Correspondence *Department of Medical Oncology, Vrije Universiteit Medical Center, 1117 De Boelelaan, HV 1081 Amsterdam, Netherlands

Email g.giaccone@vumc.nl

Introduction

Lung cancer is the leading cause of cancer-related death in the Western world and the mortality rate is rapidly increasing in Asia; 1.2 million cancer deaths worldwide were from lung cancer in the year 2002.¹ There are primarily two major types of lung cancer: non-small-cell lung cancer (NSCLC) and small-cell lung cancer. More than 50% of NSCLC patients are candidates for systemic treatment with chemotherapy, either for advanced disease, or as adjuvant or neoadjuvant treatment, in addition to local therapy. Chemotherapy has, however, modest activity in NSCLC and, in the past few years, several drugs that are more specific for cancer cell targets have shown activity in NSCLC.^{2, 3} There are two molecular-targeted agents approved for the treatment of advanced NSCLC: gefitinib (Iressa^®, AstraZeneca, Wilmington, DE) and erlotinib (Tarceva^®, OSI Pharmaceuticals Inc, Melville, NY). Both agents are small molecules that belong to the quinazolinamine class and inhibit the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) by competing with ATP for the ATP-binding site.⁴ Besides these two rather selective tyrosine kinase inhibitors (TKIs) of EGFR, other TKIs with a broader spectrum of activity, and monoclonal antibodies to the extracellular domain of the receptor, are also being tested in advanced NSCLC.⁴ Among broader spectrum TKIs are lapatinib and canertinib, which have activity on more members of the ErbB family of receptors, and ZD6474 and AEE788, which inhibit the vascular endothelial factor receptor in addition to EGFR. After failure of chemotherapy, gefitinib and erlotinib are able to induce major objective responses in approximately 10% of Caucasian patients and 25–30% of Japanese patients (gefitinib) with NSCLC tumors. The response rate to the EGFR monoclonal antibody cetuximab (Erbitux^®, ImClone Systems/Bristol-Myers Squibb) appears similar in the same setting, but no experience is available in Asian patients.⁴

Biological aspects of EGFR mutations associated with tyrosine kinase inhibitor responsiveness

A major breakthrough in the field of EGFR-targeted therapy was seen in 2004 with the identification of somatic mutations in the EGFR gene, which were closely associated with a favorable clinical response to gefitinib and erlotinib treatment in NSCLC patients.^{5, 6, 7} These genetic alterations consisted of small IN-FRAME DELETIONS or POINT MUTATIONS in EGFR exons 18–24, which encode the kinase domain of the protein and are clustered in two mutational 'hot spots' in the EGFR gene. The most frequently detected alterations were small deletions in exon 19 that eliminate amino acids 747–750 (Leu-Arg-Glu-Ala), located around the active site of the kinase, and point mutations in exon 21 that result in the amino acid substitution Leu858Arg, a residue located in the activation loop.^{5, 6, 7}

The in vitro analysis of the phenotypic effects associated with the expression of mutant EGFR proteins provided information on the molecular and cellular mechanisms underlying the enhanced responsiveness to gefitinib and erlotinib in patients with NSCLC tumors that have EGFR mutations. The efficiency of these drugs was significantly higher, in terms of blocking the activity of the receptor and decreasing cell viability, in those cell lines harboring mutant EGFR genes. Transfection experiments in NSCLC-derived cell lines^{5, 8, 9} revealed that, although mutant EGFRs undergo more pronounced and sustained activation upon ligand binding than the wild-type receptors, their EGFR activity is more efficiently inhibited by gefitinib and erlotinib.^{6, 7, 8} The molecular basis for such different sensitivity of mutant EGFR proteins remains to be established, as wild-type and mutant receptors do not appear to differ significantly in their ability to interact with these inhibitors in vitro.¹⁰ Importantly, binding of the ligand to the mutant EGFRs was shown to result in the selective activation of downstream signaling pathways that promote cell survival, such as the protein kinase B (Akt) and the signal transducers and activators of transcription (STAT) pathways.^{8, 9} The pro-survival signals transduced by these downstream signaling molecules appear to be essential for NSCLC cells that express mutant EGFRs, as these cells underwent massive cell death after pharmacologic inhibition of the Akt and STAT signaling or RNA INTERFERENCE-mediated specific downregulation of the mutant receptor.⁸ These observations indicate that the increased efficacy of gefitinib and erlotinib in NSCLC patients bearing EGFR mutations results, at least in part, from the abrogation of the pro-survival signals on which the tumor cells expressing a mutant receptor have become dependent.^{8, 11}

EGFR mutational status as a predictive marker of tyrosine kinase inhibitor response

The initial studies that assessed EGFR mutations selected patients with striking and long-lasting responses.^{5, 6} Mutations were most frequently detected in a subpopulation of NSCLC patients with characteristics associated with a better treatment outcome: women, non-smokers, patients of Japanese origin, and patients with adenocarcinoma histology and, in particular, bronchioloalveolar carcinoma.^{4, 12} The largest database currently available of patients treated with a single agent is from the BR.21 study, a randomized trial where erlotinib was compared with best supportive care in patients with advanced NSCLC who had failed one or two lines of chemotherapy. This study demonstrated that erlotinib significantly improved survival, although the response rate was only 8.8%.¹³ This modest response rate might not be solely attributable to such an important survival gain. It should be noted that a large proportion of patients in this study had either a minor response or stable disease; considering that one of the major effects of EGFR inhibitors observed in preclinical studies was the ability to reduce cell proliferation,¹⁴ this might indeed be an important effect. In NSCLC cell lines, apoptosis was only observed when both the extracellular signal-regulated kinase and the Akt pathways were blocked by EGFR inhibition,^{14, 15} whereas in most cell lines this was not possible and only inhibition of proliferation was observed. Of the 731 patients included in the BR.21 study, 197 were assessed for EGFR status using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and mutation analysis. In multivariate analysis, objective response was significantly associated with adenocarcinoma histology, never-smoking status, and expression of EGFRs by IHC.¹³ Univariate analysis showed that survival was significantly longer in the erlotinib arm when EGFR expression was high and tumor cells had a high number of copies of EGFR genes, detected by FISH. However, the influence of the EGFR status determined by any of the three methods used was not significant by multivariate analysis. This study casts some doubts on the validity of the mutational status of EGFR as a predictor of response and survival on larger populations of patients. In this study, however, a large number of mutations were identified (24/45, 53%) that were previously unreported. These data, therefore, might need further confirmation, perhaps by carrying out a cross-validation analysis. The functional implications of the novel mutations should also be confirmed.

The proportion of patients who benefited from anti-EGFR therapy in this study¹³ and other large clinical studies^{16, 17} exceeded the proportion of NSCLC patients expected to harbor a mutant EGFR gene, based on the prevalence of EGFR mutations determined in the initial analyses. Approximately 40–50% of patients in two large phase II studies of gefitinib derived clinical benefit, which was similar to the number of patients who experienced a major response plus stable disease.^{16, 17} These observations indicated that, although the presence of an EGFR mutation can reliably predict a favorable response to gefitinib and erlotinib treatment, a subset of patients who would benefit from these drugs would not be selected on the basis of EGFR mutational status.

The close relationship between the presence of mutant EGFR genes and the clinical response to TKIs has been consistently confirmed (Table 1). However, as the number of NSCLC patients undergoing EGFR mutation analysis increases, it has become clearer that a small, but sizable, subset of patients with mutations do not respond to TKI treatment. Conversely, it is also evident that some patients who respond to gefitinib and erlotinib therapy have wild-type EGFRs. These observations might suggest that procedures for evaluating EGFR mutational status need to be optimized, and additional markers need to be established to accurately identify all of the NSCLC patients who can benefit from anti-EGFR therapy. Similar to findings in patients with gastrointestinal stromal tumors that displayed mutations in the gene C-KIT, it appears that different EGFR mutations might confer different sensitivities to TKIs in NSCLC. In two reports from Asia (Taiwan and Japan), where the incidence of EGFR mutations is much higher than in the Western world, mutations in exon 19 of the EGFR gene appear to confer sensitivity more often than point mutations in exons 20 and 21.^{18, 19} Given the relative infrequency of EGFR mutations in Caucasians, it is difficult at present to extend these findings beyond Asian patients.

Table 1 Relationship between the presence of EGFR gene mutations and the response to gefitinib and erlotinib treatment in NSCLC patients.

Full tableFigures & Tables indexDownload Power Point slide (394K)

Optimizing EGFR mutation analysis

It has been postulated that the presence of undetected EGFR mutations might explain some of the favorable responses to TKIs observed in NSCLC patients bearing seemingly wild-type EGFRs.^{12, 20} Although several of the NSCLC series analyzed for the presence of EGFR mutations consist, wholly or in part, of frozen tissue samples (especially from resected patients), the most common type of sample available for analysis is DNA extracted from formalin-fixed, paraffin-embedded tissues. As a result of the fixation procedure, these samples usually yield low-quality, degraded DNA that can be difficult to analyze. On the other hand, in patients with advanced lung cancer, often only small diagnostic material is available, and it is often cytology that makes it difficult to implement direct sequencing, because of insufficient number of tumor cells present in the specimens. In addition, wild-type EGFR DNA sequences from the normal cells in the sample can mask the presence of mutations. In this regard, there is some evidence that the most commonly used method of polymerase chain reaction (PCR) amplification followed by direct sequencing might not detect mutations that can be identified using a different analysis method, such as SINGLE-STRAND CONFORMATION POLYMORPHISM.²¹ Furthermore, the analysis could be improved by the use of novel techniques based on denaturing high-performance liquid chromatography, which appear to be more sensitive than direct sequencing for the detection of EGFR mutations,²² as well as by developing methods to specifically determine the most prevalent types of mutations based on simple PCR and restriction enzyme digestion. Moreover, novel data indicate that the use of LASER CAPTURE MICRODISSECTION to select tumor cells might lead to the identification of EGFR mutations that are undetectable when the non-microdissected tumor tissue is analyzed.²³ However, most of these additional procedures are technically demanding and might be difficult to implement in many clinical laboratory settings. In summary, the possibility that some EGFR mutations might be difficult to detect using easy-to-implement, technically straightforward approaches highlights the importance of establishing additional predictive markers, in particular those that would not depend on PCR-based analysis of DNA extracted from tumor tissue.

Additional markers to predict response to EGFR tyrosine kinase inhibitors in NSCLC

In other molecular-targeted approaches to therapy, such as the treatment of metastatic breast cancer with the anti-human epidermal growth factor receptor (anti-HER2) antibody trastuzumab (Herceptin^®, Genentech Inc., San Francisco, CA), overexpression of the target protein (i.e. HER2) is a critical marker that is used to select patients who will benefit from treatment using EGFR TKIs.²⁴ In contrast, initial studies on NSCLC patients indicated that gefitinib treatment outcome was no different in patients who overexpressed both HER2 and EGFR, compared with those who had no HER2 overexpression.^{25, 26} Similar results were obtained in the first phase II study of erlotinib.²⁷ In a more recent report,²⁸ EGFR status was determined by using three different methods: direct sequencing to investigate the presence of mutations, FISH to assess EGFR gene copy number, and IHC to evaluate EGFR protein expression levels. In this study, 'EGFR-positivity' in any of these assays was significantly associated with a better response to gefitinib, but a better survival was found to be significantly associated only with a high EGFR gene copy number in multivariate analysis. These results are, generally, in concordance with the results obtained in erlotinib-treated patients in the BR.21 study.²⁹ Consistent results supporting the predictive value of determining EGFR gene copy number in gefitinib-treated patients have been obtained in independent studies using either FISH³⁰ or a quantitative PCR assay.²³ Interestingly, a significant correlation between the presence of EGFR mutations and a high EGFR gene copy number was seen,^{23, 28, 31} suggesting that the mutant allele of the EGFR gene is selectively amplified in tumors, as it has been observed in EGFR-mutant NSCLC cell lines.^{9, 23} Although potentially more appealing, the increased gene copy number was based on a six-category scoring system using FISH analysis and an arbitrary cut-off value by reverse transcription PCR. These assays will need validation in prospective studies and confirmation by other laboratories.

Members of the ErbB family of receptors (other than EGFR) that might heterodimerize with the EGFR, such as HER2, have also been investigated as potential predictive markers for anti-EGFR therapy. Similarly, signaling molecules that function downstream of the EGFR pathway such as K-ras and Akt, have been implicated to function as predictive markers. Although the response to gefitinib appears to be independent of HER2 levels, as determined by IHC,²⁵ the presence of somatic mutations that alter the protein structure of the HER2 kinase domain has been recently reported in NSCLC patients.^{32, 33} These mutations consist of small insertions and duplications that target a region of HER2 analogous to exon 19 deletions in the EGFR gene. HER2 mutations seem to be more frequent in patients with clinical characteristics similar to the subpopulation of patients bearing EGFR mutations but, remarkably, they never occur simultaneously with EGFR mutations. The prevalence and functional relevance of HER2 mutations should be investigated further to determine their value as markers for anti-EGFR therapy.

Interestingly, mutations in EGFR or HER2 genes have been found to be mutually exclusive with K-ras mutations.^{34, 35, 36, 37} Furthermore, the presence of K-ras mutations is associated with a lack of treatment response to gefitinib and erlotinib.³⁵ Therefore, the presence of a K-ras gene mutation, which is detected in approximately 30% of NSCLC,³⁸ is likely to constitute a useful marker for selecting those patients who will not benefit from anti-EGFR therapy. In a large, randomized study of chemotherapy with or without erlotinib as first-line therapy for advanced NSCLC patients,³⁹ EGFR mutations, detected in 13% of tumors, were associated with longer survival irrespective of treatment.⁴⁰ K-ras mutations, detected in 21% of tumors, were associated with significantly decreased time to progression and survival in patients treated with erlotinib plus chemotherapy. These results suggest that EGFR mutation might be a positive prognostic factor for survival, independent of treatment with erlotinib, and that the combination of EGFR inhibition with chemotherapy should be avoided in patients with K-ras mutations. Finally, it was reported that activation of Akt, determined using IHC and phosphorylation-specific antibodies, was associated with a better response to gefitinib compared with the response in patients who were negative for P-Akt (phosphorylated Akt).⁴¹ There was a statistically significant association between P-Akt positivity status and female gender, a history of never-smoking, and with bronchioloalveolar carcinoma histology, suggesting that the activation of this pathway might be associated with the presence of EGFR mutations. It should be noted, however, that detection of P-Akt in the absence of EGFR expression could represent the transduction of an EGFR-independent pro-survival signal, which might render anti-EGFR therapy less effective. In fact, P-Akt-positive staining in 'EGFR-negative' NSCLC (i.e. tumors lacking mutation or amplification of the EGFR gene or expressing low levels of EGFR protein) could predict a worse treatment outcome,⁴¹ whereas P-Akt-positive staining in 'EGFR-positive' cases (i.e. tumors containing a mutation, high gene copy number, or high EGFR protein levels) could identify a subgroup of NSCLC patients particularly sensitive to gefitinib. Collectively, these results indicate that the decision regarding treatment with anti-EGFR agents should ideally be based on the combined use of several markers that would be evaluated using different techniques (e.g. PCR-direct sequencing, IHC, and FISH) in different types of samples (e.g. tissue sections and extracted DNA).

Secondary mutations in the EGFR gene and acquired resistance to EGFR inhibitors

In addition to the primary resistance of anti-EGFR therapy associated with the presence of K-ras mutation,³⁵ resistance eventually develops in most NSCLC patients who initially responded to gefitinib and erlotinib but harbor EGFR mutations, leading to disease progression during treatment. Acquired resistance to EGFR inhibitors has been shown to be associated with the occurrence of an additional EGFR mutation.^{42, 43} One study described the occurrence of a secondary mutation in a patient with NSCLC following successful gefitinib treatment.⁴³ While the first mutation, a deletion in exon 19, conferred sensitivity to the treatment, a second point mutation in exon 20, leading to the substitution Thr790Met, induced resistance to several EGFR inhibitors. Another group identified the same Thr790Met mutation in two of five patients with acquired resistance to gefitinib or erlotinib⁴² and a sixth patient whose tumor progressed when treated with adjuvant gefitinib after complete resection. The mutation could not be detected in untreated tumor samples. Biochemical analysis of transfected cells and growth inhibition studies with lung cancer cell lines confirmed that the Thr790Met mutation conferred resistance in tumors with EGFR mutations that are usually sensitive to gefitinib or erlotinib.⁴² Two reports from Asia demonstrated that the Thr790Met mutation can be present from diagnosis and confer resistance to EGFR TKIs.^{44, 45}

These findings closely resemble the experience with imatinib in gastrointestinal stromal cell tumors and chronic myeloid leukemia, where secondary mutations in the C-KIT and BCR–ABL genes, respectively, have been associated with a major cause of acquired resistance to imatinib.^{46, 47} Interestingly, the EGFR protein bearing the second mutation was sensitive to CL-387,785, a specific and irreversible anilinoquinazoline TKI of EGFR, suggesting that second-generation TKIs of EGFR might have a role in the treatment of NSCLC. In this respect, agents undergoing clinical development for inhibition of the gefitinib-resistant and erlotinib-resistant tumors with two EGFR mutations (Leu858Arg and Thr790Met) were screened in vitro. This screening identified two drugs, EKB-569 and CI-1033, which have broader spectrums for ErbB family members than erlotinib and gefitinib, and showed activity against the resistant mutant.⁴⁸ EGFR mutations are much more frequent in East Asia, where the incidence exceeds 40%, and the higher incidence of these mutations is correlated with a higher response rate. Secondary mutations have been reported in Asians, but their role in modifying sensitivity to EGFR inhibitors has not yet been elucidated.^{18, 49} It has been reported that deletions in exon 19 might more accurately identify sensitive tumors than point mutations, which were found in some patients whose tumors progressed on treatment with gefitinib.¹⁸ It is quite possible that some point mutations detected in exons 20 and 21 might confer resistance rather than sensitivity. Moreover, the presence of mutations in the ATP-binding site probably does not predict response to anti-EGFR therapy using monoclonal antibodies, such as cetuximab.^{50, 51} Interestingly, it has recently been reported that two patients responded to gefitinib after failure of several chemotherapy regimens and cetuximab.⁵² This finding could indicate that monoclonal antibodies and TKIs have different mechanisms of action and might be effectively combined in order to broaden their spectrum of activity.

Selection of patients for treatment with EGFR inhibitors

The association of EGFR mutations with particular patient characteristics related to drug sensitivity, such as female gender, adenocarcinoma histology, never-smoking status, and Asian race, is striking. However, the selection of patients for treatment with these agents based solely on the presence of EGFR mutations is controversial, because not all patients who benefit from the treatment harbor a mutation, and a few patients with mutations are resistant to TKIs. It might seem that deletion mutants would confer sensitivity more consistently than point mutations, which are also harder to confirm by direct sequencing. Unlike deletion mutations, some point mutations can affect the protein conformation in a way that prevents optimal enzyme inhibition by EGFR TKIs. In addition, other factors, including amplification of the EGFR gene and the activity of molecules downstream of EGFR, such as P-Akt, and mutations of K-ras, might play a role in the definition of sensitivity to EGFR inhibitors. On the basis of available data, limiting treatment only to patients harboring an EGFR mutation might not be justified, although patients with mutations appear to have a very high chance of responding to TKI treatment. The occurrence of secondary mutations that alter the conformation of the ATP-binding pocket should be investigated, and the development of second-generation EGFR inhibitors that overcome this resistance is warranted.

Prospectively conducted clinical trials of patients selected on the basis of biological characteristics and/or clinical features need to be performed. For the time being, it might be inappropriate to exclude treatment using a TKI based solely on EGFR mutational status. The assessment of patient features, such as smoking status, gender and histology, might help guide the choice of therapy in patients with advanced NSCLC. A first-line treatment with an EGFR TKI in patients with EGFR mutations or in patients who are never-smokers, female, and with adenocarcinoma histology is certainly reasonable either as single agent or in combination with chemotherapy. An EGFR TKI might be preferable in second- and third-line therapy in patients with these characteristics, instead of chemotherapy, given the lower toxicity profile and the ease of administration. Several studies that take into account EGFR mutations and clinical features for patient selection are now underway or being planned. In light of the appearance of secondary mutations conferring resistant to TKIs, clinical studies should try to incorporate repeated biopsies in an attempt to help guide treatment decisions.

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