Lung cancer is ranked as the most common cause of cancer

BRIEF REPORT Compound EGFR Mutations and Response to EGFR Tyrosine Kinase Inhibitors Susumu Kobayashi, MD, PhD, Hannah M. Canepa, BA, Alexandra S. Bailey, MD, Sohei Nakayama, MD, PhD, Norihiro Yamaguchi, MD, MPH, Michael A. Goldstein, MD, Mark S. Huberman, MD, and Daniel B. Costa, MD, PhD Background: Non small-cell lung cancers (NSCLCs) containing EGFR mutations are exquisitely sensitive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). This is the case of the most common EGFR mutations affecting exon 18 (G719X), 19 (inframe deletions), and 21 (L858R and L861Q). However, the frequency of compound (i.e., double or complex) EGFR mutations where an EGFR TKI sensitizing or other mutation is identified together with a mutation of unknown clinical significance and their pattern of response/resistance to EGFR TKIs are less well described. Methods: We analyzed the EGFR mutation pattern of 79 cases of NSCLC harboring EGFR mutations and compiled the genotyperesponse data for patients with NSCLCs with compound EGFR mutations treated with EGFR TKIs. Results: Of the 79 EGFR-mutated tumors identified, 11 (14%) had compound mutations. Most involved the EGFR TKI sensitizing G719X (n = 3, plus S768I or E709A), L858R (n = 4, plus L747V, R776H, T790M, or A871G), L861Q (n = 1, plus E709V), and dell747_ T751 (n = 1, plus R776H). Eight patients received an EGFR TKI: three cases with G719X plus another mutation had partial responses (PRs) to erlotinib; of three cases with L858R plus another mutation, two displayed PRs and one (with EGFR-L858R+A871G) progressive disease (PD) to erlotinib; one NSCLC with EGFR-L861Q+E709A and one with dell747_t751+r776s had PRs to EGFR TKIs. Conclusion: Compound EGFR mutations comprised 14% of all mutations identified during routine sequencing of exons 18 21 of EGFR in our cohort. Most patients with an EGFR TKI sensitizing mutation (G719X, exon 19 deletion, L858R, and L861Q) in addition to an atypical mutation responded to EGFR TKIs. Reporting of the genotype-response pattern of NSCLCs with EGFR compound and other rare mutations, and the addition of this information to searchable databases, will be helpful to select the appropriate therapy for EGFR-mutated NSCLC. *Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. Disclosure: Dr. Costa received consulting fees from Pfizer, Roche, and AstraZeneca. The authors declare no other conflict of interest Address for correspondence: Daniel B. Costa, MD, PhD, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. E-mail: dbcosta@bidmc.harvard.edu Copyright 2012 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/12/0801-118 Key Words: Lung cancer, Non small-cell lung cancer, Epidermal growth factor receptor, EGFR mutation, Erlotinib, Gefitinib, Tyrosine kinase inhibitor, L858R, L861Q, G719X, Exon 19 deletion compound, Exon 19 deletion double, Exon 19 deletion complex. (J Thorac Oncol. 2013;8: 118 122) Lung cancer is ranked as the most common cause of cancer death in the United States and many developed countries. Non small-cell lung cancers (NSCLCs) are the most prevalent form of this disease, and the understanding of its genetic diversity has led to the potential for molecularly targeted interventions. In particular, mutations in the EGFR gene and translocations involving the ALK or the ROS1 genes play a pivotal role in carcinogenesis of some lung cancers. Alterations in these actionable kinases predict the clinical effectiveness of tyrosine kinase inhibitors (TKIs) that target these aberrant oncogenes. 1 There are a multitude of somatic mutations in the EGFR that have been described in NSCLC samples, and most cluster within the tyrosine kinase domain of this receptor tyrosine kinase. The most frequent and clinically significant mutations include inframe deletions involving amino acids LREA of exon 19 and the exon 21 L858R. Together, these classic mutations account for almost 85% of all EGFR mutations. Gefitinib and erlotinib, oral epidermal growth factor receptor (EGFR) TKIs, have been extensively studied in clinical trials that included NSCLCs with classic EGFR mutations, and both drugs lead to superior response rates (RRs) and progression-free survivals (PFSs) when compared with standard platinum-doublet cytotoxic chemotherapies. 1,2 RRs to gefitinib/erlotinib exceed 60% to 70% in these trials, with median PFSs of more than 9 to 10 months and overall survival times beyond 20 months. 1 Other EGFR mutations have also been associated with enhanced effects of EGFR TKIs. These include the less-prevalent exon 18 G719X mutations (~3% of reported EGFR mutations) and the exon 21 L861Q (~2% of all EGFR mutations). In aggregate, G719X and L861Q mutated NSCLCs have been described to have RRs that exceed 50% and PFSs of more than 5 months in gefitinib/erlotinib-treated patients. 3 On the contrary, other classes of EGFR mutations can be associated with lack of response to gefitinib or erlotinib. This is the case of the most prevalent EGFR exon 20 inframe insertion mutations (~5% of EGFR mutations) following the regulatory C-helix of EGFR. 4 118 Journal of Thoracic Oncology Volume 8, Number 1, January 2013

Journal of Thoracic Oncology Volume 8, Number 1, January 2013 Compound EGFR mutations Other EGFR mutations and tumors with multiple EGFR mutations have not been completely characterized. This is the case of compound EGFR mutations where an EGFR TKI sensitizing mutation (such as G719X, exon 19 deletions, L858R or L861Q) coexists with uncommon mutations involving other residues of the tyrosine kinase domain of EGFR. In this study, we report the frequency of and responses to EGFR TKIs of our center s cohort of compound EGFR-mutated NSCLCs and provide a review of the literature on the pattern of response to EGFR TKIs of these mutation types. The date presented here will enhance efforts, such as Vanderbilt University s DNA-mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT) database (http://www.mycancergenome.org), to compile a searchable database for oncologists treating patients genotyped for EGFR mutations and other genetic alterations. 5 PATIENTS AND METHODS Patient Selection Patients with a diagnosis of NSCLC and whose tumors were genotyped for EGFR mutations up to August 1, 2012, were identified through an ongoing Institutional Review Board approved protocol at Beth Israel Deaconess Medical Center (BIDMC2009-P-000182). Tumor Genotype EGFR mutation analysis was performed using standard DNA sequencing techniques with direct sequencing of exons 18 to 21 of EGFR. In brief, DNA was isolated from the sample, quantified and amplified by polymerase chain reaction (PCR) using primers to exons 18 to 21 of EGFR. 6 PCR products were analyzed by bidirectional direct DNA sequencing. Tumor genotype was performed in baseline diagnostic specimens before patient exposure to EGFR TKIs. Data Collection We reviewed the BIDMC electronic medical records and radiographic studies to obtain clinical and pathologic information such as EGFR mutation status and response to EGFR TKIs. Response was calculated using Response Evaluation Criteria In Solid Tumors version 1.1. Study data were collected and managed using REDCap electronic data capture tools hosted at BIDMC. Statistical Methods The reporting of parameters involving clinical, pathologic, radiographic, response data, and tumor genotypes used descriptive methods. RESULTS Frequency of EGFR Mutations Table 1 summarizes the frequency of EGFR mutations identified during routine clinical genotype of our patients tumor specimens. Most mutations were single mutations (67 of 79, 84.75%) with exon 19 deletions (34 of 79, 43%) and L858R (24 of 79, 30.5%) being the most prevalent mutation types. EGFR exon 20 insertion mutations (6.3%), plus single mutations involving G719X (1.25%) and L861Q (2.5%) were less frequent. Of the 79 unique tumors, 11 (14%) had compound mutations (Table 1). EGFR Compound Mutations Most (9 of 11, 81.8%) EGFR compound mutations involved the EGFR TKI sensitizing G719X (n = 3), L858R (n = 4), L861Q (n =1), and dell747_t751 (n = 1) mutations (Table 1). The partner mutation identified during sequencing was either an exon 18 atypical mutation (E709X, n = 2), an exon 19 atypical mutation (L747V, n = 1), an exon 20 atypical mutation (S7681, n = 2; R776S, n = 2; T790M, n = 1), or an exon 21 atypical mutation (A871G, n = 1). Table 1 summarizes how these combinations paired in each specific tumor. TABLE 1. Frequency of Different EGFR Mutations Detected in NSCLC Samples by Direct Sequencing of Exons 18 21 of EGFR EGFR Mutation Type No. % of Total Single mutations 67 84.75 exon 18 mutations 2 2.5 E709_T710delETinsD 1 1.25 G719A 1 1.25 exon 19 deletions 34 43 dele746_a750 26 33 dele746_s752insv 2 2.5 dell747_a750insp 1 1.25 dell747_t751 3 3.75 dell747_s752 1 1.25 dell747_a753insq 1 1.25 exon 20 insertions 5 6.30 A763_Y764insFQEA 1 1.25 D770delDinsGY 1 1.25 P772_H773dupH 2 2.5 P772_H773dupPH 1 1.25 exon 21 mutations 26 33 L858R 24 30.5 L861Q 2 2.5 Compound mutations 11 14 G719A+S768I 2 2.5 G719S+E709A 1 1.25 dell747_t751+r776s 1 1.25 S768I+V769L 1 1.25 H773L+V774M 1 1.25 L858R+L747V 1 1.25 L858R+R776H 1 1.25 L858R+T790M 1 1.25 L858R+A871G 1 1.25 L861Q+E709V 1 1.25 Other mutation 1 1.25 G724fs 1 1.25 Total 79 100 Copyright 2012 by the International Association for the Study of Lung Cancer 119

Koboyashi et al. Journal of Thoracic Oncology Volume 8, Number 1, January 2013 Response to EGFR TKIs of EGFR-Mutated NSCLCs Of the 11 patients with tumor genotype revealing a compound EGFR mutation, eight received an EGFR TKI during their clinical course. Table 2 summarizes seven patients who only received erlotinib. Of the three cases with G719X plus another mutation, all had PR to erlotinib (at concentrations of 100 150 mg/day). However, in none did the PFS exceed 8 months, and in none did the survival exceed 12 months. Out of the three cases with L858R plus another mutation, the patients whose tumors had L858R+L747V and L858R+R776H displayed PRs that are ongoing (Table 2). The patient (who had a poor performance status at diagnosis) whose tumor had L858R+A871G had rapid PD and died within 3 months of starting erlotinib 150 mg per day (Table 2). One patient with an exon 19 deletion (dell747_ T751)+R776S mutated NSCLC, who had her initial clinical course detailed previously, 7 had a prolonged response to a low dose of erlotinib with a PFS of 20 months (Table 2). The final patient (a 68-year-old white, never-smoker, with Eastern Cooperative Oncology Group performance status of 0 and stage IV adenocarcinoma) with an NSCLC harboring L861Q+E709A had a prolonged PR (decreased in 48% of target lesions) to an experimental irreversible EGFR TKI (as firstline systemic therapy) that lasted for 25 months, followed by an additional 7 months of erlotinib at a dose of 150 mg per every other day until further progression at month 32 of EGFR TKIs. DISCUSSION EGFR mutations are considered the most robust predictive biomarker for the clinical and radiographic responses attained by EGFR TKIs in clinical practice. Single EGFR mutations or multiple mutations can be identified during sequencing of exon 18 to 21 of EGFR. The clinical prevalence and significance of multiple mutations have not been completely ascertained. One of the largest cohorts of EGFR-mutated NSCLCs disclosed that approximately 7% of the tumors harbored compound mutations involving the EGFR TKI sensitizing mutations G719X, exon 19 deletions, L858R, and L861Q in combination with other EGFR mutations in Taiwanese patients. 3 Others have reported frequencies lower than 4% for these mutations. 8 The method of EGFR mutation detection (direct sequencing of exon 18 21 [as used in this report], mutant-enriched PCR, peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR, PCR clamp, allelespecific reactions, high-resolution melting analysis, Applied Biosystems Prism SNaPshot Multiplex system, among others) and the potential introduction of artifacts generated in the PCR step may account for the unequal prevalence of compound mutations in prior series. 5,9 The cohort reported here indicated a higher prevalence of EGFR compound mutations in 14% of our 79 cases. Therefore, it seems that the frequency of EGFR compound mutations is not insignificant. The genotype-clinical/radiographic response pattern of EGFR compound mutations has not been completely defined and neither has there been a concerted effort to determine whether these compound mutations occur in cis or in trans within EGFR alleles. We will summarize the available literature below. NSCLCs with G719X mutations in addition to other mutations are commonly reported. As an example, in the aforementioned Taiwanese cohort, eight of the 15 NSCLCs with G719X harbored compound G719X plus other mutations and the RR for G719X mutations (single + compound) was 53.3% with a PFS of 8.1 months. 3 In our data set, three fourths (75%) G719X mutations were compound. The EGFR-G719A+S768I mutation, which we identified in two patients with PR to erlotinib, has been reported by others in two NSCLCs that had lack of responses to gefitinib, 10,11 and EGFR-S768I alone has been reported in a gefitinib-responsive NSCLC. 12 The in vitro characterization of G719S+S768I showed that this EGFR is as sensitive to gefitinib as G719S alone. 13 An NSCLC with G719S+E709A, which we identified in one patient with PR to erlotinib, has been previously reported as having a PR to the irreversible EGFR TKI neratinib. 14 The in vitro characterization of G719C+E709A demonstrated that the resulting EGFR protein is only slightly less sensitive to gefitinib than G719C alone, 15 which may not be significant at clinical doses used for erlotinib (at 150 mg/day, the serum trough exceeds 1.5μM) or irreversible EGFR TKIs. 16 Exon 19 deletions are less frequently identified as compound mutations, outside the setting of acquired resistance to gefitinib/erlotinib where they are frequently found with EGFR-T790M that shifts the ensuing proteins into resistant patterns to clinically achievable doses of gefitinib and erlotinib. 17 It seems other compound exon 19 mutations, such as the erlotinib responsive EGFR-delL747_T751+R776H reported by our group, 7 retain their responsiveness to EGFR TKIs. L858R mutations are frequently reported as a part of compound mutations, with approximately 10% of L858R mutated NSCLC in association with other mutations. 3 We identified five of 29 (17.25%) of our EGFR-L858R as compound mutations. How these compound mutations affect the response to gefitinib and erlotinib of L858R alone is not completely known. Our group has characterized in vitro EGFR- L858R+L747S and shown that this compound mutation shifts the sensitivity curve to EGFR TKIs in a manner that would make it resistant to trough serum concentrations of a dose of gefitinib 250 mg/day (<0.5μM) but not to serum troughs (>0.5 1μM) of erlotinib at doses above 50 to 100 mg per day. 18 The same seems true for L858R+D761Y. 19 We and others have also shown that EGFR-L858R+T790M is resistant to gefitinib and erlotinib at their maximum tolerated doses. 16,18 The in vitro characterization of L858R+E709A, L858R+E709G, and L858R+L838V showed that these EGFRs are as sensitive to gefitinib as L858R alone. 13 In this report, we show that NSCLCs with L858R+L747V and L858R+R776H had PRs to erlotinib. A prior NSCLC with L858R+R776H has also been described as having PR to gefitinib. 11 The case of EGFR-L858R+A871G NSCLC reported here as having PD to erlotinib 150 mg per day may indicate that this compound mutation may confer resistance to EGFR TKIs; an assertion that will require in vitro modeling. EGFR-L858R+H870R in vitro is less sensitive to gefitinib than L858R alone. 15 L861Q mutations can be part of compound mutations in more than half of EGFR-L861Q positive cases identified. We observed one third (33.3%) of cases of L861Q as 120 Copyright 2012 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 8, Number 1, January 2013 Compound EGFR mutations TABLE 2. Clinical, Pathologic, Molecular Characteristics, and Response to Erlotinib of NSCLCs with Compound EGFR Mutations EGFR Mutation Sex/Age (yr)/ Ethnicity/PS Smoking History (pack-yr) Histology EGFRTKI (Line of Therapy) Dose (for >50% Course) Response RECIST % Change Target Lesion(s) PFS (mo) Total Time on EGFR TKI (mo) OS (from Start of EGFR TKI) G719A+S768I Woman/63/ Woman/78/ Asian/1 Woman/55/ black/1 Woman/65/ white/0 Woman/66/ Asian/3 Woman/64/ Woman/81/ Former (23) NSCLC NOS Erlotinib Never(0) Adenocarcinoma Erlotinib never(0) Adenocarcinoma Erlotinib Former (30) Adenocarcinoma Erlotinib Never (0) NSCLC NOS Erlotinib Never (0) Adenocarcinoma Erlotinib Never (0) Adenocarcinoma Erlotinib 100 mg/day PR 69.3 5 5 8 G719A+S768I 100 mg/day PR 33.3 7 7 10 G719S+E709A 150 mg/day PR 54.5 8 8 11 dell747_ T751+R776S a L858R+A871G 25 mg/every PR 41.1 20 40 b 40 b other day 150 mg/day PD +91.6 2 2 3 L858R+L747V L858R+R776H 150 mg every PR 37.3 6 b 6 b 6 b other day 100 mg/day PR 61.3 3 b 3 b 3 b a Initial clinical course for this patient had been detailed previously (ref 7 ). b Ongoing response, PFS, or OS. EGFR, epidermal growth factor receptor; PS, Eastern Cooperative Oncology Group performance status; TKI, tyrosine kinase inhibitor; NSCLC, non small-cell lung cancer; NOS, not otherwise specified; RECIST, Response Evaluation in Solid Tumors version 1.1; PD, progressive disease; PFS, progression-free survival; OS, overall survival. a compound mutation. Others have reported RRs as high as 60% for single or compound mutant L861Q bearing tumors with PFSs of 6 months. 3 We report a L861Q+E709A mutation that had a prolonged PR to an experimental irreversible EGFR TKI followed by additional disease control with erlotinib. In summary, compound EGFR mutations comprised 14% of all mutations identified during routine sequencing of exons 18 to 21 of EGFR in our cohort. Most tumors with an EGFR sensitizing mutation (G719X, exon 19 deletions, L858R, and L861Q) in addition to an atypical mutation responded to EGFR TKIs. Reporting of the genotype-response pattern of NSCLCs with EGFR compound and other rare mutations will help define the complete spectrum of how EGFR genotype affects the response to EGFR TKIs of NSCLCs. ACKNOWLEDGMENTS This work was supported in part by fellowships from the American Society of Clinical Oncology Conquer Cancer Foundation (DBC), an American Cancer Society grant RSG 11 186 (DBC), National Institutes of Health grants CA090578 (DBC, SK), CA126026 (SK), and William F. Milton Fund (SK). The funding agencies provided financial research support and were not involved in the writing of this article. REFERENCES 1. Gaughan EM, Costa DB. Genotype-driven therapies for non-small cell lung cancer: focus on EGFR, KRAS and ALK gene abnormalities. Ther Adv Med Oncol 2011;3:113 125. 2. Rosell R, Carcereny E, Gervais R, et al.; Spanish Lung Cancer Group in collaboration with Groupe Français de Pneumo-Cancérologie and Associazione Italiana Oncologia Toracica. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 2012;13: 239 246. 3. Wu JY, Yu CJ, Chang YC, Yang CH, Shih JY, Yang PC. Effectiveness of tyrosine kinase inhibitors on uncommon epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer. Clin Cancer Res 2011;17:3812 3821. 4. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications. Lancet Oncol 2012;13:e23 e31. 5. Yatabe Y, Pao W, Jett JR. Encouragement to submit data of clinical response to EGFR-TKIs in patients with uncommon EGFR mutations. J Thorac Oncol 2012;7:775 776. 6. 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;350:2129 2139. 7. Yeo WL, Riely GJ, Yeap BY, et al. Erlotinib at a dose of 25 mg daily for non-small cell lung cancers with EGFR mutations. J Thorac Oncol 2010;5:1048 1053. 8. Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res 2004;64:8919 8923. 9. Pao W, Ladanyi M, Miller VA; Lung Cancer Oncogenome Group. Erlotinib in lung cancer. N Engl J Med 2005;353:1739 41; author reply 1739. 10. 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;14:4877 4882. 11. Wu SG, Chang YL, Hsu YC, et al. Good response to gefitinib in lung adenocarcinoma of complex epidermal growth factor receptor (EGFR) mutations with the classical mutation pattern. Oncologist 2008;13:1276 1284. 12. Masago K, Fujita S, Irisa K, et al. Good clinical response to gefitinib in a non-small cell lung cancer patient harboring a rare somatic epidermal growth factor gene point mutation; codon 768 AGC > ATC in exon 20 (S768I). Jpn J Clin Oncol 2010;40:1105 1109. 13. Chen YR, Fu YN, Lin CH, et al. Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants. Oncogene 2006;25:1205 1215. 14. Sequist LV, Besse B, Lynch TJ, et al. Neratinib, an irreversible pan-erbb receptor tyrosine kinase inhibitor: results of a phase II trial in patients with advanced non-small-cell lung cancer. J Clin Oncol 2010;28:3076 3083. 15. Tam IY, Leung EL, Tin VP, et al. Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations. Mol Cancer Ther 2009;8:2142 2151. Copyright 2012 by the International Association for the Study of Lung Cancer 121

Koboyashi et al. Journal of Thoracic Oncology Volume 8, Number 1, January 2013 16. Nguyen KS, Kobayashi S, Costa DB. Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancers dependent on the epidermal growth factor receptor pathway. Clin Lung Cancer 2009;10:281 289. 17. 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;352:786 792. 18. Costa DB, Halmos B, Kumar A, et al. BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS Med 2007;4:1669 1679; discussion 1680. 19. 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;12:6494 6501. 122 Copyright 2012 by the International Association for the Study of Lung Cancer