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Optimization of Patient Selection for Gefitinib in Non–Small Cell Lung Cancer by Combined Analysis of Epidermal Growth Factor Receptor Mutation, K-ras Mutation, and Akt Phosphorylation

Authors' Affiliations: Departments of ¹ Internal Medicine and ² Pathology, Seoul National University Hospital, and ³ Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea

Requests for reprints: Yung-Jue Bang, Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul, 110-744, Korea. Phone: 82-2-2072-2390; Fax: 82-2-762-9662; E-mail: bangyj{at}plaza.snu.ac.kr.

	Abstract

Top Abstract Patients and Methods Results Discussion References

 
Purpose: Mutations in epidermal growth factor receptor (EGFR) are strongly predictive of gefitinib efficacy in non–small-cell lung cancer. However, the presence of EGFR mutant nonresponses and nonmutant responses points out the need for more comprehensive analysis.

Patients and Methods: For 69 non–small-cell lung cancer patients treated with gefitinib, we have extended our analysis to EGFR gene copy number by fluorescence in situ hybridization, mutations in K-ras, HER2, and exon 20 of EGFR by direct sequencing, and phosphatase and tensin homologue expression by immunohistochemistry, in addition to EGFR exons 18, 19, and 21, and phosphorylations of Akt and extracellular signal–regulated kinase reported previously.

Results: EGFR mutation and high gene copy number were associated with better objective response in univariate analysis. However, only gefitinib-sensitive EGFR mutation was independently predictive of both response (P = 0.011) and survival (P = 0.002) in multivariate analysis. No patients with K-ras mutation, including two EGFR mutants, showed response. In EGFR nonmutants, patients with either K-ras mutation or p-Akt overexpression exhibited poor response and time-to-progression whereas patients with high gene copy number tended to have better outcomes in univariate analysis. In multivariate analysis of time-to-progression in EGFR nonmutants, K-ras mutation or p-Akt overexpression was associated with shorter time-to-progression (P = 0.017). No patient with HER2 mutation showed response to gefitinib. Reduced phosphatase and tensin homologue expression was not associated with gefitinib sensitivity.

Conclusion: Gefitinib-sensitive EGFR mutation is the single most important predictor of gefitinib sensitivity. In addition to EGFR mutation, K-ras mutation and Akt phosphorylation aid in better prediction of gefitinib responsiveness in non–small-cell lung cancer.

Recent reports have shown a significant association between EGFR tyrosine kinase domain mutation and gefitinib responsiveness (8–12). Due to the benefits conferred by EGFR mutations, gefitinib should be considered as a treatment option in such patients. However, not all patients who carry an EGFR mutation respond to gefitinib and, conversely, there are patients who respond to gefitinib without an EGFR mutation, making patient selection complicated. Increased EGFR gene copy number has recently been reported to be significantly associated with the presence of EGFR mutation and gefitinib sensitivity (13–15).

We have previously shown that EGFR-independent activation of the phosphatidylinositol 3'-kinase/Akt and the Ras/Raf/mitogen-activated protein kinase/extracellular signal–regulated kinase (Erk) kinase/Erk pathways may contribute to an unfavorable treatment outcome (11). In addition, preclinical evidence indicates that loss of phosphatase and tensin homologue (PTEN) leads to the constitutive activation of Akt and resistance to gefitinib, and K-ras mutation to Erk activation (6, 7, 16). Interestingly, mutations in the tyrosine kinase domain of HER2, the preferential heterodimer of EGFR, were recently discovered in non–small-cell lung cancer (17, 18). However, their clinical significance in terms of gefitinib responsiveness has not been elucidated.

Therefore, to further identify gefitinib-sensitizing and resistance mechanisms, we have investigated EGFR gene copy number, HER2 and K-ras mutations, and loss of PTEN expression in non–small-cell lung cancer patients who had been treated with gefitinib, in addition to EGFR mutation and phosphorylations of Akt and Erk reported previously.

	Patients and Methods

Top Abstract Patients and Methods Results Discussion References

 
Patients. Of 90 patients included in our previous study (11), 69 cases had adequate tissue for further analysis of the following three molecules: EGFR mutation (exon 20), K-ras mutation (exon 2), and PTEN expression. EGFR gene copy number was assessable in 66 patients and HER2 mutation (exons 19 and 20) in 57 patients. Baseline patient characteristics did not differ from those of the previous report (Table 1 ; ref. 11). All patients had pathologically proven locally advanced or metastatic non–small-cell lung cancer. Treatment consisted of 250 mg of gefitinib daily continued until disease progression, intolerable toxicity, or patient refusal. Patients initiated gefitinib treatment from February 2002 to July 2004. Survival status was collected until the end of January 2005. Median duration of follow-up was 17.9 months (range, 7.0-37.5 months).

DNA sequencing. Sequencing analysis was done as previously described (11). Additional primer pairs used were EGFR exon 20, F: 5'-ccctgtgctaggtcttttgc, R: 5'-cacactgagcactcaataaagagaa; K-ras exon 2, F: 5'-ggtggagtatttgatagtgta, R: 5'-ggtcctgcaccagtaatatgca; HER2 exon 19, F: 5'-ggtgaaggatgtttggagga, R: 5'-agagaccagagcccagacct; and exon 20, F: 5'-tccaggctggtactttgagc, R: 5'-cagcaagagtccccatccta. Mutations were confirmed with independent duplicate analyses.

Fluorescence in situ hybridization. EGFR gene copy number was analyzed by fluorescence in situ hybridization. Briefly, representative 4-µm sections of tumor block were incubated overnight at 56°C. After deparaffinization and dehydration, sections were incubated in 0.2 mol/L HCl for 20 minutes and washed with 2x SSC (pH 7.0). Sections were then boiled in a microwave in citrate buffer (pH 6.0) for 5 minutes and incubated in 1 mol/L sodium sulfocyanate for 35 minutes at 80°C, followed by washes in 2x SSC. Sections were subsequently immersed in pepsin solution (0.0625% in 0.01 N HCl protease buffer) for 25 minutes at 37°C. After serial washes and dehydration, dual-probe hybridization was done using LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen Probe set (Vysis, Downers Grove, IL). The probe was applied, then appropriately covered and sealed. The slides were incubated in humidified atmosphere (Hybrite, Vysis) at 73°C for 5 minutes and at 37°C for 19 hours. Slides were then washed by immersing in 2x SSC/0.3% NP40 solution at room temperature and at 73°C for 5 minutes. After drying, nuclei were counterstained with 4',6'-diamidino-2-phenylindole. Archival tissue identified to have high gene copy number was included as positive control and adjacent normal tissue served as negative control. Scoring was done by a single pathologist (Y.K.J.) blinded to any clinical information or other molecular markers. At least 100 cells were evaluated in each sample. Gene copy number was scored as previously reported by others: (a) high gene copy number which includes high polysomy (4 copies in 40% of the cells) and gene amplification (tight gene clusters and a ratio of EGFR gene to chromosome of 2 or 15 copies per cell in 10% of the cells); (b) low gene copy number in the remaining cases (13).

Immunohistochemistry. Immunohistochemical determination of p-Akt and p-Erk has previously been described (11). For determination of PTEN expression, antigen retrieval was done by microwaving in 0.01 mol/L citrate buffer (pH 6.0) for 25 minutes at 650 W. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol for 15 minutes. After incubation with blocking solution for 10 minutes, sections were incubated with rabbit polyclonal PTEN antibody Ab-2 (1:50 dilution; Lab Vision, Fremont, CA) at 4°C for 12 hours, followed by 10 minutes of incubation with biotinylated secondary antibody and with streptavidin-horseradish peroxidase. Staining was done with 3,3'-diaminobenzidine chromogen and counterstaining with Mayer's hematoxylin. Blocking solution, secondary antibody, streptavidin-horseradish peroxidase, and 3,3'-diaminobenzidine chromogen were from Cap-Plus Kit (Zymed Laboratories, San Francisco, CA). PTEN expression was scored using an immunoreactive score (19). Staining intensity was defined as 3, strong; 2, moderate; 1, weak; and 0, negative. Adjacent normal cells including vascular endothelial cells were used as internal positive control. Percentage of positive cells was scored as 0, 0%; 1, 1% to 10%, 2, 11% to 50%; 3, 51% to 80%; and 4, 81% to 100% positive cells. Immunoreactive score was calculated by multiplying staining intensity and positive cell scores. An immunoreactive score of <9 was considered as reduced expression (19).

Statistical analysis. The statistical analyses of categorical variables were done using the Pearson's ² test or the Fisher's exact test where appropriate. The median durations of overall survival and time-to-progression were calculated using the Kaplan-Meier method. Comparisons between different groups were made using the log-rank tests. Multivariate analyses were done using a logistic regression model for response and stepwise Cox regression models for time-to-progression and overall survival to identify independent biomarkers and to adjust for baseline characteristics. Two-sided P < 0.05 was considered significant. All analyses were done using SPSS for Windows, version 12.0 (SPSS, Inc., Chicago, IL).

	Results

Top Abstract Patients and Methods Results Discussion References

 
EGFR mutation. Gefitinib-sensitive mutations in exons 18, 19, and 21 of EGFR tyrosine kinase domain had previously been reported (11). Among the 69 patients included herein, deletions in exon 19 were found in six patients, L858R in four patients, and G719A in three patients (one patient had E709K and G719A simultaneously). Four mutations were found in exon 20. Among them, two were found in patients with sensitive mutations: S768I with G719A and V819A with L858R. The former patient showed partial response and the latter showed stable disease lasting 13.8 months. Other two mutations in exon 20 were insertions (D770-N771insSVQ and D770-N771insG, N771T) found in two other patients. These two patients harboring insertions were female never-smokers with adenocarcinoma. Despite their favorable clinical characteristics, they all showed progressive disease, suggesting that these insertions may not be gefitinib-sensitive mutations. The overall response rate of 15 patients with an EGFR mutation was 53.3%, compared with 14.8% in 54 patients with no mutation (P = 0.004; Table 2 ). EGFR mutants had significantly longer time-to-progression (P = 0.0005; median, 7.9 versus 1.7 months in nonmutants) and overall survival (P = 0.0001; median, not reached versus 7.4 months in nonmutants; Fig. 1 ). Response rate among patients with gefitinib-sensitive mutations (G719A, deletion in exon 19, and L858R) was 61.5% (8 of 13) and median time-to-progression was 13.8 months.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Kaplan-Meier plots of time-to-progression (A and B) and overall survival (C and D) according to EGFR mutation and gene copy number (GCN). *, log-rank test.

K-ras mutation. Nine patients (15.9%) harbored mutations in K-ras exon 2. Four patients exhibited G12D, two with G12V, two with G12A, and one with G13C. K-ras mutations were more frequently found in males [20.5% (8 of 39) versus 3.3% (1 of 30) in females; P = 0.067] and in smokers [20.6% (7 of 34) versus 5.7% (2 of 35) in never-smokers; P = 0.084] and were less frequently found in adenocarcinomas [9.6% (5 of 52) versus 23.5% (4 of 17) in others; P = 0.21]. All but one patient with K-ras mutation showed positive p-Erk expression (Table 3 ).

Of 54 patients without an EGFR mutation, 7 patients carried a K-ras mutation. Again, no patient with a K-ras mutation responded whereas the response rate in EGFR nonmutants without a K-ras mutation was 17.0% (P = 0.58; Table 4 ). Time-to-progression and overall survival did not differ significantly according to K-ras mutational status (data not shown).

PTEN, p-Akt, and p-Erk expression. PTEN expression was reduced (immunoreactive score < 9) in 22 patients (31.9%). No significant association was found between reduced PTEN expression and p-Akt overexpression (Table 3). Response rate did not differ according to PTEN expression (Tables 2 and 4). Time-to-progression and overall survival were not affected by PTEN expression (data not shown). Among the EGFR nonmutants, no patient with p-Akt overexpression showed response to gefitinib and 8.6% of patients with positive p-Erk expression exhibited partial response (Table 4). Time-to-progression was better in patients with p-Akt (–/+) compared with p-Akt (2+) (P = 0.037; median, 2.1 and 1.2 months, respectively), and in patients with p-Erk (–) compared with p-Erk (1+/2+) (P = 0.052; median, 2.7 and 1.4 months, respectively).

Combined analysis according to EGFR genetic status, K-ras mutation, and p-Akt. In multivariate analyses including sex, histology (adenocarcinoma versus others), and smoking history (never versus ever), which are the clinical predictors of gefitinib responsiveness, and biomarkers of gefitinib-sensitive EGFR mutation and EGFR gene copy number (high versus low) as covariates, gefitinib-sensitive EGFR mutation was independently predictive of response to gefitinib [adjusted odds ratio, 7.05; 95% confidence interval (95% CI), 1.56-31.9; P = 0.011] whereas EGFR gene copy number was not included in the final model. Gefitinib-sensitive EGFR mutation was also independently predictive of prolonged time-to-progression (adjusted hazard ratio, 0.32; 95% CI, 0.14-0.77; P = 0.011) and overall survival (adjusted hazard ratio, 0.11; 95% CI, 0.03-0.45; P = 0.002) whereas EGFR high gene copy was not. Thus, in the present patient population, gefitinib-sensitive EGFR mutation is the single most important biomarker associated with objective benefit from gefitinib. Among EGFR mutants, patients with gefitinib-sensitive mutations without a K-ras mutation had significantly better response (response rate, 72.7%; 8 of 11) compared with the remaining EGFR mutant patients (0.0%; 0 of 4; P = 0.026) and better time-to-progression (P = 0.079; median, 13.8 versus 2.3 months).

In patients without an EGFR mutation, patients with high EGFR gene copy number had better responses (P = 0.10; Table 4). As a resistance mechanism, no patient simultaneously exhibited K-ras mutation or p-Akt overexpression. Patients without these resistance mechanisms had significantly better response (P = 0.016; Table 4). As of time-to-progression, patients with either K-ras mutation or p-Akt overexpression showed shorter time-to-progression (P = 0.007). In contrast, patients with high EGFR gene copy number had longer time-to-progression although it was not statistically significant (P = 0.20; Fig. 2 ). None of the markers tested was significantly associated with overall survival (data not shown). In multivariate analysis of time-to-progression which included the three clinical covariates, EGFR gene copy number and K-ras and p-Akt status, presence of K-ras mutation or p-Akt overexpression was significantly associated with increased risk of disease progression (adjusted hazard ratio, 2.1; 95% CI, 1.1-4.0; P = 0.017). High EGFR gene copy number was removed from the final model.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Kaplan-Meier plots of time-to-progression according to EGFR gene copy number (A) and K-ras mutation and p-Akt expression (B) in patients without EGFR mutation. *, log-rank test.

	Discussion

Top Abstract Patients and Methods Results Discussion References

Increased gene copy number of EGFR has also been shown to be associated with the efficacy of gefitinib and has been reported to be superior to EGFR mutation in the prediction of gefitinib efficacy by Cappuzzo et al. (13–15). However, in the present study, high EGFR gene copy number was associated with objective response to gefitinib but failed to show significant association with prolonged time-to-progression or overall survival. Moreover, high gene copy number was removed from the multivariate regression model for testing association between biomarker and response to gefitinib. This may possibly be due to the different mechanism of gefitinib sensitivity in different ethnic populations or the small sample of patients included in the present study. The role of genetic gain of EGFR in the prediction of gefitinib responsiveness, especially in patients without EGFR mutation, should be further investigated in larger scaled studies.

K-ras mutations are associated with poor prognosis in non–small-cell lung cancer (28). Our results show that K-ras mutant patients also have poor outcome when treated with gefitinib. No patient with K-ras mutation responded to gefitinib, and all but one patient showed positive p-Erk expression, which suggests resistance via constitutive activation of Ras/Raf/mitogen-activated protein kinase/Erk kinase/Erk pathway. A recent report also shows that K-ras mutation may partly explain the gefitinib resistance seen in non–small-cell lung cancer (29). On the other hand, only 18.1% (8 of 44) of patients with p-Erk-positive tumors harbored K-ras mutation (Table 3). Identification of other molecular mechanisms leading to p-Erk activation and gefitinib resistance is mandatory.

Previous reports have shown that EGFR and K-ras mutations are mutually exclusive, suggesting the presence of different pathways of lung carcinogenesis (20–22). However, our data show that K-ras mutation may coexist with EGFR mutation in some patients. Moreover, these patients failed to respond to gefitinib, which shows that gefitinib sensitivity conferred by gefitinib-sensitive mutations may be incapacitated by the downstream pathway activation by K-ras mutation.

HER2 mutation in tyrosine kinase domain was recently reported in a small subset of non–small-cell lung cancer (17, 18). In the present study, we have found four patients with HER2 mutation and none responded to gefitinib. This observation raises the possibility that HER2 mutation may have no effect on gefitinib sensitivity or may serve as a resistant mechanism, which needs to be clarified in the future.

Loss of PTEN has repeatedly been associated with Akt activation (30). In addition, loss of PTEN was associated with in vitro resistance to gefitinib and was also associated with resistance to trastuzumab (6, 7, 19). However, we failed to find significant associations between PTEN expression and p-Akt overexpression or resistance to gefitinib. It may possibly be due to the limitation of immunohistochemical method used herein or insufficient sample size to detect such a correlation. However, recent reports showing absence of inverse correlation between p-Akt and PTEN expression in a large series of breast cancer and lack of association between PTEN expression and gefitinib sensitivity in non–small-cell lung cancer cells support our finding (31, 32). These observations suggest that the simple linear model of association between PTEN and the phosphatidylinositol 3'-kinase/Akt pathway may not apply in non–small-cell lung cancer and that factors other than reduced PTEN expression contribute to Akt activation and gefitinib resistance. Investigations into other mechanisms of Akt activation, such as PTEN or phosphatidylinositol 3'-kinase mutation, are warranted, together with development of better method for detection of activated Akt considering its limited stability, inhomogeneous distribution, and conflicting results as a predictive marker (11, 33–35).

In conclusion, gefitinib-sensitive EGFR mutation is the single most important predictor of benefit from gefitinib treatment. Moreover, our molecular analysis shows that patient selection for gefitinib may be optimized by combined analysis of K-ras mutation and Akt phosphorylation, which are the negative predictors of gefitinib responsiveness, in addition to EGFR mutation. Patients with HER2 mutations were not sensitive to gefitinib. Future studies should include these molecules together with other molecules possibly associated with resistant or sensitive mechanisms to further improve patient selection.

	Acknowledgments

 
We thank Prof. Byung Joo Park (Department of Preventive Medicine, Seoul National University College of Medicine) for his helpful discussion in statistical analysis.

	Footnotes

 
Grant support: Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ10-PG13-GD01-0002).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Presented in part at the AACR-National Cancer Institute-European Organization for Research and Treatment of Cancer International Conference on Molecular Targets and Cancer Therapeutics, November 14-18, 2005, Philadelphia, Pennsylvania.

Received 12/29/05; revised 2/ 5/06; accepted 2/15/06.

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