Department of Hematology-Oncology, Istituto Clinico Humanitas, Rozzano, Italy

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1 The Oncologist Lung Cancer Understanding the New Genetics of Responsiveness to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors LUCA TOSCHI,FEDERICO CAPPUZZO LEARNING OBJECTIVES Department of Hematology-Oncology, Istituto Clinico Humanitas, Rozzano, Italy Key Words. Epidermal growth factor receptor Tyrosine kinase inhibitors Gefitinib Erlotinib Non-small cell lung cancer After completing this course, the reader will be able to: 1. Select a patient candidate for a tyrosine kinase inhibitor therapy. 2. Describe the mechanism of action of gefitinib and erlotinib. 3. Discuss the role of clinical and biological factors as determinants for sensitivity or resistance to tyrosine kinase inhibitors in lung cancer. CME Access and take the CME test online and receive 1 AMA PRA Category 1 Credit at CME.TheOncologist.com ABSTRACT The epidermal growth factor receptor (EGFR) is implicated in cancer progression and development and, being overexpressed in a variety of human malignancies, is an attractive target for selective anticancer therapy. EGFR tyrosine kinase inhibitors (TKIs) have been demonstrated to produce dramatic and durable responses in a fraction of non-small cell lung cancer patients. During the last few years, clinical and biological predictors for TKI sensitivity have been identified. Among clinical features, never-smoking history seemed the most critical factor, probably because of the different spectrum of molecular abnormalities associated with cigarettesmoking exposure. Among biological predictors, several studies indicate that EGFR mutations and increased EGFR gene copy number are implicated in response to TKI therapy, with conflicting results in survival. Mutations in the EGFR gene as well as in K-ras and HER2 genes seemed to impair TKI effects, leading to TKI resistance. Because most available data come from retrospective studies, there is an urgent need to validate these results in prospective trials. Several studies have been recently completed, and these data could indicate how to properly select patients who are candidates for TKI therapy. The Oncologist 2007;12: INTRODUCTION Recent advances in cancer biology have led to the identification of several potential molecular targets that play a key role in cancer development and progression. Selective targeting of cancer cells on the basis of their molecular phenotype can provide effective anticancer activity, avoiding the commonly experienced side effects induced by chemotherapy. Among these targets, the epidermal growth factor receptor (EGFR) has become widely investigated in different human malignancies due to its critical role in cancer proliferation and survival. The EGFR superfamily includes four distinct, closely Correspondence: Federico Cappuzzo, M.D., Istituto Clinico Humanitas IRCCS, via Manzoni 56, Rozzano, Italy. Telephone: 39 (0) ; Fax: ; federico.cappuzzo@humanitas.it Received September 28, 2006; accepted for publication October 29, AlphaMed Press /2007/$30.00/0 doi: /theoncologist The Oncologist 2007;12:

2 212 Understanding Responsiveness in EGFR TKIs related transmembrane receptors: EGFR/erbB-1, HER2/ erbb-2, HER3/erbB-3, and HER4/erbB-4 [1]. EGFR is normally found on the surface of epithelial cells and has been found to be commonly overexpressed in a variety of human malignancies [2 4]. Different ligands can lead to EGFR activation and subsequent signal transduction, including the epidermal growth factor, the transforming growth factor, and neuregulins. After ligand binding to the extracellular receptor domain, EGFR undergoes homo- or heterodimerization and autophosphorylation of its intracellular tyrosine kinase domain. These autophosphorylation events trigger a cascade of downstream signals that ultimately result in an increase of cellular motility, proliferation and invasion, and a block of apoptosis, contributing to cancer development and progression. Strategies aimed at inhibiting the EGFR pathway include different classes of compounds, with tyrosine kinase inhibitors (TKIs) and monoclonal antibodies being the most widely investigated agents. This review focuses on molecular predictors for sensitivity and resistance to EGFR TKIs and possible clinical implications. EGFR TYROSINE KINASE INHIBITORS Gefitinib (ZD 1839, Iressa; AstraZeneca, London) and erlotinib (OSI 774, Tarceva; Genentech, South San Francisco, CA) are the first EGFR TKIs to have reached clinical trial testing, but several other agents, including lapatinib, EKB-569, CI-1033, PKI-166, and AEE-788, are currently under investigation. Both gefitinib and erlotinib are orally active, selective EGFR TKIs that demonstrated antitumor activity in a variety of human cancer cell lines overexpressing EGFR [5, 6]. Phase I studies were conducted in patients affected with different solid tumors, including non-small cell lung cancer (NSCLC) and ovarian, breast, colorectal, and head and neck cancers [7 14]. Gefitinib showed promising antitumor activity at doses of at least 150 mg/day, with dose-limiting toxicity observed at 800 mg/day or 1,000 mg/ day. Main side effects, which occurred in approximately 70% 80% of the patients, and especially above 600 mg/ day, were diarrhea and skin rash, both dose-dependent and reversible. On the basis of these studies, 250 mg/day and 500 mg/day were the fixed doses chosen for phase II studies. Erlotinib showed a similar activity and toxicity pattern, with 150 mg/day emerging as the optimal dose for phase II trial testing [13]. In the Iressa Dose Evaluation in Advanced Lung Cancer (IDEAL)-1 and -2 studies, a total of 426 NSCLC patients progressing after one or more chemotherapy regimens were randomized to receive 250 mg/day of gefitinib versus 500 mg/day [15, 16]. These trials showed response rates of 10% 19%, with a clear symptom improvement in approximately 40% of the patients, leading to U.S. Food and Drug Administration (FDA) gefitinib approval for use in NSCLC. Subgroup analyses indicated that response to gefitinib was associated with Asian ethnicity, adenocarcinoma histology (especially in the presence of bronchioloalveolar features), female sex, and neversmoking history [15, 16]. Similar results were observed with erlotinib. In a phase II study, the drug produced a 12.3% response rate, with no grade 4 toxicity and minimal grade 3 side effects [17]. As for gefitinib, high response rate was observed in nonsmokers and adenocarcinomas with bronchioloalveolar features (37% and 30%, respectively). The promising EGFR TKI single-agent activity observed in these studies has not been confirmed in subsequent phase III combination trials. Four large prospective studies, including more than 4,000 patients with advanced NSCLC (Iressa NSCLC Trail Assessing Combination Treatment [INTACT]-1 and -2, Tarceva Responses in Conjunction with Paclitaxel and Carboplatin [TRIBUTE], Tarceva Responses in Conjunction with Cisplatin and Gemcitabine [TALENT]), randomly assigned untreated patients to standard chemotherapy plus a TKI, or the same chemotherapy regimen plus placebo [18 21]. All trials failed to demonstrate any survival advantage for patients receiving the TKI, probably because of the lack of patient selection. Unplanned subgroup analyses showed that neversmokers receiving the TKI had a significant survival improvement compared with never-smokers treated with placebo, supporting the relevant role of smoking history for TKI sensitivity [21]. Recently, two large randomized phase III studies assessed gefitinib and single-agent erlotinib in advanced NSCLC patients who experienced treatment failure with at least one chemotherapy regimen [22, 23]. The Iressa Survival Evaluation in Lung cancer (ISEL) trial included 1,692 patients who were randomized to 250 mg/day of gefitinib or placebo [22]. Despite higher response rate and longer time to progression in the gefitinib arm, the study concluded that the drug produced no survival advantage over best supportive care in advanced NSCLC (5.6 months vs. 5.1 months; p.09). Conversely, the BR21 trial, in which 731 previously treated advanced NSCLC patients were randomized to receive 150 mg/day of erlotinib or placebo, succeeded in meeting its primary endpoint, with patients in the experimental arm experiencing a statistically significant improvement in overall survival with a hazard ratio of 0.70 [23]. Results from these large randomized trials led to erlotinib approval by the FDA and to restrictions for gefitinib use, limiting drug administration to patients currently receiving gefitinib and benefiting from the agent or patients who have previously received and benefited from gefitinib. Although the ISEL and the BR21 trials pro-

3 Toschi, Cappuzzo 213 duced different results, both studies showed that neversmokers treated with a TKI had a significantly longer survival compared with never-smokers who received placebo, with no survival difference in smokers irrespective of the treatment [22, 23]. The relevance of smoking history in TKI sensitivity has been confirmed in a recently published phase II trial of gefitinib as first-line treatment in Korean never-smokers with lung adenocarcinoma [24]. In this study, authors observed a 69% response rate, with an estimated 1-year survival of 73%. These results are remarkable, supporting further studies comparing a TKI with standard chemotherapy in selected cohorts of NSCLC. In summary, erlotinib is the only TKI that has been demonstrated to significantly improve survival versus best supportive care in pretreated NSCLC patients, with no survival benefit in combination with chemotherapy in untreated NSCLC. Among clinical features, smoking history is probably the most relevant predictor for TKI sensitivity, and both gefitinib and erlotinib have been demonstrated to significantly prolong survival in never-smokers in large phase III trials. EGFR PROTEIN EXPRESSION Although clinical characteristics could be useful for identifying patients more likely to have a benefit, selection of patients who are candidates for TKI therapy should be performed on the basis of biological characteristics (Tables 1, 2). That is because clinical features associated with TKI efficacy are not important per se but are relevant only because they reflect particular biological aspects of the disease. EGFR expression assessed by immunohistochemistry (IHC) has been the first biological marker to be retrospectively explored in cohorts of NSCLC patients treated with TKIs. Analyses conducted among patients who received gefitinib in the IDEAL-1 and -2 trials showed no correlation between EGFR levels and tumor response [25]. Similarly, when retrospectively analyzing tumor tissue from 43 patients who received 250 mg/day of gefitinib, we found no impact of EGFR expression in predicting sensitivity to the drug [26]. Recently, Miller et al. [27] reported molecular analyses performed within a prospective phase II study of erlotinib in bronchioloalveolar carcinoma (BAC). Sixtytwo patients have undergone EGFR IHC. No association between EGFR expression and sensitivity to erlotinib in terms of response rate, time to progression, or survival was observed [27]. Similarly, Villaflor et al. [28] observed that EGFR IHC status was not predictive for survival in 87 patients treated with gefitinib in the Expanded Access Program. Conversely, two studies suggested that EGFR IHC Table 1. Clinical and biological predictors for tyrosine kinase inhibitor sensitivity in non-small cell lung cancer patients Predictive for response Predictive for survival Clinical Gender Smoking history Histology Response to prior therapy Smoking history Performance status Histology Exposure to prior platinum Skin rash Biological EGFR gene mutations EGFR gene gain EGFR gene mutations HER2 gene gain EGFR gene gain Akt activation Abbreviation: EGFR, epidermal growth factor receptor. Table 2. Biological determinants of EGFR-tyrosine kinase inhibitor resistance Intrinsic resistance Acquired resistance k-ras mutations Loss of PTEN HER2 mutations IGFR-1 overexpression EGFR exon 20 insertion EGFR exon 20 mutations mutations EMP-1 overexpression EMP-1 overexpression Abbreviations: EGFR, epidermal growth factor receptor; EMP-1, epithelial membrane protein-1; IGFR-1, insulinlike growth factor receptor-1; PTEN, phosphatase and tensin homolog deleted on chromosome 10. assessment could be useful for identifying patients who could achieve a survival improvement [29, 30]. In the BR21 study, 325 tumor samples underwent IHC staining to assess EGFR protein expression. EGFR IHC-positive patients treated with erlotinib had a significant survival improvement compared with EGFR IHC-positive patients who received placebo (hazard ratio [HR], 0.68; p.02), whereas no difference was observed among EGFR IHC-negative patients irrespective of the treatment [29]. These conflicting results suggest that IHC is probably not an optimal method for patient selection. Nevertheless, the relevant data from the BR21 study indicated that TKI therapy should not be denied to EGFR IHC-positive individuals. EGFR GENE MUTATIONS In 2004, three groups showed that mutations in the TK domain of EGFR were associated with response of NSCLC to gefitinib [31 33] or erlotinib [33]. These mutations were

4 214 Understanding Responsiveness in EGFR TKIs somatic and more frequently observed in patients with clinical features known to be associated with TKI sensitivity, such as female sex, adenocarcinoma histology, Asian ethnicity, and never-smoking history. Pham et al. [34] recently observed that the likelihood of harboring EGFR mutations in lung adenocarcinomas decreases as the exposure to tobacco smoke increases. The most common EGFR-sensitizing mutations, accounting for 85% of all EGFR mutations in NSCLC, include deletions in exon 19 and L858 substitutions in exon 21. Such EGFR mutations increase sensitivity to TKIs, most likely through induction of critical structural modifications of the ATP-binding site in the tyrosine kinase domain [31, 32]. Several other EGFR gene mutations have been described, but their role is not clear, and it is not possible to exclude that some of them are artifacts [35]. The threonine-to-methionine substitution at amino acid position 790 (T790M) in exon 20 of the EGFR gene has been reported in progressing lesions after gefitinib or erlotinib therapy [36, 37]. In vitro experiments confirmed that the presence of the T790M substitution confers resistance to EGFR mutant cell lines usually sensitive to either gefitinib or erlotinib, probably modifying the structure of the EGFR tyrosine kinase domain [36 38]. Interestingly, this mutation might be present in a small fraction of tumor cells before drug treatment and might have germline transmission, resulting in inherited lung cancer susceptibility and intrinsic TKI resistance [39 41]. Most studies showed a significant association of EGFR mutations, particularly exon 19 deletion, and response to TKIs [42 51]. Recent prospective studies confirmed that responders to gefitinib or erlotinib harbored an EGFR gene mutation [52 57]. In a phase II study conducted by the Spanish Lung Cancer Group, 297 chemotherapy-naïve, advanced NSCLC patients were screened for exon 19, 20, and 21 mutations [52]. Overall, the incidence of mutated EGFR was 12.5%, with 25 exon 19 deletions, 11 L858R substitutions, and no exon 20 mutation. Among the 21 patients evaluable, response rate was 90%. In a similarly designed trial with gefitinib in a chemotherapy-naïve Japanese population, Inoue et al. [53] observed a 33% incidence of EGFR mutations in the 75 tumors screened. Sixteen EGFRmutated patients received 250 mg/day of gefitinib, with an overall response rate of 75%. Although available data indicate that patients with EGFR mutations respond to TKIs, the impact on survival is unclear because of the possible prognostic rather than predictive value of such mutations (Table 3) and the possible different role of exon 19 rather than exon 21 mutation in TKI sensitivity. Survival analysis of the largest trials with TKIs showed no survival benefit for patients harboring EGFR mutations receiving a TKI [29, 58, 59]. Bell et al. [58] observed longer survival for EGFR-mutant patients treated with chemotherapy alone in the INTACT trials compared with patients with wild-type EGFR (19.4 months vs. 9.2 months; HR, 0.48), suggesting a possible better natural history for EGFR mutation-positive tumors. Similar results have emerged from retrospective molecular analyses from the TRIBUTE and TALENT trials, where patients with EGFR mutations had a better outcome than did patients with wild-type EGFR regardless of the treatment received [59, 60]. Shigematsu et al. [61] evaluated the possible prognostic value of EGFR mutations in a cohort of surgically resected NSCLC patients not exposed to TKIs. This important study showed no difference in survival for patients with EGFR mutations compared with individuals with wild-type EGFR. Nevertheless, individuals with exon 19 deletion had worse survival than did patients with an L858R substitution, suggesting a different prognostic value for exon 19 and exon 21 EGFR mutations. Recent studies evaluated the impact on survival of different EGFR mutations in patients treated with TKIs [62 64]. Although the retrospective nature precludes any firm conclusion, these studies suggest that patients treated with TKIs harboring an EGFR exon 19 mutation have a longer survival compared with those with exon 21 mutation. Prospective trials are needed to further define the prognostic and predictive roles of different EGFR mutations. EGFR GENE COPY NUMBER Available data indicate that a significant fraction of patients with EGFR mutations (12% 84%) do not respond to TKIs [29, 30, 42]. Retrospective analyses of large phase III trials [29, 58] did not show any significant improvement in response rate for patients with EGFR mutations treated with a TKI. Moreover, in the BR21 study, response to TKI was not confined to patients with mutations, and some individuals with wild-type EGFR experienced dramatic response [29]. These findings suggest that other mechanisms are involved in TKI sensitivity, and recent findings indicate that increased copy number of the EGFR gene is relevant for TKI sensitivity. Several studies evaluated EGFR gene copy number by fluorescent in situ hybridization (FISH) [29, 30, 65, 66]. In the Cappuzzo et al. study [30], individuals with EGFR high polysomy or gene amplification (defined as EGFR FISH-positive) had a significantly higher response rate and a significantly longer time to progression and survival than did patients with no EGFR gene gain (defined as EGFR FISH-negative). Importantly, EGFR FISH-positive status was significantly associated with certain clinical and biological characteristics predictive for TKI sensitivity, such as female sex, never-smoking history, and the presence of EGFR mutations. Tsao et al. [29] successfully per-

5 Toschi, Cappuzzo 215 Table 3. Main studies assessing EGFR status and survival in non-small cell lung cancer patients treated with tyrosine kinase inhibitors Study n Method for EGFR assessment EGFR (%) IDEAL-1 and -2 [58] 78 Mutation 18.0 Not significant 312 Mutation 10.2 Not significant INTACT-1 and -2 [58] formed EGFR FISH analysis in 125 patients who participated in the BR21 trial. Increased gene copy number was detected in 45% of the patients and was not associated with the presence of EGFR mutations. This large study showed that EGFR FISH-positive patients treated with erlotinib had a higher response rate and longer survival than did EGFR FISH-positive patients treated with placebo (HR, 0.44; p.008), whereas there was no advantage determined by the drug in EGFR FISH-negative patients. Hirsch et al. [65] performed EGFR FISH analysis in patients enrolled in the ISEL trial. This study confirmed the better outcome in terms of response rate and survival for EGFR FISH-positive patients treated with gefitinib than for EGFR FISH-positive patients treated with placebo, with no survival difference in EGFR FISH-negative patients irrespective of the treatment. In the Southwest Oncology Group S0126 trial, where patients with BAC were treated with 500 mg/day of gefitinib, Hirsch et al. [66] observed longer survival for EGFR FISH-positive patients over those who had no gene gain (HR, 2.02; p.042). Other methods have been used for EGFR gene copy number assessment. In the study performed by Miller et al. [27] in patients with bronchioloalveolar carcinoma, individuals with increased EGFR gene copy number detected by chromogenic in situ hybridization had a significantly better response rate (38% vs. 14%; p.03) and time to progression (8.8 months vs. 2.3 months) and a trend toward longer survival than did EGFR-negative individuals. EGFR gene copy number was evaluated by quantitative Association with survival (p value) TRIBUTE [59] 228 Mutation 13.0 Not significant TALENT [20] 233 Mutation 9.8 Not significant BR21 [29] 177 Mutation 23.0 Not significant Takano et al. [44] 66 Mutation Mitsudomi et al. [51] 59 Mutation ISEL [65] 370 FISH Cappuzzo et al. [30] 102 FISH Hirsch et al. [66] 81 FISH BR21 [29] 125 FISH Abbreviations: EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; IDEAL, Iressa Dose Evaluation in Advanced Lung Cancer; INTACT, Iressa NSCLC Trial Assessing Combination Treatment; ISEL, Iressa Survival Evaluation in Lung cancer; TALENT, Tarceva Responses in Conjunction with Cisplatin and Gemcitabine; TRIBUTE, Tarceva Responses in Conjunction with Paclitaxel and Carboplatin. polymerase chain reaction (PCR) in 90 patients from the IDEAL trials and 453 tumors of the INTACT trials [58]. Amplification of the EGFR gene was detected in 8% and 7% of the cases, respectively, and was associated with female sex (p.02). However, no association with adenocarcinoma histology or with never-smoking status was observed. In the IDEAL trials, patients with more than a four-fold amplification had a response rate to gefitinib not significantly higher than did those with no gene gain (29% vs. 15%; p.39). When considering patients with EGFR gene amplification in the INTACT trials, no advantage in terms of response to chemotherapy plus gefitinib was observed over those with no gene gain (56% vs. 53%). A trend toward improved time to progression (7.3 months vs. 4.6 months) and survival ( 20 months vs months) irrespective of gefitinib addition was observed for patients with EGFR amplification, raising questions about a possible prognostic role for EGFR gene gain in chemotherapytreated NSCLC patients. In the study performed by Takano et al. [44], EGFR gene copy number was assessed by quantitative real-time PCR in 66 gefitinib-treated Asian patients. EGFR gene gain ( 3.0 genes per cell) was found in 29 patients (44%), and it was associated with the presence of EGFR-sensitizing mutations (p.014). In particular, high EGFR gene copy number ( 6.0 genes per cell) was observed only in patients with a high proportion ( 60%) of mutant alleles. In this study, increased EGFR gene copy number was significantly

6 216 Understanding Responsiveness in EGFR TKIs associated with a higher response rate and longer time to progression. In a recently published trial, Dziadziuszko et al. [67] evaluated EGFR mrna expression and EGFR gene dosage by quantitative PCR in a cohort of NSCLC patients who received gefitinib for advanced disease. Responding patients had a significantly higher median relative EGFR mrna expression compared with nonresponders (p.012), whereas no difference in terms of gene dosage was observed. Moreover, patients with high EGFR mrna expression, but not those with high gene dosage, had significantly longer progression-free survival compared with individuals with low EGFR mrna expression (p.028), with no advantage in survival. Of note, EGFR mrna expression but not EGFR gene dosage significantly correlated with FISH status, suggesting that EGFR mrna expression might be interchangeably used with FISH to select patients who might benefit from treatment with TKIs. In summary, although prospective studies are needed, available data suggest that EGFR FISH analysis is an accurate predictor for gefitinib sensitivity and that this method should be used for selection of patients candidate for TKI therapy. Moreover, FISH technology is well established and has a short turnaround in clinical cytogenetics and molecular pathology laboratories, and an EGFR FISH probe is already commercially available. Other methods for EGFR gene copy number assessment produced conflicting results and are not recommended outside clinical trials. OTHER BIOLOGICAL MARKERS HER2 Several biological markers that play a key role in the EGFR downstream pathway have been investigated in the last few years as potential predictors for TKI sensitivity. HER2 is the major partner of EGFR because activated heterodimers containing HER2 are more stable than complexes containing other members of the EGFR family [68]. Overexpression of HER2 in various cancer cell lines or xenografts increases antitumor effects of gefitinib [69 72]. Recently, Hirata et al. [73] demonstrated that the effects of gefitinib are independent of EGFR levels but are influenced by HER2 expression in one NSCLC cell line transfected with the HER2 gene. As for EGFR, the method for HER2 assessment is of great relevance. In a small study, the HER2 protein was evaluated by IHC in 43 NSCLC patients treated with gefitinib [26]. This study did not demonstrate any association of HER2 protein expression and gefitinib sensitivity in terms of response rate, time to progression, and survival. Nevertheless, only eight patients showed strong HER2 positivity (3 ), and FISH analysis was performed in a limited number of cases. To further investigate the role of HER2 in patients exposed to TKIs, we evaluated HER2 gene copy number by FISH in 101 NSCLC patients treated with gefitinib [74]. Using the same score system adopted for EGFR [30], and considering HER2 FISH-positive patients with gene amplification as well as patients with high polysomy, we observed that HER2 FISH-positive individuals had significantly higher response rate (34.8% vs. 6.4%; p.001) and time to progression (9.1 months vs. 2.7 months; p.02), with a trend toward longer survival (20.8 months vs. 8.4 months; p.056), compared with HER2 FISH-negative individuals. Interestingly, HER2 FISH-positive patients were frequently EGFR FISH-positive and EGFR mutation-positive, a phenomenon not previously reported and probably relevant for tumor cell survival. Independent of the method for EGFR assessment, EGFRpositive patients who also had increased HER2 gene copy number had a better outcome in terms of response and survival, suggesting that HER2 gene gain increases sensitivity to gefitinib only in presence of EGFR. In fact, EGFRnegative patients with HER2 FISH-positive status had the same outcome as did patients negative for both markers. Mutation of the HER2 gene was reported in lung adenocarcinoma, offering the potential for additional therapy targeted at the altered protein [75]. HER2 mutations, which mainly consist of exon 20 insertions/duplications, target the same corresponding region as do EGFR mutations [75, 76]. These mutations were more frequently observed in neversmokers and in adenocarcinomas [75, 76], and recent findings indicated that NSCLC presenting these mutations remains sensitive to HER2-targeted therapies but insensitive to EGFR TKIs [77, 78]. Han et al. [79] observed no response to gefitinib in four NSCLC patients harboring an HER2 mutation, suggesting that detection of such mutation could help in patient selection. Overall, available data suggest that HER2 gene assessment might be a complementary tool to EGFR assay in selecting patients who are candidates for TKI treatment, providing the rationale for exploring anti-her2 strategies, alone or in combination with EGFR inhibitors, in selected NSCLC populations. K-Ras K-ras is a critical downstream effector of the EGFR pathway that has been found to be mutated in about 15% 30% of lung adenocarcinomas [80, 81]. Activating mutations of k-ras usually occur in codons 12 and 13 in exon 2 [82, 83] and have been reported to be associated with unfavorable outcome [84 87]. Mutations in k-ras are commonly associated with history of tobacco smoke exposure and are hardly found in never-smokers [75, 84]. Several studies showed that EGFR-sensitizing

7 Toschi, Cappuzzo 217 mutations and k-ras mutations are mutually exclusive [39, 59, 61, 63, 81, 88 91]. NSCLC patients harboring k-ras mutations are insensitive to TKI therapy [90]. Pao et al. [90] investigated the possible role of k-ras mutations in 60 patients with lung adenocarcinomas who have shown to be sensitive or refractory to either gefitinib or erlotinib; k-ras mutations were identified in 9 (24%) of 38 patients refractory to either drug, whereas no mutations were found in the 21 sensitive patients (p.02). Conversely, 17 (77%) of 22 responding patients carried EGFR mutations, whereas none were found in the 38 refractory tumors. All 17 tumors harboring EGFR mutations responded to TKIs, whereas none of the nine tumors with k-ras mutations showed drug sensitivity. In the BR21 study, patients with k-ras mutation had lower response rate and significantly shorter survival (HR, 1.63; p.03) compared with those with wild-type k-ras [92]. Those data indicated that k-ras mutations are associated with intrinsic TKI resistance, and k-ras gene sequencing could be useful for selecting candidates for TKI therapy. This concept is reinforced by the provocative results of the TRIBUTE trial. In that study, patients with k-ras mutations had significantly shorter survival when treated with chemotherapy plus erlotinib, suggesting a possible detrimental effect REFERENCES of TKIs in patients harboring such mutations [59]. As for the previously presented biomarkers, prospective studies for k-ras are warranted to better define the relevance of such mutations for patient selection. CONCLUSION Novel targeted therapies directed against the EGFR signaling pathway offer new hope to cancer patients. EGFR TKIs have emerged as particularly effective drugs in subsets of NSCLCs, with never-smoking history, EGFR mutations, and increased EGFR gene copy number as the major predictors for sensitivity to these agents. 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