Oncogene (2007) 26, 5693–5701. doi:10.1038/sj.onc.1210383; published online 12 March 2007

EGFR kinase domain mutations – functional impact and relevance for lung cancer therapy

D Irmer1, J O Funk1 and A Blaukat1

1Oncology Research Darmstadt, Merck KGaA, Darmstadt, Germany

Correspondence: Dr A Blaukat, Oncology Research Darmstadt, Merck KGaA, Frankfurter Str. 250, A25/501, 64293 Darmstadt, Germany. E-mail:

Received 20 October 2006; Revised 15 December 2006; Accepted 1 January 2007; Published online 12 March 2007.



In 2004 remarkable clinical responses in non-small-cell lung cancer (NSCLC) patients treated with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib were reported to correlate with the presence of certain somatic EGFR kinase domain mutations in tumors. Since then, a surge of enthusiasm has been encountered in the field of molecular and clinical oncology. Beyond the promise of a tailored medicine, questions about the molecular mechanisms underlying the observed effects have arisen. In vitro analysis of NSCLC cells with endogenous EGFR mutations, recombinant expression of EGFR variants by transfection of several cell lines and the generation of transgenic mice expressing mutant EGFR were applied to study the impact of these genetic alterations on cellular signaling and cell fate. This review outlines the current mechanistic knowledge derived from such studies and discusses the relevance of EGFR kinase domain mutations for EGFR-directed therapies, including monoclonal antibodies.


EGFR, mutation, NSCLC, gefitinib, erlotinib, cetuximab


EGFR and cancer

The ErbB family encompasses four members, all of which are receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), ErbB2/Neu/Her2, ErbB3/Her3 and ErbB4/Her4. They consist of an extracellular ligand-binding domain connected by a short transmembrane stretch with an intracellular tyrosine kinase domain. ErbBs are activated by secreted growth factors, such as EGF. Their binding induces structural changes of the receptors inducing homodimer and heterodimer formation, followed by an increase of the kinase activity and subsequent phosphorylation of intracellular tyrosine residues. These phosphotyrosines recruit specific partner proteins that trigger intracellular signaling pathways (Pawson et al., 2001). ErbB2 forms dimers with all other ErbB family members, yet cannot bind any known natural ligand by itself, whereas ErbB3 has a nonfunctional kinase domain and can only signal in heterodimeric complexes. Signaling pathways activated by ErbB family receptors, include phosphatidyl-inositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades (Yarden, 2001).

Overexpression of EGFR together with ligand secretion and concomitant receptor activation provides multiple advantages to tumors promoting cell proliferation, survival, angiogenesis, invasion and metastasis (Normanno et al., 2005). In addition to ligand stimulation and transactivation by G-protein-coupled receptors, deregulated ErbB signaling may be caused by mutations. The most common alteration is a deletion of EGFR encompassing residues 6–273, called EGFRvIII. Despite being unable to bind growth factors, this variant is constitutively phosphorylated and elicits downstream signaling qualitatively and quantitatively different from ligand-activated wild-type EGFR (Pedersen et al., 2001). EGFRvIII confers enhanced tumorigenicity (Damstrup et al., 2002) and has been detected, for example, in gliomas (57–86%) and in non-small cell lung cancer (NSCLC) (5–39%) (Moscatello et al., 1995; Okamoto et al., 2003; Ji et al., 2006).

Since the 1980s when cancer treatment via EGFR inhibition was suggested for the first time, a variety of EGFR-directed therapeutics have been developed and some of them are in clinical use today (Sato et al., 1983; Harari, 2004). These drugs belong to two major classes, monoclonal antibodies and small-molecule tyrosine kinase inhibitors (TKIs). TKIs usually bind to the ATP-binding pocket of the kinase domain, thereby blocking EGFR autophosphorylation and activation leading to inhibition of cell proliferation, induction of apoptosis, delay in cell cycle progression and anti-angiogenic effects (Noble et al., 2004). Monoclonal antibodies inhibit receptor activation by interference with growth factor binding, but may also accelerate receptor internalization and eventually degradation (Oliveira et al., 2006). Another important aspect of antibodies is their interaction with the host immune system that can result in antibody-dependent cellular cytotoxicity (Mellstedt, 2003). EGFR inhibitors show favorable tolerability with manageable adverse events and laboratory as well as clinical studies have demonstrated significant antitumor effects, especially together with radiation or chemotherapy. By 2006 five anti-EGFR drugs have been approved for cancer treatment: the TKIs gefitinib and erlotinib, and the monoclonal antibodies cetuximab, panitumumab and nimotuzumab (Supplementary Table 1). Furthermore, several additional therapeutics are clinically tested, including bi-specific EGFR/ErbB2 as well as EGFR/VEGFR inhibitors and several monoclonal antibodies (Heymach et al., 2006).

Lung cancer is an important indication for EGFR-directed therapies. Clinically, four main histologic types of lung cancer are distinguished and classified in small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), the latter including adenocarcinomas, squamous and large-cell carcinomas. Adenocarcinomas are the most common type, especially in females and never-smokers. Lung cancer is often diagnosed not before appearance of clinical symptoms, which may be due to primary disease, metastasis or formation of neoplasms (Fong et al., 2003). This implies that drugs for NSCLC treatment must be efficient enough to counteract tumors of advanced stages.


EGFR kinase domain mutations

In May 2004 two groups independently described EGFR kinase domain mutations in NSCLC patients with clinical response to gefitinib (Paez et al., 2004; Lynch et al., 2004a), suggesting the discovery of the first molecular response marker for TKI treatment. Meanwhile, the initial findings are substantiated, providing strong evidence that the presence of certain EGFR mutations in NSCLC positively correlates with tumor sensitivity to gefitinib. More than 15 alterations at different sites in or close to the EGFR kinase domain have been described so far, small deletions in exon 19 encompassing the leucin-avginin-glutamate-alanin (LREA) motive (approx48% of mutations), point mutations, such as L858R (approx38%), and in-frame deletions/insertions in exon 20 (approx6%) being most abundant (Janne et al., 2005; Pao and Miller, 2005) (Figure 1). They are referred to as the classical or conventional EGFR kinase domain mutations to underline that they confer comparable responses towards TKIs, whereas other alterations also depicted in Figure 1 display variable impacts (Chen et al., 2006). The fact that EGFR mutations on their own do not affect binding affinities of TKIs (Fabian et al., 2005) triggered a variety of laboratory analyses using different in vitro and animal models with the aim to better understand the molecular basis of EGFR mutations in tumor homeostasis.

Figure 1.
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EGFR kinase domain mutations in NSCLC. Structural organization of EGFR and localization of clinically relevant alterations in the kinase domain (yellow boxes). Frequencies are indicated and mutations that may confer resistance to gefitinib (T790M) or erlotinib (T790M, E884K) are highlighted (orange boxes).

Full figure and legend (140K)


In vitro studies

Well-characterized laboratory cell lines provide convenient and controllable models for mechanistic and pharmacological studies. Lynch et al. initially showed in transiently transfected COS-7 cells that gefitinib inhibited the EGF-induced phosphorylation of L858R and delL747-P753insS mutants more efficiently than that of wild-type EGFR. These basic findings have been reproduced using a variety of cell lines, either transiently or stably expressing mutant EGFR (Supplementary Table 2). Similar results are described for erlotinib, suggesting that the observed sensitization towards TKIs by specific EGFR mutations could be a general phenomenon not limited to a certain chemical inhibitor structure (Pao et al., 2004; Amann et al., 2005; Pao and Miller, 2005). Yet, not all mutants display the same degree of sensitivity in the various recipient cells. Although gefitinib effectively inhibits growth of cells transfected with classical EGFR mutants or EGFR-G719S, it is much less active on cells expressing for example, EGFR-S768I or -E709G (Chen et al., 2006). Furthermore, anchorage-independent growth of clones with classical mutations was sensitive to gefitinib while cells expressing the nonclassical mutant EGFR-D770-N771insNPG were rather resistant to this treatment (Greulich et al., 2005; Jiang et al., 2005).

How mutant receptor activation is altered in comparison to wild-type EGFR is not yet fully understood. Although some studies suggest an overall lower or equivalent growth factor-independent activation of classical EGFR mutants (Pao et al., 2004; Amann et al., 2005), others found a constitutive activation (Arao et al., 2004; Sordella et al., 2004; Lynch et al., 2004a) that may depend on whether or not DNA is stably integrated into the genome (Greulich et al., 2005). Mechanistic studies have shown that individual phosphorylated tyrosine residues in RTKs trigger different downstream signaling pathways via recruitment of specific adaptor proteins (Pawson et al., 2001). Consistent with this model, a link between increased phosphorylation of certain tyrosines of mutant EGFR and constitutive activation of Akt and signal transducer and activator of transcription 3/5 (STAT3/5) was frequently observed, whereas the MAPK pathway does not seem to be affected in transfected cells (Sordella et al., 2004; Amann et al., 2005; Greulich et al., 2005). A potential role of the PI3K/Akt pathway in mutant EGFR signaling was also derived from a clinical NSCLC study that suggested a correlation of response to gefitinib with high basal activation of Akt (Cappuzzo et al., 2004). Differences between mutant and wild-type EGFR signaling were less evident during longer time periods of EGF stimulation and quantitatively as well as quantitatively different in individual cell lines (Amann et al., 2005; Chen et al., 2006). Obviously, the cellular background has an impact, with secretion of growth factors, overexpression of EGFR and co-expression of ErbB2/3 as potentially important parameters (Worthylake et al., 1999; Engelman et al., 2005; Singh and Harris, 2005). When interpreting studies with transfected cells it should be kept in mind that forced overexpression of tyrosine kinases, such as EGFR, favors promiscuous effector interactions and subsequent signaling events that may differ from endogenous EGFR (Jones et al., 2006).

In contrast to wild-type receptor, expression of mutant EGFR in transfected NIH3T3 cells was depleted upon treatment with an Hsp90 inhibitor, suggesting that, similar to many other oncogenes, the stability of mutant EGFR critically depends on chaperone function (Shimamura et al., 2005). Furthermore, transfection studies with H1299 cells revealed prolonged EGF-induced phosphorylation of Y1045 in EGFR-L858R and -delE746-A750. Phosphorylated Y1045 recruits Cbl, an ubiquitin ligase that labels EGFR for degradation by the proteasome and indeed challenge with EGF led to enhanced ubiquitination of these mutants as compared to wild-type EGFR (Chen et al., 2006) (Figure 2).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Major differences between NSCLC cells expressing wild-type and mutant EGFR. Wild-type EGFR (right) is activated by ligands like EGF (green) through homodimerization and subsequent autophosphorylation of tyrosines (blue). Activation of mutant EGFR (left) may occur in the absence of ligand but can be further augmented. EGF-induced activation of signaling cascades (e.g. PI3K/Akt, STAT, MAPK) depends on cellular context and is enhanced with mutant EGFR. Although sensitivity to TKIs (yellow) is context-dependent in wild-type background, cells expressing mutant EGFR are sensitized. Whereas ubiquitin-mediated (Ubi; orange) degradation of wild-type EGFR is ligand-induced, mutant EGFR displays constitutively elevated ubiquitinylation and its maturation critically depends on Hsp90 (red cross).

Full figure and legend (274K)

Studies with cultured NSCLC cells carrying mutant EGFR endogenously may more closely reflect the pathophysiology of NSCLC, but as drawbacks many of these cell lines are incompletely characterized, unevenly distributed in the scientific community preventing independent confirmation of data and sometimes hard to transfect making them less accessible for dominant-negative approaches and RNA interference. Furthermore, NSCLC cells display variable genetic backgrounds, and although this reflects the molecular heterogeneity of the disease, it also limits the value of mechanistic conclusions based on comparisons of a limited number of cell lines. On the other hand, in a given genetic background, EGFR mutations may have a more pronounced functional impact, for example, on cell growth or survival (Sordella et al., 2004).

The fundamental observation in NSCLC cells resembles the one described in transfected cells: mutant EGFR sensitizes towards TKIs (Supplementary Table 2 and 3). But there are exceptions that may put this dogmatic view into perspective. HCC2279 cells expressing EGFR-del746-750 are rather resistant to gefitinib-mediated signaling inhibition (Fujimoto et al., 2005), while H292 cells with wild-type EGFR are exquisitely sensitive (Janmaat et al., 2006). An additional implication from NSCLC cell data is that the EGF-induced MAPK activation by mutant EGFR could be more sensitive to gefitinib and geldanamycin treatment (Paez et al., 2004; Amann et al., 2005; Mukohara et al., 2005; Shimamura et al., 2005). However, it is important to note that many wild-type EGFR NSCLC cell lines used for comparison behave similarly, questioning whether all observed signaling alterations are indeed causally linked to EGFR mutations (Tracy et al., 2004; Amann et al., 2005).

Gefitinib resistance of NSCLC cells with mutant EGFR and accumulating data about additional determinants suggest that EGFR mutations alone may not be sufficient to explain the observed TKI sensitivity. ErbB3 has been described as a candidate and interestingly two selected gefitinib-hypersensitive cells with EGFR mutations do co-express ErbB3. However, forced expression of ErbB3 in gefitinib-resistant cells does not render them sensitive, indicating that also the presence of ErbB3 and its activation are not enough for sensitization. It was suggested that expression levels of other ErbB family members conjoint with their ligands could be stronger indicators for gefitinib response than EGFR mutations alone (Amann et al., 2005; Engelman et al., 2005; Fujimoto et al., 2005). Microarray profiling of a panel of NSCLC cells identified increased ErbB3, E-cadherin and TACSTD2 levels as potential indicators for cellular response to gefitinib (Coldren et al., 2006). More systematic and unbiased studies with NSCLC cell lines not only focusing on a handful of potential candidates should help to better understand the complex biology behind TKI sensitization.

In contrast to the constantly increasing dataset for TKIs, only a few studies have addressed implications for monoclonal antibody therapies. Two gefitinib-sensitive NSCLC cell lines with EGFR mutations showed only moderate response to cetuximab, whereas three wild-type EGFR expressing cell lines and one with EGFR-delE746-A750 reacted comparably to both drugs (Amann et al., 2005; Mukohara et al., 2005). It therefore appears that in vitro effects of gefitinib and cetuximab are similar on wild-type EGFR and that EGFR mutations do not sensitize towards cetuximab. Because immunogenic effects elicited by monoclonal antibodies cannot be addressed, future studies focusing on more relevant in vivo models are needed to substantiate these findings.


In vivo models and clinical aspects

As EGFR mutations were initially described in NSCLC patients with good response to gefitinib, their potential importance was immediately evident. Subsequent analysis of various clinical studies substantiated the initial data. The majority of investigators did find a statistically significant correlation between the presence of EGFR mutations in NSCLC and a survival benefit for patients treated with gefitinib, either used upfront or in combination with conventional chemotherapy (Supplementary Table 4), while only a few studies failed to confirm such an association (Bell et al., 2005a; Cappuzzo et al., 2005a). Recently, encouraging data on TKI monotherapy as first-line regimen in chemo-naïve NSCLC have been reported: pre-selection of patients based on EGFR mutations increased TKI response rates from 22.7–30 to 75–90% (Asahina et al., 2006; Giaccone et al., 2006; Niho et al., 2006; Paz-Ares et al., 2006). As is evident from these data, the availability of reliable tests for detection of EGFR mutations in tumor biopsies and serum samples could be fundamental for future NSCLC therapy (Nagai et al., 2005).

EGFR mutations appear to be more frequent in females, never-smokers and in patients with adenocarcinomas, of whom those with bronchioalveolar carcinoma (BAC) seem to display the highest TKI sensitivity (Supplementary Tabel 4). Furthermore, Asians show a much higher prevalence for EGFR mutations (20–40%) than Caucasians (<10%) (Pao and Miller, 2005; Calvo and Baselga, 2006). However, there are also several reports about patients resistant to gefitinib regimen despite the presence of EGFR mutations, whereas others with wild-type EGFR responded well in the same study. Interestingly, EGFR gene amplification has also been positively correlated with NSCLC response to TKI regimen, but it is still not clear whether it translates to increased EGFR protein levels detectable by immunohistochemistry (Takano et al., 2005; Hirsch et al., 2006).

Upto now, EGFR kinase domain mutations have only been sporadically described in other tumors, such as colorectal (approx0.3%) and head and neck cancer (0–7%) suggesting that response rates to cetuximab seen in these diseases cannot correlate with their presence (Barber et al., 2004; Lee et al., 2005; Sihto et al., 2005). Furthermore, metaplastic breast carcinomas overexpress EGFR and are sensitive to TKIs, but in 47 patient samples no EGFR mutation was detected (Robertson et al., 2003; Reis-Filho et al., 2006). Altogether, the frequency of EGFR kinase domain mutations in cancers other than NSCLC appears to be too low to explain clinical responses observed, but further clinical studies are needed to corroborate this impression.

Interestingly, current data seem to point to a generally good prognosis of tumors with EGFR kinase domain mutations, independent of TKI regimen. In the TRIBUTE trial chemo-naïve NSCLC patients with EGFR mutations showed longer overall survival irrespective of the treatment, a finding also supported by the INTACT study conducted with prior chemotherapy (Bell et al., 2005a; Eberhard et al., 2005). Another investigation suggested that NSCLC patients with EGFR-L858R mutations generally survive longer than those with exon 19 mutations (Shigematsu et al., 2005a).

In a laboratory study with transgenic mice expressing EGFR-L858R and EGFR-delL747-S752 histopathological differences and variable tumor growth was observed. Although EGFR-L858R mice rapidly develop diffuse BAC, EGFR-delL747-S752 animal display longer tumor latency. In both cases tumor development and maintenance is dependent on mutated EGFR and significant tumor shrinkage is achieved upon treatment with erlotinib (Politi et al., 2006). In a similar study with mice transgenic for EGFR-L858R and an EGFR exon 19 deletion no differences in histopathology and tumor latency were observed (Ji et al., 2006). In xenograft experiments with NR6 cells stably transfected with EGFR variants deceased tumor latency of EFGR-del746-752 as compared wild-type or EGFR-L858R was seen (Carey et al., 2006). Also clinical studies suggest differences in the biology of these two conventional EGFR mutations (Mitsudomi et al., 2005; Jackman et al., 2006). For example, median survival upon TKI treatment was longer for patients with EGFR exon 19 deletions as compared to those carrying a L858R mutation (34 vs 8 months) (Riely et al., 2006).

Despite similar key pharmacological, erlotinib did not improve survival in NSCLC patients with EGFR kinase domain mutations in the BR.21 trial (Tsao et al., 2005). Albeit non-classical mutations identified in this trial could be sequencing artefacts an adjacent analysis including only biopsies from patients with classical mutations supported the original finding (Marchetti et al., 2006). The TRIBUTE erlotinib/chemotherapy combination trial did provide evidence for a better objective response for NSCLC patients with EGFR mutations, but again failed to prove any significant survival benefit (Eberhard et al., 2005). However, other clinical data did show comparable response and survival of NSCLC patients with EGFR mutations treated with erlotinib or gefitinib (Pao et al., 2004, 2005b; Riely et al., 2006). Reasons for these partially contradicting data may be racial diversities of patients, predominance of different types of mutations or sensitivity and accuracy of methods used to identify mutations in patients' material (Johnson and Janne, 2005; Takano et al., 2005; Shepherd and Tsao, 2006). An interesting case study about a NSCLC patient that developed erlotinib resistance and subsequently responded well to gefitinib treatment revealed an EGFR-L858R/E884K mutation. Laboratory studies confirmed that the EGFR-E884K confers sensitivity to gefitinib, but resistance to erlotinib (Choong et al., 2006). It is still unclear how frequent EGFR-E884K occurs in NSCLC, because the corresponding exon 22 was often not covered in previous studies.

Upto now, only very limited data are available for cetuximab: three patients with EGFR kinase domain mutations who relapsed after initial disease control by cetuximab showed all partial response to subsequent gefitinib treatment (Mukohara et al., 2005). In a phase II single agent trial seven of 33 NSCLC patients achieved stable disease and two had a partial response, both expressing wild-type EGFR (Lynch et al., 2004b). Another study including 38 NSCLC patients that received cetuximab monotherapy revealed 13 patients with stable disease three of them carrying classical EGFR mutations (Tsuchihashi et al., 2005). These preliminary data indicate that clinical response to cetuximab in NSCLC does not seem to correlate with the presence of EGFR mutations.


Mechanisms of resistance and clinical relapse

Resistance against TKIs is common and may either be intrinsic or can be acquired during drug treatment. This has been impressively shown for Bcr-Abl kinase in chronic myeloid leukemia where mutations conferring imatinib resistance can arise during treatment, but may also be detected at low frequency in patients who had not received the drug before (Roche-Lestienne et al., 2002).

EGFR mutations in NSCLC may not necessarily be evenly distributed throughout cell populations. Only in 11 of 30 common NSCLC cell lines a homogenous EGFR mutation status was observed, whereas in additional 12 examples just subpopulations had mutant EGFR (Nagai et al., 2005). This finding points to genetic instability of NSCLC tumors and heterogeneity of EGFR within them. Further, it may also explain the variability of the clinical responses of NSCLC to TKI treatment.

The recently identified EGFR-T790M mutation may occur in addition to other alterations and confers resistance to TKIs (Gow et al., 2005; Kobayashi et al., 2005a; Pao et al., 2005a). Intriguingly, it was initially generated in laboratory studies when findings from imatinib-resistant Bcr-Abl were projected to EGFR (Blencke et al., 2003). However, current clinical data suggest a low frequency of this alteration in NSCLC and the incidence of EGFR-T790M appears to be independent from treatment, though subpopulations of corresponding cells may be enriched during TKI regimen (Pao et al., 2005a, Toyooka et al., 2005; Inukai et al., 2006). Effects of gefitinib on H1975 cells with the L858R-T790M mutation were variable either described as moderate or equal to wild-type EGFR cells (Sordella et al., 2004; Pao et al., 2005a). Interestingly, irreversible EGFR inhibitors such as HKI-292 or CL-387 785 seem to overcome resistance in H1975 as well as in transfected BaF3 cells (Kwak et al., 2005; Kobayashi et al., 2005b). Furthermore, treatment of H1975 xenografts with cetuximab showed marked tumor regression, whereas gefitinib was basically inactive (Perez-Torres et al., 2006). Of note, it was suggested that EGFR-T790M could be associated with inherited susceptibility to lung cancer (Bell et al., 2005b).

Systematic approaches to the problem of relapse of NSCLC patients that initially responded to TKI therapy are rare. To address this issue, resistant clones of cells either carrying EGFR-delE746-A750 or -L858R mutations were generated by long-term treated with gefitinib. These cells displayed various molecular alterations at the EGFR level (e.g. increased internalization, loss of sensitizing mutations, gain-of-resistance conferring T790M mutation, receptor downregulation) as well as downstream of EGFR (e.g. increased Akt and decreased necrosis factor kappaB activation, loss of phosphatase and tensin homolog (PTEN) and reduced sensitivity to apoptosis induction) (Ando et al., 2005; Kokubo et al., 2005; Kwak et al., 2005; Engelman et al., 2006) (Supplementary Table 5). In future, these findings should be substantiated in studies with gefitinib-resistant human NSCLC specimen.



The discovery of EGFR kinase domain mutations in NSCLC biopsies and the correlation with clinical response to gefitinib was certainly a breakthrough in biomarker research and a first step towards a personalized cancer treatment with EGFR inhibitors. The described mutations do not alter binding properties of the drugs to their target, but rather seem to result in a moderate amplification of EGFR signaling pathways with PI3K/Akt and STAT being predominantly affected. Despite substantial experimental efforts it is still unclear how this mechanistically translates into an increased TKI sensitivity, yet the mutant EGFR scenario in NSCLC resembles to what has been described as 'oncogene addiction' (Gazdar et al., 2004).

The picture is further complicated by initial evidence that the mutations do not seem to affect response to monoclonal antibodies, such as cetuximab that should more specifically inhibit EGFR. A very provocative deduction could be that off-target effects of the not entirely EGFR-specific TKIs contribute to the observed clinical benefits in patients with mutant receptor. The fact that a significant number of patients (less than or equal to20%) despite carrying these mutations do not respond to gefitinib further complicates the scenario. It is also still unclear which factors account for the difference between stable disease and partial response and why patients with EGFR mutations do not show total tumor regression. Obviously, there are additional variables other than EGFR mutations that are important for lung cancer tumorigenesis and that are not hit by EGFR-directed therapy.

Due to the limited availability of patient samples exploratory in vitro studies are indispensable. In particular systematic experiments not only focusing on single candidates, but being completely unbiased or combining different response markers to study their relevance in concert are necessary. Such analyses may finally also help to define appropriate combination regimens for EGFR-directed therapeutics. Using in vitro models, it should be kept in mind that several properties of tumors are not reflected, including angiogenesis, oxygenic stress, physical barriers limiting drug uptake, but also defence against challenges conveyed by the patients' immune system. However, to optimize TKI therapy for individual cancer patients and to identify additional biomarkers, clinical studies that obligatory include the collection of tumor biopsies, blood and eventually other specimen are indispensable.



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We offer our apologies to scientists whose work we could not appreciate due to space limitation. We are grateful to Merck KGaA for supporting our work and to all colleagues who critically reviewed this manuscript.

Supplementary Information accompanies the paper on the Oncogene website (