.7 months versus 4.9 months), and longer overall survival time (median, 24.7 months versus 12.3 months) after gefitinib treatment. Comparing patients harboring complex EGFR mutations with a classical mutation pattern with those harboring single classical mutations, there were no statistical differences in the response rate (83% versus 73%), progression-free survival time (median, 12.7 months versus 8.1 months,) or overall survival time (median, 24.7 months versus 16.4 months). Conclusion. Patients with complex EGFR mutations with the classical mutation pattern had the same response rate, progression-free survival duration, and overall survival time as those with single classical mutations. EGFR TKIs may be the choice of treatment for this type of lung adenocarcinoma. The Oncologist 2008; 13: Correspondence: Jin-Yuan Shih, M.D. Ph.D., Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan. Telephone: ; Fax: ; jyshih@ntu.edu.tw Received April 14, 2008; accepted for publication November 10, 2008; first published online in THE ONCOLOGIST Express on December 5, AlphaMed Press /2008/$30.00/0 doi: /theoncologist The Oncologist 2008;13:

2 Wu, Chang, Hsu et al INTRODUCTION Epidermal growth factor receptor (EGFR) is a member of the ErbB receptor family and is a transmembrane glycoprotein encoded by a gene located in the short arm of chromosome 7 [1]. Activation of EGFR controls cell proliferation, antiapoptosis, angiogenesis, differentiation, and invasion [2], which are regulated via the phosphorylation of several tyrosine kinase residues after ligands bind to the extracellular domain of EGFR. The expression of EGFR is correlated with poor prognosis and is seen in 50% of non-small cell lung cancer (NSCLC) cases on immunohistochemistry [3]. The EGFR tyrosine kinase inhibitors (TKIs) gefitinib (Iressa ; AstraZeneca, Wilmington, DE) and erlotinib (Tarceva ; Genentech, South San Francisco, CA) are used to treat NSCLC. A higher response to EGFR TKIs is noted in specific subgroups that include females, never smokers, patients with adenocarcinoma histology, and East Asians [4, 5]. Higher EGFR mutation rates are also noted in these subgroups [6] and are also related to a better response to EGFR TKIs and longer survival [7 9]. The in-frame deletion in exon 19 (del-19) accounts for 45% of mutations, while the point mutation L858R in exon 21 accounts for around 40% 45% of EGFR mutations in lung cancer [3, 7, 8, 10 13]. Patients with both the del-19 and the L858R mutations have good responses to EGFR TKIs and these are termed classical mutations [11]. Patients with these mutations exhibit objective response rates in the range of 75% 95% [7 9, 14 16]. In addition to the classical mutations, other EGFR mutations in exons have also been reported, although quite rarely. Patients with these nonclassical mutations have variable responses to EGFR TKIs [12, 17, 18]. Most studies on the relationship between EGFR mutations and response to EGFR TKIs show a single site mutation. However, two or more concomitant sites of EGFR mutations have also been detected despite the low patient number. Chen et al. [19] showed that the frequency of double EGFR mutations is around 6% in all EGFR mutations of lung cancer by an analysis of the literature. Concomitant EGFR mutations have been termed complex mutations [20]. A second mutation may substantially alter the biological properties of the mutant EGFR and sensitivity to TKIs [21]. For example, T790M is a noteworthy acquired EGFR mutation after EGFR TKI treatment in patients with L858R or del-19, which confers resistance to EGFR TKI treatment [22, 23]. Although most studies have shown that patients with EGFR mutations have a good response to gefitinib, it remains uncertain whether patients with complex EGFR mutations have a similarly good response. This study investigated the clinical characteristics and response to gefitinib of patients with lung adenocarcinoma and complex EGFR mutations. Because T790M is a well-known gefitinib resistance mutation, we excluded those patients with T790M from this analysis. A literature review of all published reports on complex EGFR mutations and EGFR TKI responsiveness is also provided. MATERIALS AND METHODS Patients and Tissue Procurement The study group included lung adenocarcinoma patients diagnosed at the National Taiwan University Hospital between June 2004 and July Tumor specimens obtained by either surgical or needle biopsy/aspiration procedures, including primary lung tumors, malignant effusion cell blocks, and other distant metastases, were sequenced for mutational analysis. Written informed consent for use of the tissue in a molecular analysis was acquired from the patients at the procurement of the tumor specimens. This study was approved by the institutional review board of the National Taiwan University Hospital. Lung cancer histology was defined according to the World Health Organization pathology classification [24]. A complete lung cancer staging workup was performed as routine practice, which included bronchoscopy, computed tomography (CT) of the head, chest, and abdomen, and whole-body bone scintigraphy. The date of diagnosis, all chemotherapy received, and responsiveness to the chemotherapy were recorded. All patients with stage IIIb or IV NSCLC who received gefitinib were identified from the records of the Department of Pharmacy in the hospital. Their clinical data, including demographic information, cancer cell type, smoking status, and imaging studies, were collected. Patients who had smoked 100 cigarettes in their lifetime were categorized as nonsmokers. Those who had smoked within 1 year of the diagnosis were categorized as current smokers. The rest were categorized as former smokers. Response Evaluation of Lung Adenocarcinoma Patients Gefitinib was taken orally at a dose of 250 mg daily. No concurrent chemotherapy or radiotherapy for the lung tumors was given during gefitinib therapy. Chest radiography every 2 4 weeks and a chest CT scan (including the liver and adrenal glands) every 2 3 months were performed as routine clinical practice, and as needed to confirm response and disease progression. The unidimensional method was used according to the Response Evaluation Criteria in Solid Tumors (RE-

3 1278 Complex EGFR Mutations with a Classical Mutation Pattern CIST) guidelines for measuring solid tumors [25]. Complete response (CR), partial response (PR), and stable disease (SD) were confirmed by a sustained four-week follow-up. Only patients with CR and PR were regarded as responders. The progression-free survival duration was calculated from the date of initiation of gefitinib treatment to the date of disease progression, death, last follow-up, or the final follow-up day of the study. The overall survival duration after gefitinib was calculated from the date of initiation of gefitinib treatment to the date of death, last follow-up, or the final follow-up day of the study. Sequencing of EGFR Exons Tumor specimens, including frozen tissues or paraffin blocks, were collected. DNA was derived from tumors embedded in paraffin blocks using the QIAmp DNA Mini Kit (Qiagen, Valencia, CA) for EGFR mutation analysis as described previously [26]. The tyrosine kinase domain of the EGFR coding sequence, exons 18, 19, 20 and 21, was amplified by independent polymerase chain reaction (PCR) amplifications. RNA was extracted from frozen tissue with a QIAmp RNA Mini Kit (Qiagen) according to the manufacturer s protocol. Spectrophotometry was used to measure the amount of extracted RNA, according to the protocol of Mitsudomi et al. [9]. Exons of the EGFR gene were amplified from cdna with the forward primer 5 - AGCTTGTGGAGCCTCTTACACC-3 and the reverse primer 5 -TAAAATTGATTCCAATGCCATCC-3. A Qiagen OneStep reverse transcription (RT)-PCR kit (Qiagen) was used for the RT-PCR, as described previously [9]. PCR amplicons were sequenced with an ABI PRISM 3100 (Applied Biosystems, Foster City, CA) in both the sense and antisense directions. Specimens with EGFR mutations were confirmed twice. Only specimens with the same results identified in both rounds were recorded as mutation-positive. Mutations were also checked against the single nucleotide polymorphism database. The single-site EGFR mutations inframe del-19 or point mutation L858R in exon 21 were defined as classical mutations. Other mutations were infrequently detected, and were defined as nonclassical EGFR mutations. When two or more concomitant different EGFR mutations were detected, the mutation types were defined as complex EGFR mutations. The term classical mutation pattern was used when the complex EGFR mutation had either an in-frame del-19 or a point mutation L858R in exon 21. Statistical Analysis All of the categorical variables were analyzed with Fisher s exact test because of the small study size. A univariate analysis of the patient characteristics was used for the predictive factor of EGFR TKI response. The overall survival curve and progression-free survival curve were plotted using the Kaplan Meier method and compared by a log-rank test. Two-sided p-values.05 were considered significant. All analyses were performed using SPSS software (version 13.0 for Windows; SPSS Inc., Chicago, IL). RESULTS Clinical Characteristics and EGFR Mutations of Lung Adenocarcinoma Patients A total of 725 NSCLC patients received gefitinib treatment between June 2004 and October Specimens from 339 lung adenocarcinoma patients who had measurable disease and received gefitinib therapy for stage IIIb or IV lung adenocarcinoma were collected for EGFR sequencing. Five patients tissue samples were inadequate for EGFR sequencing. Finally, 334 gefitinib-treated patients (124 men and 210 women) provided EGFR mutation data. Among them, 211 patients had tumors harboring EGFR mutations (63.2%, 211 of 334). One hundred sixty-eight patients had tumors with classical mutations (83 with del-19 and 85 with L858R), and 43 patients had tumors with nonclassical EGFR mutations, including complex EGFR mutations. These patients have been reported in our previous studies [16, 17, 26 28]. In the present study, we update the follow-up data and focus on those patients who had complex mutations. Clinical Characteristics and EGFR Mutations of Lung Adenocarcinoma Patients with Complex EGFR Mutation Patterns Because patients with T790M mutations have been shown to have a high resistance to EGFR TKIs [23, 29, 30], three patients with T790M were excluded from this study. There was a total of 19 lung adenocarcinoma patients who had undergone gefitinib treatment and had complex EGFR mutations (Tables 1 and 2). The median age at which lung cancer was diagnosed was 62.8 years (range, years), with four smokers and 15 nonsmokers. The distribution of lung cancer stages was one stage IIIA patient, one stage IIIB patient, and 17 stage IV patients. The patient with stage IIIA refused surgical treatment and took systemic chemotherapy according to patient preference. Twelve patients had the classical mutation pattern of L858R or del-19. No concurrent del-19 and L858R mutations were observed.

4 Wu, Chang, Hsu et al Table 1. Clinical features and response to gefitinib of 19 lung adenocarcinoma patients with complex EGFR mutations Gefitinib n Gender Age Smoker Stage response Progression PFS (mos) Status OS (mos) Mutation 1 F 53.5 N IV PR 14.3 Dead 14.4 Del-19 E804K 2 F 65.4 N IV PR 8.1 Dead 24.7 Del-19 A871V 3 M 73.6 Current IV PR 6.0 Dead 13.9 L858R E758G 4 F 45.5 N IV PR 19.3 Alive 19.3 L858R S768I 5 M 54.6 N IV PR 16.0 Alive 31.4 L858R R776H 6 F 76.1 N IV PR 18.8 Dead 22.4 L858R G779S 7 F 64.0 N IV PR 12.7 Alive 23.1 L858R G779S 8 F 59.3 N IV PR 26.1 Alive 42.7 L858R V834L 9 F 57.5 N IIIB PR 14.8 Alive 14.8 L858R K860I 10 F 66.4 N IV PR 5.3 Dead 13.5 L858R L861F 11 M 46.6 Former IV SD 3.5 Alive 21.6 L858R H850D 12 M 50.0 N IV PD 1.6 Dead 9.9 L858R H850D 13 M 62.8 N IV PR 8.1 Dead 24.8 G719A S720F 14 M 78.3 Current IV PR 6.3 Alive 6.3 G719C S768I 15 F 66.1 N IV PD 0.6 Dead 1.8 G719A S768I 16 F 47.7 N IIIA SD 3.1 Dead 14.8 G719S L861Q 17 F 65.9 N IV SD 1.8 Dead 6.7 R776H L861Q 18 F 63.2 N IV SD 4.9 Dead 6.7 R831H L861Q 19 M 58.1 Current IV SD 4.2 Dead 12.3 K860I L861Q Abbreviations: EGFR, epidermal growth factor receptor; N, nonsmoker; OS, overall survival; PD, progressive disease; PFS, progression-free survival; PR, partial response; SD, stable disease. Response of Adenocarcinoma Patients with Complex EGFR Mutations Treated with Gefitinib Among the 19 patients with complex EGFR mutations, 13 had gefitinib as first-line treatment. Two and four patients took gefitinib as second-line and third-line treatment, respectively. The maximal responses of the previous chemotherapies were one PR, two SD, two progressive disease (PD), and one unevaluated. Among the 19 patients, 12 had the classical mutation pattern. There were no differences in gender, smoking status, age ( 65 or 65 years), lung cancer stage, and prior chemotherapy use between patients with complex EGFR mutations with the classical mutation pattern and those with complex EGFR mutations without the classical mutation pattern. Patients with the classical mutation pattern had a higher gefitinib response rate (83%, 10 of 12) than those without the classical mutation pattern (29%, 2 of 7) (p.045) (Table 2). PROGRESSION-FREE SURVIVAL AND OVERALL SURVIVAL AFTER EGFR TKI TREATMENT Patients with nonclassical EGFR mutations (n 43) had a lower gefitinib response rate (44% [19 of 43] versus 73% [122 of 168]; p.001), shorter progression-free survival time (median, 4.8 months versus 8.1 months; p.009), and shorter overall survival (median, 13.5 months versus 16.4 months; p.239) than patients with classical EGFR mutations (n 168). Among the 19 patients with complex EGFR mutations, the median progression-free survival and overall survival times after gefitinib treatment were 8.1 months (95% confidence interval [CI], months) and 22.4 months (95% CI, months), respectively. Patients with complex mutations with the classical mutation pattern had a longer progression-free survival time than those without the classical mutation pattern (median, 12.7 months; 95% CI, months versus. 4.9 months; 95% CI, months; p.048, by the log-rank test) (Fig. 1A). Patients with complex mutations with the classical mutation pattern also had a longer overall survival time after gefitinib treatment than those without the classical mutation pattern (median, 24.7 months; 95% CI, months versus 12.3 months; 95% CI, months; p.027, by the log-rank test) (Fig. 1B). Comparing patients with complex EGFR mutations with the classical mutation pattern (n 12) with those harboring single classical mutations (n 168), there were no

5 1280 Complex EGFR Mutations with a Classical Mutation Pattern Table 2. Clinical characteristics of gefitinib-treated patients with complex EGFR mutations with or without the classical mutation pattern Variable Complex EGFR mutations Classical mutation pattern, n (%) p-value a Total n (63) Gefitinib treatment Responder (83).045 Nonresponder 7 2 (29) Sex Male 7 4 (57) Female 12 8 (67) Age, years (57) (67) Smoking status Nonsmoker (67).603 Former or current 4 2 (50) smoker Stage III 2 1 (50) IV (65) Prior chemotherapy (62) (67) a Analyzed with Fisher s exact test. Abbreviation: EGFR, epidermal growth factor receptor. statistical differences in the response rate (83%, 10 of 12 versus 73%, 122 of 168; p.52), progression-free survival (median, 12.7 months versus 8.1 months; p.39), or overall survival (median, 24.7 months versus 16.4 months; p.170). DISCUSSION Prior studies on EGFR mutations in NSCLC patients have mostly shown single EGFR mutations and very few complex EGFR mutations. The responses to EGFR TKIs in these patients were not clear. Here, we demonstrate the characteristics of 19 lung adenocarcinoma patients with complex EGFR mutations and their responses to gefitinib. Patients with complex EGFR mutations with the classical mutation pattern had the same response rate, progressionfree survival time, and overall survival time as those with single classical mutations. Patients with complex EGFR mutations with the classical mutation pattern had a better response to gefitinib than those without the classical mutation pattern. Longer progression-free survival and overall Figure 1. Kaplan Meier survival curve of progression-free survival (A) and overall survival (B) after gefitinib treatment in gefitinib-treated adenocarcinoma patients with complex epidermal growth factor receptor (EGFR) mutations with the classical mutation pattern (solid line) and without the classical mutations pattern (dashed). Differences in progression-free survival and overall survival between patients with complex EGFR mutations with and without the classical mutation pattern were statistically significant (p.048 and p.027, respectively, by the log-rank test). survival times after gefitinib therapy were also noted in patients with the classical mutation pattern. The present study is the first to determine and assert that patients with complex EGFR mutations with the classical mutation pattern (excluding those with T790M) have the same response rate, progression-free survival time, and overall survival time as patients with single classical mutations (L858R or del-19) after gefitinib treatment. The response rate to gefitinib, 63% (12 of 19), in the patients with complex mutations is also similar to that in patients with

6 Wu, Chang, Hsu et al Table 3. Comparison of lung adenocarcinoma of complex EGFR mutation studies related to EGFR TKI responsiveness Responder Nonresponder Study Response Complex mutation Study Response Complex mutation Pao et al. [10] PR a L858R R776C Mitsudomi et al. [9] PD L858R E709H Zhang et al. [21] CR Del-19 L858R Hsieh et al. [20] PD W731Stop H773R PR Del-19 L858R PD L838P E868G Hsieh et al. [20] PR F712S D855G E868G Han et al. [42] SD L858R V819A PR 3 b V765M L798H K806E L814P SR Del-19 L861Q Asahina et al. [44] PD S768I V769L Ichihara et al. [45] NC L858R D761Y Han et al. [43] PR G719A S768I NC L858R G721S Taron et al. [46] PR Del-19 L861Q Jackman et al. [38] SD a Del-19 L861Q Takano et al. [32] PR L858R S768I Tokumo et al. [47] PD L858R D761Y PR G719C S768I Chou et al. [48] NPD Del-19 R803W van Zandwijk et al. [49] PR E709A G719A PD G719S L861Q Han et al. [31] PR E709K G719A Pallis et al. [50] PD T847A G863S Kosaka et al. [51] PR L858R L833V SD L858R V843I PR L858R R776H Choong et al. [52] PR L858R E884K (exon 22) Pugh et al. [53] PR Del-19 V774L PR S768I L815L Kimura et al. [54] PR L858R V689L Oshita et al. [55] PR Del-19 F856L a Treated with erlotinib. Abbreviations: CR, complete remission; EGFR, epidermal growth factor receptor; NC, no change; NPD, nonprogressive disease; PD, progressive disease; PR, partial response; SD, stable disease; SR, serum response; TKI, tyrosine kinase inhibitor. b Three patients. single EGFR mutations in other studies [31, 32]. Among the patients with single classical mutations (L858R or del-19), Takano et al. [32] and Riely et al. [33] also showed a progression-free survival duration of 12 months and a median survival time of 20 months. Their survival data were similar to those from patients with complex mutations with the classical mutation pattern in our study. Therefore, the addition of a second mutation, other than T790M, to and exon 19 deletion or L858R substitution does not alter the sensitivity to EGFR TKIs, and it is reasonable to suggest that patients with complex EGFR mutations with the classical mutation pattern can still benefit from gefitinib. Complex EGFR mutations can appear before treatment [7, 8, 13] or be induced by TKI sequentially [21 23]. For example, a novel high resistance mutation, T790M, is an acquired mutation after EGFR TKI treatment [22, 23]. However, a primary somatic mutation of T790M has also been found [30]. Complex EGFR mutations change not only the amino acid sequence but also the molecular conformation. The characteristics of the biological properties related to complex EGFR mutations have not yet been clarified. Chen et al. [34] used the H1299 cell line stably transfected with various EGFR mutant constructs. They showed that different complex EGFR mutations had different gefitinib responsiveness in engineered cell line studies, but in vitro biochemical and growth inhibition cannot be extrapolated to in vivo tumor response in patients. In a review of the English medical literature up to 2007, there was a total of 216 papers on gefitinib and EGFR mutation and 146 papers on erlotinib and EGFR mutation. We collected data on lung adenocarcinoma patients with complex EGFR mutations from these published reports. Moreover, the patients had records on their response to gefitinib or erlotinib. Consistently, the enrolled patient number was relatively small in each report (Table 3). A to-

7 1282 Complex EGFR Mutations with a Classical Mutation Pattern tal of 34 lung adenocarcinoma patients could be included from the published data, and two of the 34 were treated with erlotinib. Different responses to EGFR TKIs were noted with different complex EGFR mutations. There were 19 patients with a PR to EGFR TKIs, one with a CR, and one with a serum response (SR). There were seven patients who had PD despite EGFR TKIs. The response rate of patients harboring complex EGFR mutations with the classical mutation pattern (12 of 20, 60%) is the same as those without the classical mutation pattern (9 of 14, 64%) (Table 3). The data pooled from the literature are quite different from our study data. The discrepancy might be caused by several reasons. First, the criteria of tumor response to EGFR TKIs were different in the different studies. Seven studies adopted the RECIST [25], five studies used the World Health Organization criteria [35], and two studies used the Southwest Oncology Group standard response criteria [36]. Some authors used other response criteria, such as SR, no change (NC), or nonprogressive disease NPD). SR was defined as a 50% decrease in serum carcinoembryonic antigen levels. Some studies did not define their criteria. We classified patients with both NC and NPD as nonresponders, including the three patients harboring the classical mutation pattern. Second, of the enrolled patients from the other reference papers, two patients received erlotinib, not gefitinib. The drug effect for lung cancer may be different between erlotinib and gefitinib [37]. Only a small number of patients was reported in each of the reviewed reports. No conclusion could therefore be made from the pooled data of the literature review. Our study had the largest group of patients with complex EGFR mutations. After combining all 34 patients with complex EGFR mutations from the other studies and the present study, there was a total of 53 lung adenocarcinoma patients with complex EGFR mutations. There were some common complex mutations among the 53 patients, such as three with V765M, L798H, K806E, and L814P; three with del-19 and L861Q; two with del-19 and L858R; two with L858R and R776H; two with L858R and S768I; two with L858R and G779S; and two with G719C and S768I (Table 4). Patients with all the above complex EGFR mutations had good responses (CR, PR, or SR) to EGFR TKIs. Among the three patients with del-19 plus L861Q, two had a PR to gefitinib and one had SD with erlotinib [38]. Patients with the other common complex mutations, including two with L858R plus D761Y and two with L858R plus H850D, had SD or PD with EGFR TKIs. D761Y has been reported as a secondary resistance mutation, like T790M, to EGFR TKIs [39]. It would be interesting to verify whether patients with H850D also have a high resistance to EGFR TKIs. Two patients with G719A plus S768I had different Table 4. Lung adenocarcinoma patients with the same complex EGFR mutations and response to EGFR TKIs n of patients Mutation type 3 V765M L798H K806E L814P Response to EGFR TKI PR 3 Del-19 L861Q 1 PR, 1 SR, 1 SD a 2 Del-19 L858R 1 CR, 1 PR 2 L858R R776H PR 2 L858R S768I PR 2 L858R G779S PR 2 G719C S768I PR 2 G719A S768I 1 PR, 1 PD 2 G719S L861Q 1 PR, 1 PD 2 L858R D761Y 1 NC, 1 PD 2 L858R H850D 1 SD, 1 PD Abbreviations: CR, complete remission; EGFR, epidermal growth factor receptor; NC, no change; PD, progressive disease; PR, partial response; SD, stable disease; SR, serum response; TKI, tyrosine kinase inhibitor. a Treated with erlotinib. maximum responses to EGFR TKIs. One patient had a PR, whereas the other had PD. A similar condition was also noted in two patients with G719S plus L861Q. Although the patients with complex EGFR mutations with the classical mutation pattern had a better response to gefitinib, for those without the classical EGFR mutation pattern or with the T790M mutation, irreversible EGFR TKIs might have some effect. In preclinical cell culture studies, irreversible EGFR inhibitors can potentially provide a solution to the T790M TKI resistance problem [40]. For example, HKI-272, an irreversible dual inhibitor of both EGFR and human epidermal growth factor receptor 2, can suppress EGFR signaling in the context of an EGFR complex mutation with T790M [40, 41]. Another irreversible EGFR TKI, BIBW 2992, also shows the effect in erlotinib-resistant cell lines [42]. Clinical studies are necessary to prove the effect of irreversible EGFR TKIs for patients with complex mutations. Although our study had the largest group of patients with complex EGFR mutations, it had limitations in that the sample size was still too small to draw any firm conclusions because we were comparing two groups of 12 and seven patients. Even after adding the data from the other studies, there are still limitations in the analysis because of the different tissue sampling methods and treatment response criteria.

8 Wu, Chang, Hsu et al CONCLUSION We demonstrated a larger series of lung adenocarcinoma patients with complex EGFR mutations. Patients with complex EGFR mutations with the classical mutation pattern had a better response, longer survival time, and longer progression-free survival time after EGFR TKI therapy than those without the classical mutation pattern. EGFR TKIs may still be the best choice of treatment for these patients. ACKNOWLEDGMENTS This study was supported by a research grant from the National Science Council, Taiwan (NSC B MY3). The authors would like to thank Dr. Gene REFERENCES 1 Davies RL, Grosse VA, Kucherlapati R et al. Genetic analysis of epidermal growth factor action: Assignment of human epidermal growth factor receptor gene to chromosome 7. Proc Natl Acad Sci U S A 1980;77: Scagliotti GV, Selvaggi G, Novello S et al. The biology of epidermal growth factor receptor in lung cancer. Clin Cancer Res 2004;10:4227s 4232s. 3 Sharma SV, Bell DW, Settleman J et al. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007;7: Kris MG, Natale RB, Herbst RS et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: A randomized trial. JAMA 2003; 290: Fukuoka M, Yano S, Giaccone G et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced nonsmall-cell lung cancer (The IDEAL 1 Trial) [corrected]. J Clin Oncol 2003; 21: Amann J, Kalyankrishna S, Massion PP et al. Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer. Cancer Res 2005;65: 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 2004;350: Paez JG, Jänne PA, Lee JC et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 2004;304: 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 2005;23: Pao W, Miller V, Zakowski M et al. EGF receptor gene mutations are common in lung cancers from never smokers and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad SciUSA2004; 101: Yatabe Y, Mitsudomi T. Epidermal growth factor receptor mutations in lung cancers. Pathol Int 2007;57: Gu D, Scaringe WA, Li K et al. Database of somatic mutations in EGFR with analyses revealing indel hotspots but no smoking-associated signature. Hum Mutat 2007;28: Shigematsu H, Lin L, Takahashi T et al. Clinical and biological features Alzona Nisperos and Dr. Malcolm Higgins for editing this manuscript. The authors also thank the Department of Medical Research in the National Taiwan University Hospital and NTU Center for Genomic Medicine, National Taiwan University College of Medicine. AUTHOR CONTRIBUTIONS Conception/design: Shang-Gin Wu, Jenn-Yu Wu, Jin-Yuan Shih Administrative support: Yih-Leong Chang, Jenn-Yu Wu, Jin-Yuan Shih, Pan-Chyr Yang Provision of study materials: Yih-Leong Chang, Chi-Hsin Yang, Chong-Jen Yu, Meng-Feng Tsai, Jin-Yuan Shih Collection/assembly of data: Shang-Gin Wu, Jin-Yuan Shih Data analysis: Shang-Gin Wu, Ya-Chieh Hsu, Jin-Yuan Shih Manuscript writing: Shang-Gin Wu, Jin-Yuan Shih Final approval of manuscript: Shang-Gin Wu, Yih-Leong Chang, Ya-Chieh Hsu, Jenn-Yu Wu, Chi-Hsin Yang, Chong-Jen Yu, Meng-Feng Tsai, Jin-Yuan Shih, Pan-Chyr Yang associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97: Huang SF, Liu HP, Li LH et al. High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res 2004;10: Tokumo M, Toyooka S, Kiura K et al. The relationship between epidermal growth factor receptor mutations and clinicopathologic features in nonsmall cell lung cancers. Clin Cancer Res 2005;11: Yang CH, Yu CJ, Shih JY et al. Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy. J Clin Oncol 2008; 26: Wu JY, Wu SG, Yang CH et al. Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clin Cancer Res 2008;14: Greulich H, Chen TH, Feng W et al. Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants. PLoS Med 2005;2:e Chen Z, Feng J, Saldivar JS et al. EGFR somatic doublets in lung cancer are frequent and generally arise from a pair of driver mutations uncommonly seen as singlet mutations: One-third of doublets occur at five pairs of amino acids. Oncogene 2008;27: Hsieh MH, Fang YF, Chang WC et al. Complex mutation patterns of epidermal growth factor receptor gene associated with variable responses to gefitinib treatment in patients with non-small cell lung cancer. Lung Cancer 2006;53: Zhang GC, Lin JY, Wang Z et al. Epidermal growth factor receptor double activating mutations involving both exons 19 and 21 exist in Chinese non-small cell lung cancer patients. Clin Oncol (R Coll Radiol) 2007;19: Pao W, Miller VA, Politi KA et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005;2:e Kobayashi S, Boggon TJ, Dayaram T et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005;352: Travis WDBE, Muller-Hermelink HK, Harris CC. Pathology and Genetics of Tumors of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press, Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and

9 1284 Complex EGFR Mutations with a Classical Mutation Pattern Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000;92: Shih JY, Gow CH, Yu CJ et al. Epidermal growth factor receptor mutations in needle biopsy/aspiration samples predict response to gefitinib therapy and survival of patients with advanced nonsmall cell lung cancer. Int J Cancer 2006;118: Wu SG, Gow CH, Yu CJ et al. Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma. Eur Respir J 2008;32: Wu JY, Yu CJ, Yang CH et al. First- or second-line therapy with gefitinib produces equal survival in non-small cell lung cancer. Am J Respir Crit Care Med 2008;178: Kosaka T, Yatabe Y, Endoh H et al. Mutations of the epidermal growth factor receptor gene in lung cancer: Biological and clinical implications. Cancer Res 2004;64: Shih JY, Gow CH, Yang PC. EGFR mutation conferring primary resistance to gefitinib in non-small-cell lung cancer. N Engl J Med 2005;353: Han SW, Kim TY, Hwang PG et al. Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol 2005;23: Takano T, Ohe Y, Sakamoto H et al. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005;23: Riely GJ, Pao W, Pham D et al. Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin Cancer Res 2006;12: Chen YR, Fu YN, Lin CH et al. Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants. Oncogene 2006;25: Miller AB, Hoogstraten B, Staquet M et al. Reporting results of cancer treatment. Cancer 1981;47: Green S, Weiss GR. Southwest Oncology Group standard response criteria, endpoint definitions and toxicity criteria. Invest New Drugs 1992;10: Costa DB, Schumer ST, Tenen DG et al. Differential responses to erlotinib in epidermal growth factor receptor (EGFR)-mutated lung cancers with acquired resistance to gefitinib carrying the L747S or T790M secondary mutations. J Clin Oncol 2008;26: ; author reply Jackman DM, Yeap BY, Lindeman NI et al. Phase II clinical trial of chemotherapy-naive patients 70 years of age treated with erlotinib for advanced non-small-cell lung cancer. J Clin Oncol 2007;25: Balak MN, Gong Y, Riely GJ et al. Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors. Clin Cancer Res 2006;12: Kwak EL, Sordella R, Bell DW et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci U S A 2005;102: Godin-Heymann N, Ulkus L, Brannigan BW et al. The T790M gatekeeper mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor. Mol Cancer Ther 2008;7: Li D, Ambrogio L, Shimamura T et al. BIBW2992, an irreversible EGFR/ HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 2008;27: Han SW, Kim TY, Jeon YK et al. Optimization of patient selection for gefitinib in non-small cell lung cancer by combined analysis of epidermal growth factor receptor mutation, K-ras mutation, and Akt phosphorylation. Clin Cancer Res 2006;12: Asahina H, Yamazaki K, Kinoshita I et al. Non-responsiveness to gefitinib in a patient with lung adenocarcinoma having rare EGFR mutations S768I and V769L. Lung Cancer 2006;54: Ichihara S, Toyooka S, Fujiwara Y et al. The impact of epidermal growth factor receptor gene status on gefitinib-treated Japanese patients with nonsmall-cell lung cancer. Int J Cancer 2007;120: Taron M, Ichinose Y, Rosell R et al. Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas. Clin Cancer Res 2005;11: Tokumo M, Toyooka S, Ichihara S et al. Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer. Lung Cancer 2006;53: Chou TY, Chiu CH, Li LH et al. Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer. Clin Cancer Res 2005;11: van Zandwijk N, Mathy A, Boerrigter L et al. EGFR and KRAS mutations as criteria for treatment with tyrosine kinase inhibitors: Retro- and prospective observations in non-small-cell lung cancer. Ann Oncol 2007;18: Pallis AG, Voutsina A, Kalikaki A et al. Classical but not other mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with non-small cell lung cancer. Br J Cancer 2007;97: Kosaka T, Yatabe Y, Endoh H et al. Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res 2006;12: Choong NW, Dietrich S, Seiwert TY et al. Gefitinib response of erlotinibrefractory lung cancer involving meninges role of EGFR mutation. Nat Clin Pract Oncol 2006;3:50 57; quiz 51 p following Pugh TJ, Bebb G, Barclay L et al. Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefitinib response in a retrospective analysis of lung cancer patients. BMC Cancer 2007;7: Kimura H, Suminoe M, Kasahara K et al. Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA). Br J Cancer 2007;97: Oshita F, Matsukuma S, Yoshihara M et al. Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer. Br J Cancer 2006;95: