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Tumour Review

Epidermal growth factor receptor tyrosine kinase inhibitors as initial

therapy for non-small cell lung cancer: Focus on epidermal growth factor

receptor mutation testing and mutation-positive patients

Monic Roengvoraphoj

a,b,d

, Gregory J. Tsongalis

b,c,e

, Konstantin H. Dragnev

a,b,d

, James R. Rigas

a,b,⇑ a

Comprehensive Thoracic Oncology Program, Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756-0001, USA b

The Geisel School of Medicine at Dartmouth, One Rope Ferry Road, Hanover, NH 03755-1404, USA c

Department of Pathology, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756-0001, USA

a r t i c l e

i n f o

Article history:

Received 27 November 2012 Received in revised form 2 May 2013 Accepted 4 May 2013

Keywords:

Epidermal growth factor receptor (EGFR) EGFR tyrosine kinase inhibitors (TKIs) EGFRgene mutations

SomaticEGFRgene mutation testing

a b s t r a c t

Activation of the epidermal growth factor receptor (EGFR) pathway has been implicated in tumorigenesis in non-small cell lung cancer (NSCLC), the most common type of lung cancer. As a result, EGFR has become a key focus for the development of personalized therapy, with several molecular biomarkers hav-ing been investigated as potential predictors of response with EGFR tyrosine kinase inhibitors (TKIs) in NSCLC (e.g., EGFR expression,EGFRgene copy gain, andEGFRmutations). Of these, activating mutations inEGFRhave thus far given the most consistent results based on the available evidence from preclinical studies and clinical trials. In an attempt to identify patients who are most likely to benefit from treatment with EGFR TKIs,EGFRmutation testing is being increasingly utilized in clinical practice. Currently in the United States, no EGFR TKI or accompanying mutational test is approved for the identification and first-line treatment of patients with advanced NSCLC. However, the first-generation EGFR TKIs, erlotinib and gefitinib, as well as investigational ErbB family TKIs andEGFRmutation testing methods are being eval-uated in this setting. This review will discussEGFRmutation testing as a biomarker of response to EGFR TKIs and the evolution ofEGFRmutational analysis in NSCLC. Completed and ongoing clinical trials eval-uating currently available or investigational EGFR TKIs as first-line therapy in molecularly and clinically selected patients with NSCLC, with a focus on trials in patients whose tumors haveEGFRmutations, will also be reviewed.

Ó2013 The Authors. Published by Elsevier Ltd.

Introduction

Lung cancer is the leading cause of cancer-related mortality for both men and women in the United States.1Non-small cell lung

cancer (NSCLC) accounts for more than 85% of lung cancer cases, of which adenocarcinoma (40%) is the most common subtype, fol-lowed by squamous cell carcinoma (25%) and large cell carcinoma

(10%).2Only approximately 15% of patients with lung cancer

pres-ent with localized disease. For those NSCLC patipres-ents with more

ad-vanced disease, conventional systemic chemotherapy (e.g.,

platinum-based chemotherapy) has been a cornerstone of treat-ment; however, it provides only a modest benefit in survival.1,3

Not surprisingly, the overall 5-year survival rate for all stages of NSCLC is only 17%.1

The discovery of epidermal growth factor receptor (EGFR) tyro-sine kinase inhibitors (TKIs) has led to a new paradigm of lung can-cer treatment. The use of EGFR TKIs for NSCLC began in 2003. In the United States, EGFR TKIs were first administered to non-selected patients with advanced NSCLC who had failed all approved chemo-therapies.4 Remarkably, some of these individuals, particularly those with adenocarcinoma of the lung, experienced profound and durable antitumor responses.5,6As the clinical development

of EGFR TKIs progressed through large-scale global clinical trials, certain clinical characteristics, including adenocarcinoma histol-ogy, female gender, non-smoking history, and Asian ethnicity, were also associated with increased response to EGFR TKIs.7–11

Molecu-lar predictors, thought to be more inclusive and reliable than 0305-7372Ó2013 The Authors. Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.ctrv.2013.05.001

⇑ Corresponding author. Address: Comprehensive Thoracic Oncology Program, Norris Cotton Cancer Center, The Geisel School of Medicine at Dartmouth, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756-0001, USA. Tel.: +1 (603) 650 6344; fax: +1 (603) 650 9496.

E-mail addresses: monic.roengvoraphoj@hitchcock.org (M. Roengvoraphoj), gregory.j.tsongalis@hitchcock.org (G.J. Tsongalis), konstantin.h.dragnev@dart-mouth.edu(K.H. Dragnev),james.r.rigas@dartmouth.edu(J.R. Rigas).

d

Tel.: +1 (603) 650 6344; fax: +1 (603) 650 9496. e Tel.: +1 (603) 650 5498; fax: +1 (603) 650 4845.

Contents lists available atSciVerse ScienceDirect

Cancer Treatment Reviews

j o u r n a l h o m e p a g e : w w w . e l s e v i e r h e a l t h . c o m / j o u r n a l s / c t r v

Open access under CC BY-NC-ND license.

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clinical characteristics, were sought to identify patients who would benefit most from treatment with EGFR TKIs. Several molecular biomarkers, including gene mutations, protein expression, and gene copy number, have been identified. In 2004, initial reports of somatic EGFR gene mutational analysis in NSCLC patients responding to EGFR TKIs seemed most promising.7,12

Subsequently, the role of these molecular biomarkers as predic-tive and prognostic factors has been investigated in NSCLC patients treated with EGFR TKIs. On the basis of available evidence thus far, the most reliable predictors of EGFR TKI sensitivity and progres-sion-free survival (PFS) are tumor EGFR-activating gene muta-tions.9,10,13–15As a result of these findings, EGFR TKIs have been

approved by the European Commission and Asian authorities as initial therapy for patients with tumorEGFR-activating gene muta-tions.16,17As of yet, there has been no US Food and Drug

Adminis-tration (FDA) approval of an EGFR TKI in conjunction with anEGFR mutation detection method for the first-line treatment of patients with advanced NSCLC.

This article will review the importance of somaticEGFR activat-ing gene mutations as predictors of response and clinical outcomes with EGFR-TKI therapy and the current and evolving detection methods for EGFR mutation analysis employed in clinical trials and in clinical practice, and finally summarize the clinical results of the currently available EGFR TKIs and ongoing clinical trials of investigational EGFR TKIs in molecularly selected NSCLC patients withEGFR-activating mutations.

Overview of EGFR biology

EGFR, also known as human epidermal growth factor receptor-1 (HER-1), is 1 of the 4 members of the ErbB family of receptor tyro-sine kinases. It is a multiple domain glycoprotein (170 kilodaltons) that consists of an extracellular ligand-binding domain and an intracellular tyrosine kinase domain separated by a transmem-brane region.18 When endogenous ligands, such as epidermal growth factor (EGF), transforming growth factor-

a

(TGF-

a

), amphi-regulin, heparin-binding EGF, or betacellulin, bind to EGFR, recep-tor homodimerization or heterodimerization occurs at the cell surface and eventually leads to internalization of the dimerized receptor.18,19The receptor dimerization results in

autophosphory-lation of the intracytoplasmic EGFR tyrosine kinase domain.20

Sub-sequently, the phosphorylated tyrosine kinase stimulates an intracellular signal transduction cascade through several down-stream pathways, including the retrovirus-associated DNA se-quences (RAS)/v-raf 1 murine leukemia viral oncogene homolog 1 (RAF)/mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) pathway, phosphoinositide-3-nase (PI3K)/protein kiphosphoinositide-3-nase B (AKT) pathway, and Janus tyrosine ki-nase (JAK)/signal transducers and activators of transcription (STAT) pathway, which regulate apoptosis and cell proliferation (Fig. 1).21

EGFR TKIs

EGFR TKIs are small molecules that act by inhibiting EGFR auto-phosphorylation and, therefore, receptor activation and signal transduction. The first-generation TKIs, erlotinib and gefitinib, are oral bioavailable synthetic anilinoquinazolines that reversibly bind with high affinity to the intracellular tyrosine kinase domain of EGFR competitively inhibiting adenosine-50 triphosphate (ATP)

binding. A new generation of EGFR TKIs possesses new features; they are active against multiple ErbB family receptor targets and/ or bind the tyrosine kinase domain targets irreversibly.22Afatinib

(BIBW 2992), dacomitinib (PF00299804), and neratinib (HKI-272) are investigational ErbB family inhibitors that irreversibly target EGFR, HER2, and HER4.23–26

EGFRmutations as molecular biomarkers

Molecularly targeted therapy has become an exciting area of lung cancer treatment. The ability to target specific cancer cell oncogenic pathways not only improves the efficacy of the treat-ment, but also may reduce toxicities as compared to conventional cytotoxic chemotherapy. However, only 6% of unselected patients with NSCLC respond to EGFR TKIs.5Therefore, the selection of

suit-able patients that would benefit most from treatment with EGFR TKIs is crucial. While patients with certain clinical characteristics respond better to EGFR TKIs, selection of patients by using molec-ular biomarkers has been found to be more inclusive and reliable.

EGFR gene mutations

SomaticEGFRgene mutations were first described in 2004 by 3 independent studies after observation of a dramatic response to gef-itinib in a subset of unselected NSCLC patients in 2 large phase II clinical trials (IDEAL1 and -2).5–8,12This subset of patients with an

increased response to EGFR TKIs was found to harbor somaticEGFR

gene mutations in the kinase domain. The most common EGFR

mutations in patients with NSCLC include short in-frame deletions in exon 19 and a specific point mutation in exon 21 at codon 858. Both mutations account for approximately 80–90% of the EGFR mutations detected (Fig. 2). These activating mutations occur in the ATP binding cleft of EGFR where EGFR TKIs bind and impart a sensitivity to small-molecule EGFR TKIs.27,28Patients found to have

anEGFRexon 19 deletion mutation have significantly longer median survival times (up to 38 months) than those with an exon 21 L858R mutation (up to 17 months) when treated with gefitinib or erloti-nib.14,15,29There have been more than 20 different exon 19 deletions

identified in lung adenocarcinomas.30The most frequent deletions in

exon 19 encompass the amino acid string from L747 through E749 (LRE fragment), and based on data from the Somatic Mutations in EGFRDatabase, the most common LRE deletions are delE746-A750 (66.1%), followed by delL747-P753insS (56.8%), delL747-A750insP (4.0%), and delL747-T751 (3.7%).31,32delS752-I759 is the most

com-mon non-LRE deletion.32

Among patients with exon 19 deletions, those with non-LRE deletions had a lower response to EGFR TKIs compared with those harboring LRE deletions (P =0.022); however, overall survival (OS) was not significantly different among mutation position groups (P= 0.776).32Other less commonly detected

sensi-tizingEGFRmutations include the G719A/C/S and S720F mutations in exon 18, the L861Q/R mutations in exon 21, and the V765A, T783A, and S768I mutations in exon 20.28,30,33,34

ActivatingEGFRmutations are most common in patients with adenocarcinoma histology, women, never smokers, and those of Asian ethnicity, which are known clinical predictors for harboring activatingEGFRmutations, and as a result, increased response to EGFR TKIs.5The overall response rate (RR) is as high as

approxi-mately 80% in patients harboringEGFRmutations,35and the prev-alence ofEGFRmutations is approximately 20–40% among Asians and 10% among Caucasians.9,36,37Although the initial overall RR

to EGFR TKIs is high for patients withEGFRmutations, almost all patients subsequently develop acquired resistance within 10– 16 months.38 Acquired resistance to EGFR TKIs is defined as (1)

prior single-agent EGFR TKI (e.g., gefitinib, erlotinib) therapy; (2) either of the following: a tumor that harbors a drug-sensitizing EGFRmutation, or objective clinical benefit achieved with an EGFR TKI; (3) systemic disease progression (as defined by Response Eval-uation Criteria in Solid Tumors [RECIST] or World Health Organiza-tion [WHO]) while receiving continuous gefitinib or erlotinib within the last 30 days; and (4) no systemic therapy administered between cessation of gefitinib or erlotinib and beginning new treatment.39

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Several mechanisms that may lead to acquired resistance in-clude development of a secondaryEGFR mutation, amplification ofmet proto-oncogene(hepatocyte growth factor receptor;MET), or increased expression of other receptor tyrosine kinases.40The most

common mutation associated with acquired resistance is T790M, a secondary point mutation located at exon 20 that results in substi-tution of methionine for threonine at position 790.30,41T790M is

associated with over 50% of adenocarcinoma cases with acquired resistance, and is associated with primary resistance in approxi-mately 1% of untreated patients.30This mutation has been shown

to increase the affinity of EGFR for ATP relative to its affinity for EGFR TKIs.42 Among patients with acquired resistance to EGFR

TKIs, the presence of T790M is associated with a favorable progno-sis relative to acquired reprogno-sistance via other processes (median time to progression, 14 vs 11 months,P =0.10; OS, 39 vs 26 months, P= 0.007).38Other less common mutations associated with

EGFR-TKI resistance include insertion mutation in exon 19 (D761Y), insertion mutations in exon 20 (D770-N771), and point mutations in exon 20 (V769L, N771T;Fig. 2).30

EGFRmutation detection methods

The discovery ofEGFRmutations as a powerful predictive and prognostic marker for response to EGFR TKIs has led to changes

in clinical practice. The detection of somaticEGFRtyrosine kinase domain mutations at diagnosis in NSCLC has been increasingly uti-lized to identify patients who are more likely to benefit from EGFR TKIs. However, as of yet, there is no accepted consensus on which EGFRmutation method is preferable and no US FDA-approvedEGFR mutation test. In addition, controversy exists regarding which his-tological subtypes of NSCLC to test forEGFR-activating mutations. The National Comprehensive Cancer Network (NCCN) recommends EGFRmutation testing for adenocarcinoma, large cell, and NSCLC not otherwise specified (NOS) and no EGFRmutation testing for squamous cell carcinoma of the lung.3 These recommendations are in contrast to the American Society of Clinical Oncology (ASCO) guidelines.43The ASCO guidelines recommendEGFRmutation

test-ing for advanced NSCLC patients who are betest-ing considered for first-line therapy with an EGFR TKI to determine whether an EGFR TKI or chemotherapy is the appropriate first-line therapy. Three differ-ent techniques for detectingEGFRmutations will be discussed. DNA sequencing

Sanger sequencing of DNA was the earliest and has been the most widely used technique for identification ofEGFRmutations. This type of DNA sequencing is often considered the ‘‘gold stan-dard’’ for mutation detection technologies. Sequencing involves

Ligand

P P

EGFR or other ErbB family member

Cell membrane Apoptosis Cell proliferation PI3K AKT JAK STAT MEK RAF RAS MAPK

Fig. 1.The EGFR signaling pathway. Upon ligand binding, EGFR or another ErbB family member dimerizes and is activated through phosphorylation of its intracellular tyrosine kinase domain. This leads to signal transduction through several downstream pathways that regulate apoptosis and cell proliferation. Abbreviations: EGFR, epidermal growth factor receptor; ErbB, erythroblastic leukemia viral oncogene homolog; PI3K, phosphoinositide-3-kinase; AKT, protein kinase B; RAS, retrovirus-associated DNA sequences; RAF, v-raf 1 murine leukemia viral oncogene homolog 1; MEK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; JAK, Janus tyrosine kinase; STAT, signal transducers and activators of transcription.

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extraction of genomic DNA from tumor tissue followed by poly-merase chain reaction (PCR) amplification of specific gene regions and subsequent sequencing of the PCR products using dideoxy-labeled nucleotides.44

The major challenge associated with this technique is its sensitivity to detect mutant alleles.45,46 Optimal

results are obtained when the percentage of tumor cells in the sample is 50% or more,44 and standard tumor biopsy samples frequently do not contain enough high-quality DNA to be useful for direct sequencing.47Two novel DNA sequencing methodologies

are being explored: pyrosequencing and massively parallel or next-generation sequencing (NGS).48,49Pyrosequencing is a method of

DNA sequencing based on the ‘‘sequencing by synthesis’’ approach that relies on the detection of pyrophosphate release upon nucleo-tide incorporation, in contrast to Sanger sequencing, which is based on chain termination with dideoxynucleotides.49 Although

pyrosequencing is limited to individual DNA sequence in the neighborhood of 300 to 500 nucleotides, shorter than Sanger sequencing (which is limited at 800–1000 nucleotides), it is a fast method with real-time read out.49–52Massively parallel

sequenc-ing employs several high-throughput DNA sequencsequenc-ing approaches and is also called NGS or second-generation sequencing.48These

technologies use miniaturized and parallelized platforms for sequencing of millions of short nucleotides (50–400 bases).52,53

All NGS platforms have in common a technical paradigm of massively parallel sequencing via clonally amplified, spatially separated DNA templates or single DNA molecules in a flow cell.48,54 This design is very different from Sanger sequencing,

which uses capillary electrophoretic separation of chain-termina-tion sequencing of individual reacchain-termina-tion products.48One of the added

advantages of NGS over other molecular technologies is its ability to rapidly sequence known mutation hotspots in multiple genes at

the same time. This is appealing when selection of therapeutics is based on the presence or absence of activation (i.e., sensitizing) mutations and resistance mutations in the same or alternative genes of a pathway. The ability to sequence multiple hotspots within the same gene or in different genes is now possible in clinical laboratories with instruments better designed for this setting such as the MiSeq from Illumina and the Ion Torrent Personal Genome Machine (PGM) from Life Technologies.55

PCR-mediated analysis

Many mutation-specific PCR assays have been used to detect EGFRmutations. The method is rapid, sensitive, and can be per-formed using small samples. PCR followed by restriction enzyme digestion and capillary electrophoresis is one of the simplest ways to detect the most common activating mutations. Different PCR as-says are commercially available, including theEGFR29mutation kit (Qiagen),56,57peptide nucleic acid-locked nucleic acid (PNA-LNA)

PCR clamp,58–60denaturing high-performance liquid

chromatogra-phy (HPLC),61,62 MassARRAYÒ System (Sequenom),63

SNaPshotÒ

(Life Technologies),64,65 and competitive allele-specific TaqManÒ

PCR (castPCR) (Life Technologies).66

Real-time PCR technology offers the advantage of increased sensitivity and specificity when followed by hybridization with mutation-specific fluorescent probes,67,68 or high-resolution

melting temperature analysis. In addition, others have used allele-specific real-time PCR for the identification of allele-specific point mutations.

There are several commercially available EGFRmutation kits from various vendors, none of which have been approved by the FDA. One such vendor, Qiagen, offers a kit designed to detect the (688) 18 G719S (c.2155G>A) G719C (c.2155G>T) G719A (c.2156G>C) S720F (c.2159C>T) D770_N771 (insNPG) D770_N771 (insSVQ) D770_N771 (insG) V765A (c.2294T>C) T783A (c.2347A>G) S768I (c.2303G>T) T790M (c.2369C>T) V769L (c.2305G>T) N771T (c.2312A>C) Exon 19 deletion D761Y (c.2281G>T) L858R (c.2573T>G) L861Q (c.2582T>A) L861R (c.2582T>G) (728) (729) 19 20 21 (761) (762) (823) (824) (875) 7p11.2 Resistant Sensitive 5% 45% 1% 41% 3% 1% 1% 3%

Fig. 2.Frequency of mutations in exons 18–21 of theEGFRgene and the association with responsiveness to EGFR TKIs. Diagram depicting the locations and frequencies of EGFRgene mutations detected in the tumors of patients with NSCLC. The most commonEGFRmutations are exon 19 deletions and exon 21 point mutations at codon 858, which together account for approximately 80–90% of cases. Reprinted with permission from Macmillan Publishers Ltd: Modern Pathology,30Ó2012. Abbreviations: EGFR,

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29 most commonEGFRmutations against a background of wild-type genomic DNA. The test kit uses a PCR-based assay in which specific areas of DNA containing mutations are targeted by Ampli-fication Refractory Mutation System (ARMS) primers and Scorpions technology is used to amplify and detect those specific areas of DNA.69 Using this kit, a German panel for mutation testing in

NSCLC evaluated the detectability of EGFR mutations compared to direct sequencing of exons 18–21 in 40 samples of NSCLC. The panel determined that the kit failed to detect 32% of the EGFR-acti-vating mutations that had been detected by direct sequencing.56

The sensitivity of this kit is approximately 93%57; however,

detec-tion is limited to those mutadetec-tions specifically targeted by the de-signed primers.

The PNA-LNA PCR clamp system is a PCR assay that is used to detect mutations occurring at specific positions, including G719S, G719C, L858R, L816Q, and 7 different exon 19 deletions; the assay can be performed in the presence of 100- to 1000-fold wild-type alleles without the need to remove the contaminating normal cells.58–60Therefore, this method can be used to detectEGFR

muta-tions from samples such as sputum, pleural effusion, and bronchial washings that often contain many normal cells. The sensitivity and specificity of the test has been reported in the range of 97% and 100%, and the test can be completed within 2 h.58,60This method

was developed by a Japanese group58 and has been increasingly

used in clinical trials conducted in Asia.

The detection ofEGFRmutations by denaturing HPLC involves subjecting PCR products to ion-pair-reverse-phase liquid chroma-tography. The underlying principle of denaturing HPLC is that par-tially denatured heteroduplex PCR products containing mutated sequences will move through the chromatography column at dif-ferent rates as compared with homoduplex sequences that do not contain mutations.62 The sensitivity and specificity of the

method has been reported in the range of 87–100%. In one study, this method detected 4% of mutations that were not previously de-tected by direct sequencing.61

Although Sanger sequencing has been most widely used for EGFRmutation detection, one of the limitations is that it cannot de-tectEGFRmutations in individuals with approximately 10% allele frequencies. Conversely, the MassARRAYÒsystem (Sequenom)

uti-lizes PCR amplification and single-base primer extension for muta-tion detecmuta-tions, with the ability to detect low abundant and rare mutations in a heterogenous mixture of cancer and normal tis-sue.70Thomas et al. compared the sensitivity of this system to both

Sanger sequencing and a pyrosequencing method for the detection ofEGFRmutations in 22 lung tumor samples. Both mass spectro-metric genotyping and pyrosequencing detected 12 mutations, while Sanger sequencing detected only 9 mutations.63To improve

the speed and accessibility of mutation profiling by this system, Sequenom has offered the OncoCarta™ Panel, a pre-designed mul-ti-gene panel with 19 oncogenes and 238 mutations.70

The SNaPshotÒ assay (Life Technologies) consists of a

multi-plexed PCR step followed by a single-base extension reaction that generates allele-specific fluorescently labeled probes, which are subsequently analyzed by capillary electrophoresis. It performs well on formalin-fixed, paraffin-embedded (FFPE) tissue samples and is designed to detect more than 40 types of recurrent genetic alterations in lung cancer.65The advantage of this method is that

it utilizes capillary electrophoresis instruments that are present in many clinical laboratories for Sanger sequencing, and thus sub-stantial capital expenses can be avoided. Dias-Santagata et al. com-pared the SNaPshotÒ assay to other platforms.64 While both

SNaPshotÒ assay and Sanger sequencing detect the same

muta-tions, the SNaPshotÒ assay requires 3–6 times less tumor DNA.

The comparison of SNaPshotÒassay and MassARRAYÒwas

some-what limited due to lack of optimization of the assay. However, it was concluded that the MassARRAYÒ system is more labor

intensive and expensive, and requires more tissue sample than the SNaPshotÒassay. A limitation of the SNaPshotÒassay is the

detection of only point mutations at a specific site and not ampli-fications, insertions, or deletions.65

The TaqManÒ Mutation Detection Assay is a real-time PCR

method for detection of somatic mutations.66,71It is powered by

castPCR technology (Life Technologies) that combines allele-spe-cific TaqMan qPCR with allele-speallele-spe-cific blockers to suppress ampli-fication of the wild-type allele, resulting in better specificity.71The

assay is capable of detecting 0.1% mutation in 10,000 copies of wild-type genomic DNA (gDNA) background.71Didelot et al.

eval-uatedKRAS,BRAF, andEGFRgenotyping by this method on subop-timal FFPE colon and lung cancer samples. Of 63 samples, 60 were correctly genotyped.66One major drawback of this method is that

it does not identify the T790M mutant due to reduced sensitivity. Immunohistochemistry

Immunohistochemistry (IHC) is a widely used method in the diagnosis of cancer that uses specific antibodies to bind antigens. In 2009, Yu et al. first developed 2 mutation-specific rabbit anti-bodies against E746-A750del and the L858R point mutation and evaluated them in 340 paraffin-embedded NSCLC tumor samples by Western blotting, immunofluorescence, and IHC. Compared with direct DNA sequencing, the sensitivity of the IHC assay was 92% with a specificity of 99%.47Other studies examined the

pres-ence ofEGFRmutations in NSCLC by IHC using the same antibodies and reported sensitivity ranging from 36%–100% and specificity ranging from 92%–100%.72–77Although these antibodies seem to

be reliable for detection of the most common EGFR mutations,

other uncommon EGFR mutations, as well as the resistance

mutation T790M in exon 20, cannot be detected.77 In addition,

IHC requires a validated standardization process due to multiple variable factors, including type of antibodies, tissue fixation methods, and methods of interpretation.33

Summary of EGFR mutation detection methods

In summary,EGFRmutations can be detected using a variety of methods. The strengths and weaknesses of each method are sum-marized inTable 1. To date, there is still no universally accepted consensus or recommendation to facilitate implementation of standardized testing forEGFR mutations in clinical practice. The most appropriate testing method forEGFRmutations to predict re-sponse with EGFR TKIs still needs to be addressed and validated. It is important to note thatEGFRmutation testing for the purpose of directing therapeutic selection must be performed in a Clinical Laboratory Improvements Amendments (CLIA)-certified clinical laboratory. Thus, different technologies may be more suitable for this type of environment and FDA clearance/approval may be warranted.

EGFRTKIs as initial therapy in molecularly selected NSCLC

Phase II studies of gefitinib in patients with advanced NSCLC have shown response accompanied by symptomatic improvement in approximately 10% of unselected patients.5,6In 2004, somatic

EGFRmutations as predictive molecular biomarkers for response to EGFR TKIs were noted by 3 landmark studies.7,8,12Since then,

further clinical trials have been conducted to evaluate efficacy of EGFR TKIs as first-line therapy for NSCLC in both clinically selected and molecularly selected patients.

One of the pivotal clinical trials that led to the approval of EGFR TKIs in Europe in the first-line setting was the phase III randomized Iressa Pan-Asia Study (IPASS) that evaluated the efficacy, safety,

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and tolerability of gefitinib compared to standard carboplatin/pac-litaxel chemotherapy as first-line treatment in 1217 clinically

se-lected East Asian, never or former light smokers with

adenocarcinoma histology.78,79The 12-month rate of PFS (primary outcome) was prolonged with gefitinib compared with chemother-apy in the intention-to-treat population (24.9% vs 6.7%; hazard ra-tio [HR], 0.74; 95% confidence interval [CI], 0.65–0.85;P< 0.001). In a planned subgroup analysis of patients withEGFRmutations, pro-longed PFS (HR, 0.48; 95% CI, 0.36–0.64;P< 0.001) and a higher objective RR (71.2% vs 47.3% with chemotherapy) were noted among patients who received gefitinib.78Final analysis of OS

dem-onstrated no significant benefit for gefitinib in the overall popula-tion (HR, 0.90; 95% CI, 0.79–1.02;P= 0.109) or theEGFR mutation-positive subgroup (HR, 1.00; 95% CI, 0.76–1.33;P= 0.990).79

Addi-tional biomarker analyses from the IPASS study, published with the final OS data, showed thatEGFRmutations were the strongest pre-dictive biomarker for PFS and tumor response to first-line gefiti-nib.79 Gefitinib produced significantly prolonged PFS among

patients with tumors that exhibited both high EGFR gene copy number andEGFRmutation (HR, 0.48; 95% CI, 0.34–0.67) but not

high EGFRgene copy number without EGFRmutation, in whom

PFS was significantly shorter relative to carboplatin/paclitaxel

recipients (HR, 3.85; 95% CI, 2.09–7.09). Using similar clinically se-lected patients, the First-line Single agent Iressa vs Gemcitabine and cisplatin trial in Never-smokers with Adenocarcinoma of the Lung (First-SIGNAL;N= 313) study revealed similar PFS and OS re-sults to those of IPASS.80A number of other phase II and phase III

trials have evaluated the efficacy of EGFR TKIs as first-line treat-ment in molecularly selected advanced NSCLC patients with con-firmedEGFRmutations (Table 2).

Gefitinib

Gefitinib was first approved in Europe in 2009 for use in all lines of treatment for NSCLC patients harboringEGFRmutations but is not currently approved by the US FDA (application to be with-drawn in 2011).81In the phase III NEJ002 trial (N= 230), gefitinib

was compared to carboplatin/paclitaxel in advanced NSCLC pa-tients whose tumors hadEGFRmutations. More than 90% of pa-tients had adenocarcinoma, 63% were women, and all had tested positive forEGFR-activating mutations with PNA-LNA clamp test-ing. After a planned interim analysis, this trial was interrupted due to a significantly longer median PFS (10.8 vs 5.4 months; HR, 0.30; P< 0.001) and a higher RR (73.7% vs 30.7%, P< 0.001) Table 1

Pros and Cons ofEGFRdetection methods.

Method Pros Cons

Sanger or dideoxy sequencing

Familiarity, stemming from being the earliest introduced and most widely usedEGFRmutation detection method94

Requires large amounts of tissue; not feasible for unresected tumors94

Expensive and time-consuming94

Limited sensitivity47

; some rareEGFR mutant alleles typically fall below the detection limit Next-generation or

massively parallel sequencing

Greater depth of coverage, multiple targets simultaneously sequenced, shorter turn-around time, scalable, cost effective48,49

Requires new instrumentation and expertise48

Data analysis is exclusively informatics48

Validation for multiple genes with many mutations is costly

No significant guidelines Immunohistochemistry Established technique47

36–100% sensitivity and >90% specificity47,72–77

Applicability to a variety of sample types47

Inability to detect uncommonEGFRmutations or the resistance mutation T790M77

Requires a validated standardization process33

PCR-based analysis Scorpion Amplification

Refractory Mutation System (DS)

Ability to detect 29 of the most prevalent somaticEGFRmutations, including rare alleles undetectable by direct sequencing95

>90% sensitivity57

Simple and rapid (results within 24 h)96,97

Applicability to a variety of sample types; allows for non-invasive testing95

Detection limited to those mutations specifically targeted by the designed Scorpions primers96

May be less cost-effective relative to other methods97

PNA-LNA PCR clamp Ability to detect a mutantEGFRsequence in specimens containing 100–1000 excess copies of wild-typeEGFR58

>97% sensitivity and 100% specificity58,60

Rapid (may be completed in 40 min) and inexpensive60

Applicability to a variety of sample types; allows for non-invasive testing59,60

Potential inability to detect small numbers of mutants in tumors with heterogeneity forEGFR mutations60

Denaturing HPLC Ability to detect a mutantEGFRsequence in specimens containing 1.6–6.25% mutated DNA62

Potential 100% sensitivity and >85% specificity61

Rapid and cost-effective62

Applicability to a variety of sample types61,62

Inability to detect homozygous mutations61,62

MassARRAYÒ

Ability to detect a mutantEGFRsequence in specimens containing610% mutated DNA70

More sensitive than Sanger sequencing63

High cost of instrument

SNaPshotÒ

Ability to detect a mutantEGFRsequence in specimens containing610% mutated DNA64

Availability of the necessary equipment (PCR machine and capillary sequencer) in most clinical laboratories98

Costs80% less than direct sequencing98

Inability to detect amplifications, insertions, or deletions65

TaqManÒ

mutation detection assay

Ability to detect 0.1% mutation in 10,000 copies of wild-type DNA background71

Rapid and simple66

Decreased sensitivity to detect T790M mutation66

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reported in the gefitinib arm.82However, final OS results reported no statistical significance between the 2 arms (27.7 months in the gefitinib group vs 26.6 months in the chemotherapy group, P= 0.483), likely explained by treatment crossover.83The Japanese phase III WJTOG3405 trial (N= 177) evaluated gefitinib vs cis-platin/docetaxel in patients withEGFR-activating mutations. In this

trial, most patients had adenocarcinoma, 60% were women, and all tested positive for EGFR mutations with the direct sequencing method. Patients who received gefitinib had significantly pro-longed PFS compared with patients who received cisplatin/ docetaxel (9.2 vs 6.3 months, respectively).84These results suggest

that gefitinib is superior to chemotherapy in terms of PFS and RR in Table 2

Results from phase II and III clinical trials evaluating EGFR TKIs as first-line therapy in molecularly selected NSCLC patient populations. Trial Patient population EGFRmutation

detection methods

Treatment RR % PFS, median OS, median Gefitinib

Phase II, open-label99 Japanese with EGFR-activating mutations;N= 30 PNA-LNA PCR clamp Gefitinib until disease progression 66% (90% CI, 51–80) 6.5 mo 17.8 mo

Phase II, open-label100

Japanese with EGFR-activating mutations;N= 19 PCR and mutation-allele-specific amplification Gefitinib until disease progression 63.2% (95% CI, 38.4–83.7) 7.1 mo 20.0 mo

Phase II, open-label101

Japanese with EGFR-activating mutations;N= 15 NR Gefitinib until disease progression 60% (95% CI, 36.0–80.9) 10.4 mo (95% CI, 7.2–13.6) NR

Phase II, open-label102

Japanese with EGFR-activating mutations;N= 54 NR Vinorelbine or gemcitabine vs gefitinib 19% vs 45% NR 18.9 mo vs 24.4 mo NEJ003; phase II,

open-label103 Japanese with EGFR-activating mutations;N= 31 PNA-LNA PCR clamp Gefitinib 74% (95% CI, 58–91) 13.6 mo NR

Phase II, open-label104

EGFR-activating mutations;N= 31

Direct sequencing Gefitinib until disease progression 55% (95% CI, 33–70) 9.2 mo (95% CI, 6.2–11.8) 17.5 mo (95% CI, 13.5–21.3) WJTOG3405; phase III,

randomized, open-label84 Japanese with EGFR-activating mutations; N= 177 Fragment analysis and the Cycleave method (with confirmation by direct sequencing), with protocol amendment allowing direct sequencing, PNA-LNA PCR clamp, or PCR invader Cisplatin/ docetaxel vs gefitinib 32.2% vs 62.1% (difference, 29.9%; 95% CI, 12.6–47.1; P< 0.0001) 6.3 mo vs 9.2 mo (HR, 0.489; 95% CI, 0.336–0.710; P< 0.0001) NR

NEJ002; phase III, randomized82,83 EGFR-activating mutations; N= 230 PNA-LNA PCR clamp Carboplatin/ paclitaxel vs gefitinib 30.7% vs 73.7% (P <0.001) 5.4 mo vs 10.8 mo (HR, 0.30; 95% CI, 0.22–0.41; P <0.001) 26.6 mo vs 27.7 mo (HR, 0.887; 95% CI, 0.634–1.241; P= 0.483) Erlotinib

Phase II, open-label105

Caucasian with EGFR-activating mutations;N= 46

NR Erlotinib n= 26 44+ wk 56+ wk

OPTIMAL; phase III, randomized, open-label86,106 Chinese with EGFR-activating mutations; N= 165 PCR, followed by direct DNA sequencing Carboplatin/ gemcitabine vs erlotinib 36% vs 83% (P< 0.0001) 4.6 mo vs 13.1 mo (HR, 0.16; P< 0.0001) NR

EURTAC; phase III, randomized, open-label87 European with EGFR-activating mutations; N= 173 Direct sequencing (with confirmation with an independent technique) Platinum-based chemotherapy vs erlotinib 15% vs 58% (P< 0.0001) 5.2 mo vs 9.7 mo (HR, 0.37; 95% CI, 0.25–0.54; P< 0.0001) 19.5 mo vs 19.3 mo (HR, 1.04; 95% CI, 0.65–1.68; P= 0.87) Afatinib

LUX-Lung 3; phase III, randomized, open-label89 EGFR-activating mutations; N= 345 PCR (Therascreen EGFRkit) Cisplatin/ pemetrexed vs afatinib 23% vs 56% (P< 0.0001) 6.9 mo vs 11.1 mo (HR, 0.58; 95% CI, 0.43–0.78; P =0.0004) NR Dacomitinib

Phase II, open-label92 Non-smokers/

former light smokers orEGFR -activating mutations, with adenocarcinoma;N= 74 NR Dacomitinib NR 9.30 mo (95% CI, 6.50–11.17) NR

CI, confidence interval; EGFR, epidermal growth factor receptor; HR, hazard ratio; NR, not reported; NSCLC, non-small cell lung cancer; OS, overall survival; PCR, polymerase chain reaction; PFS, progression-free survival; PNA-LNA PCR, peptide nucleic acid-locked nucleic acid polymerase chain reaction; RR, response rate; TKI, tyrosine kinase inhibitor.

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molecularly selected NSCLC patients harboring EGFR mutations. Several phase II and phase III clinical trials are evaluating gefitinib and other EGFR TKIs as first-line therapy in clinically or molecu-larly selected patients with advanced NSCLC (Table 3). Safety data with gefitinib and other EGFR TKIs in molecularly selected patients with NSCLC are summarized inTable 4.

Erlotinib (OSI-774)

Erlotinib is the only FDA-approved EGFR TKI in the United States. It is only indicated for the treatment of patients with locally advanced or metastatic NSCLC after failure of at least 1 prior

chemotherapy regimen and for maintenance treatment in patients whose disease has not progressed after 4 cycles of platinum-based first-line chemotherapy.85In a recent multicenter, open-label,

ran-domized, phase III study (OPTIMAL;N= 165) comparing erlotinib and gemcitabine/carboplatin chemotherapy as first-line treatment for patients with advancedEGFRmutation-positive NSCLC, a signif-icant PFS benefit (median 13.1 vs 4.6 months; HR, 0.16; 95% CI, 0.10–0.26;P< 0.0001) and more favorable tolerability were associ-ated with erlotinib.86In this trial, 86% of the patients had

adeno-carcinoma, 60% were women, 69% were never-smokers, and all tested positive for EGFR mutation with the PCR method. The European Erlotinib vs Chemotherapy (EURTAC;N= 174) trial was Table 3

Ongoing clinical trials evaluating EGFR TKIs as first-line therapy in clinically and/or molecularly selected patient populations with advanced NSCLC.

Trial Estimated

enrollment

Patient population Therapy Estimated

completion

Primary endpoint(s) Gefitinib

NCT00411047; phase II, open-label

30 Female, adenocarcinoma histology, non-smoker/former light smoker, or Asian ethnicity

Gefitinib December 2012 RR

NCT01017874; phase III, randomized, open-label

226 Asian ethnicity and non-smoker/former light smoker Cisplatin/pemetrexed plus gefitinib vs gefitinib October 2014 PFS NCT01203917; phase IV, open-label

107 EGFR-activating mutations Gefitinib April 2014 RR Erlotinib

NCT00126581; phase II, randomized, open-label

180 Adenocarcinoma and non-smoker/former light smoker Carboplatin/paclitaxel plus erlotinib vs erlotinib June 2010 (primary completiona) PFS NCT00445848; phase II, open-label

89 Adenocarcinoma and non-smoker Bevacizumab/erlotinib July 2013 OS NCT01153984; phase II,

open-label

30 Adenocarcinoma andEGFR-activating mutations

Erlotinib February 2015 PFS NCT01260181; phase II,

open-label

30 EGFR-activating mutations Erlotinib October 2014 RR NCT01250119; phase II,

open-label

120 EGFRexon 19 deletion or exon 21 mutation Erlotinib March 2013 Prevalence ofEGFR mutations at screening NCT01310036; phase II,

open-label

208 EGFR-activating mutations Erlotinib December 2014 PFS NCT00997334; phase II,

open-label

60 EGFR-activating mutations Erlotinib October 2012 (primary completiona) Frequency of mutations conferring resistance NCT00137839; phase II, open-label

75 Female non-smokers or former light smokers with adenocarcinoma

Erlotinib April 2013 RR

NCT01344824; phase II, open-label

40 Non-smoker/former light smoker Carboplatin/pemetrexed plus bevacizumab/ erlotinib December 2015 PFS NCT01131429; phase II, randomized, open-label

60 Adenocarcinoma andEGFR-activating mutations Cisplatin/docetaxel vs erlotinib June 2015 OS NCT00661193; phase II, randomized, open-label

98 EGFR-activating mutations Carboplatin/paclitaxel plus erlotinib vs erlotinib October 2013 (primary completiona ) PFS NCT01342965; phase III, randomized, open-label

210 EGFR-activating mutations Cisplatin/gemcitabine vs erlotinib

December 2013 PFS

NCT01287754; phase IV, open-label

24 EGFR-activating mutations Erlotinib December 2015 PFS Afatinib

NCT01121393; phase III, randomized, open-label

364 Adenocarcinoma andEGFR-activating mutations Cisplatin/gemcitabine vs afatinib May 2014 PFS NCT00949650; phase III, randomized, open-label

330 Adenocarcinoma andEGFR-activating mutations Cisplatin/pemetrexed vs afatinib December 2013 PFS Dacomitinib NCT00818441; phase II, open-label

114 Adenocarcinoma and non-smoker/former light smoker orEGFR-activating mutations

Dacomitinib March 2015 PFS

EGFR, epidermal growth factor receptor; KRAS, Kirsten rat sarcoma viral oncogene homolog; NR, not reported; NSCLC, non-small cell lung cancer; OS, overall survival; PFS, progression-free survival; RR, response rate; TKI, tyrosine kinase inhibitor.

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a prospective, randomized phase III study that compared erlotinib with platinum-based chemotherapy in chemonaive patients with

advanced NSCLC whose tumors hadEGFRmutations. Among 173

patients evaluable at interim analysis, the difference in median PFS (9.4 with erlotinib vs 5.2 months with chemotherapy; HR, 0.42; 95% CI, 0.27–0.64; P< 0.0001) was statistically significant, resulting in early closure of the trial.87As shown inTable 2, final

PFS and RR results favored erlotinib but there was no significant difference in median OS, although a high rate of crossover from chemotherapy to EGFR TKIs (most commonly erlotinib) was noted. Results from these 2 recent phase III clinical trials of erlotinib could lead to approval of erlotinib in the first-line setting for patients withEGFRmutation-positive NSCLC.

Afatinib (BIBW2992)

Afatinib is an irreversible TKI that might potentially overcome the resistance of first-generation EGFR TKIs. In preclinical studies, afatinib has demonstrated activity against mutant EGFRin lung cancer models.23The phase II LUX-Lung 2 trial evaluated afatinib

in patients with advanced NSCLC whose tumors hadEGFR muta-tions and who were previously untreated or had received 1 prior chemotherapy regimen.88Among 129 patients who received

afati-nib as first- or second-line treatment, median PFS was 10.1 months (95% CI, 8.12–13.80 months) and median OS was 24.8 months (95% CI, 21.98–38.74 months). Both PFS and OS were numerically longer with first-line treatment (12.0 months and not yet reached, respec-tively) vs second-line treatment (8.0 months and 23.3 months, respectively). Whereas median PFS was 13.7 months in patients with deletion-19 mutations (12.0 months first-line; 13.7 months second-line) as well as in patients with L858R mutations (15.6 months first-line; 8.0 months second-line), it was 3.7 months in patients with other mutations (4.5 months first-line; 3.2 months second-line). OS results followed a similar trend, with median durations of 38.7 months for deletion-19 mutations (not reached first-line; 38.7 months second-line), 31.5 months for L858R muta-tions (not reached first-line; 23.3 months second-line), and

16.3 months for other mutations (15.5 months first-line;

16.9 months second-line). LUX-Lung 3 (NCT00949650) is the larg-est prospective trial inEGFRmutation-positive lung cancer evalu-ating the efficacy and safety of first-line afatinib compared to standard chemotherapy with cisplatin and pemetrexed. Prelimin-ary data from LUX-Lung 3 show that first-line afatinib significantly prolonged PFS vs standard chemotherapy (median 11.1 months vs 6.9 months with pemetrexed/cisplatin; HR, 0.58;P= 0.0004) and

improved the RR (56% vs 23%;P <0.0001) with an associated delay in worsening of lung cancer-related symptoms (cough [P= 0.0072] and dyspnea [P= 0.0145]) inEGFRmutation-positive lung adeno-carcinoma.89LUX-Lung 6 (NCT01121393) is an ongoing phase III trial that is evaluating the efficacy and safety of first-line afatinib compared to the chemotherapy combination of cisplatin and gem-citabine in molecularly selected patients (Table 3).

Dacomitinib (PF-00299804)

The other ErbB family TKI dacomitinib has more attractive pharmacokinetic properties compared with the first-generation irreversible pan-ErbB inhibitor CI-1033 (including greater bioavail-ability, longer half-life, larger volume of distribution, and lower clearance), and it has demonstrated preclinical activity in cancer models withEGFRandErbB2mutations that are resistant to gefiti-nib.25,90,91Dacomitinib has been evaluated in an ongoing phase II

study in patients with adenocarcinoma who were either non-smokers/former light smokers or had documentedEGFRmutations. In a preliminary analysis of 27 patients withEGFR-activating muta-tions, PFS rates at 4, 6, and 9 months were 95.7%, 84.7%, and 84.7%, respectively.92 More recently, preliminary data were presented

specifically for 47 patients with exon 19 or 21 mutations, which in-cluded a PR rate of 74%, 1-year PFS of 77%, and median PFS of 17 months (similar outcomes for each individual mutation).93

Conclusion

It has been established that patients with advanced lung cancer harboring EGFR-activating mutations are more likely to benefit from EGFR TKI therapy than patients withEGFRwild-type disease. Although there is currently no consensus on the optimal EGFR mutation detection method, technologies for identifying EGFR mutations and other molecular markers are rapidly evolving. Based on the activity of EGFR TKIs in clinically and/or molecularly se-lected lung cancer patients, ongoing clinical trials are evaluating EGFR and ErbB family inhibitors in these patients as first-line ther-apy. The results of these studies may help to determine the poten-tial of these agents in the first-line setting in NSCLC.

Conflicts of interest statement

All of the authors declare no conflicts of interest. Table 4

Grade 3/4 AE profiles of EGFR inhibitors in molecularly selected NSCLC patient populationsa

. Agent Source Reported grade 3/4 AEsb,c

Reversible EGFR inhibitors Gefitinib WJTOG340584

ALT elevation (24%), AST elevation (14%), rash (2%), fatigue (2%), diarrhea (1%), nausea (1%), paronychia (1%), sensory disturbance (1%) NEJ00282 Aminotransferase elevation (26%), appetite loss (5%), rash (5%), fatigue (3%), pneumonitis (3%), diarrhea (1%), arthralgia (1%),

neutropenia (1%) Erlotinib OPTIMAL86

ALT elevation (4%), rash (2%), diarrhea (1%), stomatitis (1%), infection (1%) EURTAC87

Rash (13%), fatigue (6%), diarrhea (5%), aminotransferase elevation (2%), neuropathy (1%), arthralgia (1%), pneumonitis (1%), anemia (1%) Irreversible ErbB family inhibitors

Afatinib Yang et al88 Rash/acne (7% with 40 mg; 28% with 50 mg), diarrhea (7% with 40 mg; 22% with 50 mg)d

Dacomitinib Mok et al92

Diarrhea (15% with 45 mg; 0% with 30 mg), dermatitis acneiform (15% with 45 mg; 0% with 30 mg), stomatitis (3% with 45 mg; 0% with 30 mg)e

AE, adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer. a

Based on published phase III clinical trial data for reversible EGFR inhibitors in molecularly selected patients and available phase II clinical trial data for the irreversible EGFR inhibitors.

b All gradeP3 AEs reported. c Incidence of AEs may vary by dose. d

Ranges reported correspond with 2 doses of afatinib (40 and 50 mg). e

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Acknowledgments

This work was supported by Boehringer Ingelheim Pharmaceu-ticals, Inc (BIPI). Editorial assistance was provided by Staci Heise, PhD, of MedErgy, which was contracted by BIPI for these services. The authors meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE), were fully responsible for all content and editorial decisions, and were involved at all stages of manuscript development. The authors re-ceived no compensation related to the development of the manuscript.

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Figure

Fig. 1. The EGFR signaling pathway. Upon ligand binding, EGFR or another ErbB family member dimerizes and is activated through phosphorylation of its intracellular tyrosine kinase domain
Fig. 2. Frequency of mutations in exons 18–21 of the EGFR gene and the association with responsiveness to EGFR TKIs

References

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