Annals of Oncology Advance Access published online on February 22, 2007
Annals of Oncology, doi:10.1093/annonc/mdm003
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© 2007 European Society for Medical Oncology
Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib
1 Division of Medical Oncology
2 Division of Pathology, Department of Medicine, University of Colorado Health Sciences Center and University of Colorado Cancer Center, Aurora, CO, USA
3 Bellaria Hospital, Div. of Medical Oncology, Via Altura 3, 40139 Bologna, Italy
4 Southwest Oncology Group, 1730 Minor Ave, Ste 1900, Seattle, WA 98101-1468, USA
5 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-8593, USA
* Correspondence to: Dr P. A. Bunn Jr, University of Colorado Cancer Center, PO Box 6511, Mail Stop 8117, Aurora, CO 80010, USA. Tel: 303-724-3155; Fax: 303-724-3162; E-mail: paul.bunn{at}uchsc.edu
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Abstract |
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Background: Biological markers for optimal selection of patient to epidermal growth factor receptor (EGFR)-targeted therapies are not established in advanced non-small-cell lung cancer (NSCLC).
Patients and methods: EGFR/HER2 gene copy number by FISH, EGFR protein and pAKT expression by immunohistochemistry (IHC) and EGFR and KRAS mutations were tested in 204 gefitinib-treated NSCLC patients.
Results: Increased EGFR and HER2 gene copy number (FISH+), EGFR protein overexpression (IHC+), EGFR mutations and pAKT overexpression were all associated with significantly higher response rates (33%, 29%, 22%, 39% and 20% respectively). EGFR FISH+ (32%) and IHC+ (61%) correlated with improved survival, while EGFR mutations (27%), KRAS mutations (26%) and pAKT expression (69%) did not. In multivariate survival analysis EGFR FISH and IHC were independent predictive markers. EGFR FISH+/IHC+ patients (23%) had a median survival of 21 months versus 6 months for double-negative patients (30%).
Conclusion: Combination of EGFR FISH and IHC is effective predictor for benefit from gefitinib. Patients with double-negative results are unlikely to benefit in western NSCLC populations.
epidermal growth factor receptor, gefitinib, gene copy number, non-small-cell lung cancer, protein expression
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introduction |
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Lung cancer is the leading cause of cancer-related death in the world [1]. Non-small-cell lung cancer (NSCLC) histologies (adeno-, squamous cell- and large-cell carcinoma) account for 80%–85% of all lung cancers and have 5-year survival rate of only 16% in the United States [2]. Progress in lung cancer biology led to the identification of the epidermal growth factor receptor (EGFR) signaling pathway as a therapeutic target leading to the development of small molecule tyrosine kinase inhibitors (TKIs) including gefitinib (Iressa®, AstraZeneca, Macclesfield, UK) and erlotinib (Tarceva®, OSI/Genentech, Boulder, CO/South San Francisco, CA). Objective responses to EGFR TKIs were reported in 10%–27% of NSCLC patients after failure of chemotherapy [3–5]. A randomized phase III trial comparing erlotinib to placebo, NCIC BR-21, showed survival advantage of erlotinib [hazard ratio (HR) = 0.73, P = 0.01], while a similar study comparing gefitinib to placebo, ISEL 709, showed no statistically significant difference (HR = 0.89, P = 0.1) [6, 7]. In both of the studies, at least 30% of patients in each treatment arm died within 3 months indicating a subgroup of patients with no clinical benefit [6, 7].
Several clinical features were found to associate with increased response rates to EGFR TKIs, including Asian ethinicity, never-smoking history, female gender and adenocarcinoma histology [6–8]. Of these features, Asian ethnicity and never-smoking history were also associated with survival benefit in two phase III monotherapy studies, BR-21 and ISEL 709 [6, 7]. Survival benefit in BR-21 study, however, was also observed in patients with very low probability of response to erlotinib–i.e. smoking males with squamous cell carcinomas [9]. Thus, clinical factors indicate populations of NSCLC patients more likely to respond, but do not include all patients who derive survival benefit from the EGFR TKIs.
Specific mutations in the tyrosine kinase domain of the EGFR gene were reported to be associated with a good prognosis irrespective of therapy, and with other favorable prognostic features including Asian ethnicity, female gender, never-smoking status and adenocarcinoma histology [10–16]. In some phase II studies, EGFR mutations were associated with improved survival [12–15], while in the other study they did not [17]. The discrepancies could relate to patient features such as ethnicity and smoking status, type of mutations or study methodologies. In the randomized placebo-controlled trial comparing erlotinib to placebo, EGFR mutations did not predict for a survival benefit from the EGFR TKI [18] and in the placebo-controlled ISEL study there were too few mutations to allow for meaningful conclusions [19]. The BR-21 trial results were criticized for the high proportion of rare EGFR mutations that could represent artefacts [20]. Updated analysis of this study based only on ‘classical’ mutations (i.e. exon 19 deletions and exon 21 point mutations), however, did not change the study conclusions [21]. Multivariate analysis of the BR-21 trial showed that only clinical factors were significantly associated with survival. This analysis, however, should be interpreted with caution due to relatively limited number of patients with available data on all biomarkers.
Increased EGFR gene copy number detected by FISH demonstrated strong correlation with response, progression-free survival (PFS) and overall survival (OS) after treatment with gefitinib in our previous studies [17, 22], in the BR-21 randomized study comparing erlotinib to placebo [18] as well as in the ISEL randomized trial [19]. A similar association between genomic gain and sensitivity to EGFR TKIs has been observed in NSCLC cell lines [23, 24]. Furthermore, FISH positivity was associated with a poor outcome after placebo in the randomized trials, in surgically treated patients, and was not predictive for outcome in patients treated with chemotherapy [19, 25, 26].
The association between EGFR protein expression detected by immunohistochemistry (IHC) and clinical outcome after EGFR TKIs has been variable. In initial studies, EGFR protein expression was not associated with clinical outcome to gefitinib [27–29], whereas more recent studies, including both the placebo-controlled randomized trials, demonstrated an association with treatment response and differential effect on survival in unselected NSCLC patients [18, 19]. In no prospective clinical study, patients with negative protein expression benefited from EGFR TKIs [18, 19, 30]. Activation of AKT was demonstrated to associate with increased response to gefitinib in several studies [17, 31], whereas KRAS mutations were associated with resistance to EGFR TKIs [32].
In order to obtain a larger number of patients and to carry out further analyses, we have pooled data from two previously reported studies [17, 22]. In this report, we examine the clinical implications of using six biological markers individually and in combination for selection of NSCLC patients for gefitinib therapy. The size of current study population allowed us to better characterize predictive value of combined markers and analyze the individual EGFR mutations.
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patients
Patients with histologically confirmed NSCLC were enrolled in two clinical studies, one conducted in Italy [17] and one in the Unite States by the Southwest Oncology Group (SWOG study 0126) [22, 33]. Each study was approved by institutional review boards of participating hospitals and the participants gave written informed consent before enrollment. The SWOG study was limited to patients having advanced adenocarcinoma with bronchioloalveolar carcinoma (BAC) features or pure BACs. The Italian study used gefitinib 250 mg daily, while the SWOG study used gefitinib 500 mg daily. All patients were evaluated for response according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria [34].
Demographic and clinical data are shown in Table 1. There were 204 NSCLC patients, of which 116 (57%) were males, 162 (79%) were former or current smokers, 89% had Eastern Cooperative Oncology Group performance status (PS) of zero or one, 87% had stage IV disease and 61% had received previous chemotherapy. Adenocarcinoma was diagnosed in 99 (49%) and BAC in 63 (31%) of these patients. Tumor specimens for biomarker analysis were obtained in all patients of the Italian cohort and in 100 of 136 patients in the SWOG cohort. All biopsies were obtained before any therapy. In the SWOG study, the clinical outcome for patients with available specimens was similar to the clinical outcome for the whole patient population [22].
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FISH, IHC and mutation analyses
EGFR and HER2 gene copy numbers per cell were investigated on formalin-fixed paraffin-embedded specimens using the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe set and the PathVysion HER-2 DNA probe Kit (Abbott Molecular, Des Plaines, IL). Patients were classified in two strata: (i) FISH+ patients with tumors having high level of polysomy (four or more copies of the gene in
40% of cells) or gene amplification; (ii) FISH– patients with tumors with no or low genomic gain (four or more copies of the gene in <40% of the cells). The FISH protocol and sample classification is described in details elsewhere [17, 18]. EGFR protein expression was evaluated by IHC using methods and assessment criteria described elsewhere [17, 25] with the mouse anti-human EGFR, clone 31G7 monoclonal antibody (Zymed Laboratories, Inc., San Francisco, CA). Phospho-AKT was also detected by IHC using the rabbit anti-mouse pAKT (Ser 473) polyclonal antibody (Cell Signaling Technology, Beverly, MA) according to the manufacturer's protocol. Final score for EGFR and pAKT expression assessment was obtained by multiplying intensity (0–4) and fraction of positive cells (0–100%), scores of 0–199 were considered negative/low expression (EGFR IHC–), and scores of 200–400 were considered positive/high expression (EGFR IHC+).
Fifty nanograms of genomic DNA was isolated from the tumor paraffin sections and amplified for EGFR exons 18, 19, and 21 by touchdown heminested PCR and sequenced in both sense and antisense directions. Exons 18, 19 and 21 were examined because they harbor the majority of the EGFR mutations in NSCLC reported to date [10, 11, 35]. Automated sequencing was carried out using an ABI 3730 DNA sequencer to create tracings that were manually reviewed and analyzed using the Mutation Surveyor program (SoftGenetics, LLC, State Park, PA), and compared with a known EGFR mutation-negative sample.
Analysis for KRAS mutations in exon 2, including codons 12 and 13, was also done with the DNA from the pretreatment tumor samples. A highly sensitive, two-step restriction fragment length polymorphism-PCR assay that identifies all KRAS 12th codon mutations was adapted for these studies [36]. PCR products were sequenced from each of the specimens using an automated DNA sequencer, which allowed the specific identification of point mutations at codon 12 and any base changes at codon 13.
statistical methods
Response evaluation was carried out by RECIST criteria [34] in 168 assessable patients with measurable disease, while PFS and OS analyses included all patients. PFS was calculated as the time of study registration to either progression of disease, death from any cause or last contact. PFS and OS curves were estimated by the product-limit method and compared using the log-rank test. Cox proportional hazards regression was used to assess the influence of each biological and clinical feature on survival and to estimate HRs. Multivariate models were constructed using backward stepwise regression methods. All univariately significant covariates were included in the stepwise selection.
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clinical variables
In the whole group of patients, objective response rate (OR) was 15%, disease control rate (DCR) was 43%, median PFS was 3 months, median survival was 11 months and 1-year survival rate was 48% (Table 1). Females (43%) had a higher OR than males (25% versus 7%, P = 0.008) and longer median survival (16 months versus 8 months, P = 0.004). Never smokers had a marginally higher OR than current or former smokers (26% versus 12%, P = 0.087) and a significantly longer median survival (19 months versus 9 months, P = 0.009). Patients with PS of zero to one had improved survival compared with those with PS of two (median 13 months versus 4 months, P < 0.001). Patients with adenocarcinomas/BAC subtypes had a response rate of 17% versus 8% (P > 0.05) and a median survival of 14 months versus 6 months for the patients with squamous cell or large-cell carcinomas (P = 0.001). There was no difference in treatment outcome between patients who had previously received systemic treatment versus those without any previous chemotherapy.
molecular variables
The results comparing the six molecular markers with clinical outcome are shown in Table 2. EGFR gene copy number was assessed by FISH in 183 patients, and was positive in 59 patients (32%). There were significant differences in OR (P < 0.001), DCR (P < 0.001) and OS (log-rank P value = 0.002) between the two FISH strata (Figure 1A).
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HER2 gene copy number detected by FISH was positive in 40 of the 157 patients analyzed (25%). OR was higher in HER2 FISH+ patients compared with HER2 FISH– (P = 0.002) but OS was nonsignificantly longer (P = 0.145).
EGFR protein was overexpressed in 121 of 200 analyzed patients (61%). EGFR IHC+ patients had higher OR (P = 0.002), DCR (P < 0.001) and OS (P = 0.003) compared with the EGFR IHC– patients (Figure 1B).
Immunohistochemical examination for pAKT was positive in 127 of 184 analyzed patients (69%). The pAKT-positive patients had a significantly higher OR (P = 0.005), but not DCR (P = 0.071). Median PFS and median OS were longer in the pAKT-positive group but the differences were not significant.
EGFR mutations were present in 43 of 157 analyzed patients (27%). Thirty-one patients had exon 21 mutations and 11 patients had exon 19 deletions exclusively. One patient had mutations in both exons 19 and 21. OR in patients with EGFR mutations was significantly higher than in patients without mutations (P < 0.001) but DCR was not (P = 0.148). Median PFS was 3 months in both groups, and median OS was 13 months versus 11 months, respectively; these differences were not significant (Figure 1C). Patients with exon 19 deletions exclusively had OR of 67% versus 20% for patients with exon 21 mutation (P = 0.021), a median PFS of 15 months versus 2 months, respectively, and median OS of 26 months versus 10 months, respectively (insufficient number of patients to detect significant difference). There was a difference in PFS (11 months versus 3 months) and OS between patients with and without exon 19 mutations (26 months versus 11 months), but the statistical power to test the significance of the difference was too low to adequately test differences in survival times. No difference was apparent in PFS or OS between patients with and without exon 21 mutations.
KRAS mutations were detected in 36 of 138 analyzed patients (26%). Among KRAS mutant patients (n = 36), 8.3% were never smokers compared with 22% of the 102 KRAS wild-type patients (P = 0.084). Patients with KRAS mutations had nonsignificantly lower OR compared with those with KRAS wild-type tumors, (OR 7% versus 19%). There was no difference with regard to PFS or OS according to KRAS mutation status (P = 0.886 and P = 0.891, respectively).
association between biological and clinical markers
The association between biological markers and gender, smoking status and histology is shown in Table 3. Females had a higher frequency of EGFR mutations than males (38% versus 20%, P = 0.012) and increased HER2 copy numbers (34% versus 19%, P = 0.034), but no gender differences were seen regarding EGFR protein expression, EGFR gene copy number, pAKT expression or KRAS mutation. High EGFR copy numbers (P = 0.046) and mutations (P = 0.043) were more frequently found in never smokers than smokers. There were no statistically significant differences in EGFR and pAKT protein expression related to gender, smoking status or histology. No association between KRAS mutations and gender or smoking history was seen, however, adenocarcinomas/BAC subtypes had more KRAS mutations than other histologies (P = 0.002). With respect to survival, the results from the multivariate analysis showed that never smokers (HR = 0.37, P < 0.001), PS of zero to one (HR=0.28, P < 0.001), increased EGFR gene copy number (HR=0.54, P = 0.006), and high EGFR protein expression (HR = 0.59, P = 0.007) were independent prognostic variables. As shown in Table 1, the source of data (Italian versus SWOG cohorts) was significant univariately (P = 0.04) but dropped out of the stepwise regression analysis. Pure BAC versus others was also significant univariately (P = 0.032). However, by adding these potential confounding factors in the multivariate analyses, HRs for smoking status, PS, EGFR FISH and EGFR IHC were unchanged.
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multiplicity of predictive biomarkers
The association between markers is summarized in Table 4. EGFR FISH+ was significantly (P < 0.05) associated with HER2 FISH+, EGFR mutations and pAKT and marginally (P = 0.051) associated with EGFR IHC.
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The association with clinical outcome for different combinations of EGFR FISH and IHC is shown in Table 5. Among 179 patients evaluated for both EGFR FISH and IHC, 42 (23%) were positive for both tests, while 54 (30%) were negative for both tests. Similar outcome of patients who were FISH+/IHC– and those who were FISH–/IHC+ justified classing these patients together. Patients positive for both markers (EGFR FISH+/IHC+) had OR of 41%, a DCR of 76%, median PFS of 9 months [95% confidence interval (CI), 6–16 months], median OS of 21 months (95% CI, 15–21 months) and 1-year survival rate of 77% (Figure 1D). This clinical outcome was significantly better than for patients with double-negative tumors or those having one positive and one negative test. Patients negative for both markers (EGFR FISH–/IHC–) had a poor outcome with OR of 2%, DCR of 17%, median PFS of 2 months, median OS of 6 months and 1-year survival rate of 30%. The 83 patients with either FISH+ or IHC+ results had OR of 10%, DCR of 43%, median PFS of 3 months, median OS of 6 months and 1-year survival rate of 44% (Figure 1D). The latter results were intermediate between those EGFR FISH+/IHC+ and EGFR FISH–/IHC– patterns, but the outcome was significantly better than the double-negative group. The EGFR FISH and IHC combination remained significantly associated with survival after adjustment for clinical variables in the multivariate Cox regression model for OS (data not shown).
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EGFR FISH and mutations were evaluated in 144 patients, with 18 patients (13%) being positive for both markers, while 81 patients (56%) were negative for both. KRAS and EGFR mutation analyses were completed in 116 patients; five patients (4%) had both tests positive, while 68 patients (59%) had both tests negative. The addition of EGFR mutation, pAKT and/or KRAS mutation did not improve outcome prediction beyond that observed for EGFR FISH and IHC alone or in combination. For example, in FISH–/IHC– patients the outcome was especially poor despite presence of EGFR mutations (n = 6, OR=0%, median PFS = 2 months and median OS = 3 months), absence of KRAS mutations (n = 21, OR = 5%, median PFS = 2 months and median OS = 5 months) or pAKT expression (n = 32, OR = 4%, median PFS = 2 months and median OS = 6 months). Similarly, in the FISH+/IHC+ patients the outcome was excellent regardless of EGFR mutation, KRAS mutation or pAKT expression.
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discussion |
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Patient selection using IHC and FISH tests is used routinely for selection of hormonal therapies and HER2 antagonists for breast cancer [37]. The selection of NSCLC patients for EGFR TKIs is critically important due to the low response rate in unselected patients and the fact that at least 30% of patients have no survival benefit when compared with placebo [6, 7]. In this report, we evaluated six biologic markers in a pooled cohort from two previously reported studies to more precisely determine the associations of biomarkers and outcome. Different inclusion criteria for the two studies are responsible for imbalances in some of the clinical characteristics. Thus, our cohort includes a high proportion of patients with adenocarcinomas/BACs than would be expected in an unselected NSCLC population in clinical practice. In the SWOG 0126 study, most of the patients were treated with gefitinib as first-line therapy in contrast to the Italian cohort. The recent published data from the SWOG 0126 study, however, showed no difference in survival between patients who were previously treated with chemotherapy and those who were not [33]. Furthermore, the two studies differed in the given doses of gefitinib, but no difference in efficacy has been observed between 250 mg and 500 mg [3, 4]. The differences in the distribution of histological subtypes between the studies were taken into account in the multivariate analysis. Our analyses showed no change in HRs for smoking status, PS, EGFR FISH or EGFR IHC whether or not we adjusted for these potential confounders, justifying pooling of data from these two study cohorts.
The large number of patients included in this combined analysis adds power to previous findings that high EGFR gene copy number and EGFR protein overexpression are significantly associated with improved response and survival after gefitinib therapy and allows for analysis of test combinations [17, 18]. Both FISH and IHC markers were independent predictive factors for survival in multivariate analysis. Molecular analyses of the NCIC BR-21 randomized trial comparing erlotinib to placebo showed also an association between FISH and IHC positivity with survival [18]. The HR for survival in the BR-21 study was 0.44 for EGFR FISH+ patients and 0.68 for IHC+ patients [18]. The small sample size available for multivariate analysis of the latter study prohibited definitive conclusions on independent value of both biomarkers. Results from the randomized trial comparing gefitinib versus placebo (ISEL 709) showed HR = 0.61 in the FISH+ patients and HR = 0.71 for IHC+ patients (19). The ISEL 709 study indicates that FISH positivity is a predictive and not a prognostic marker because FISH+ patients receiving placebo had a worse survival than FISH– patients receiving placebo [19]. In addition, we previously reported that increased EGFR gene copy number detected by FISH was associated with a poor prognosis in surgically resected patients [25].
The clinical predictors of benefit to EGFR TKIs point towards different subsets of patients than the molecular markers, as demonstrated in molecular analyses of BR.21 and ISEL trials [18, 19]. In both studies, some survival advantage was observed with EGFR inhibitors across all clinical subsets, including also patients with squamous cell carcinomas. Although our data are not derived from a placebo-controlled study, the results indicate that molecular predictors of prolonged survival are independent from clinical features.
The large cohort size of the current study allowed us to evaluate the predictive role of the combination of EGFR gene copy number and EGFR protein expression. Positivity for the two markers, which identified a subset of 
25% of NSCLC patients with a much better clinical outcome, had a significant and independent association with prolonged survival. This double-positive group of patients had OR of 41%, DCR of 76% and a 1-year survival rate of 77%. Current standard first-line treatment of advanced NSCLC is platinum-based doublet chemotherapy, which provides a median survival time of 8–10 months [38, 39]. The addition of bevacizumab to standard chemotherapy improved the median survival from 10.2 months to 12.5 months in one recent randomized trial [40]. Our data in the double-positive patients who had progressed after chemotherapy appeared superior to these treatment results for advanced untreated NSCLC patients and illustrate the need for prospective trials in selected NSCLC patients without prior therapy.
The combination of EGFR FISH and IHC negativity identified a subset of 30% of NSCLC patients, who had no clinical benefit from gefitinib treatment. Patients with tumors negative for both markers had low OR (2%) and DCR (17%) with short survival (median of 6 months and 30% 1-year survival rate). These results indicate strongly that the patients with tumors negative for EGFR FISH and IHC should not be offered treatment with EGFR TKIs in any setting.
The addition of other markers, such as EGFR mutation and pAKT to the double-negative group provided no enhancement to the prediction, as the outcome was poor in any patient with FISH– and IHC– tumors. The group of patients with tumors positive for one of the markers, EGFR FISH or IHC (45% of the total population) had an intermediate clinical outcome with ORs and DCRs of 10% and 43%, respectively, and a median survival of 11 months. The outcome in this group was significantly better than in the double-negative group. Prospective trials are needed to identify the most appropriate treatment of the ‘one marker positive’ group of patients.
There are reports of survival benefit in patients carrying EGFR mutations especially in Asian populations [12–14, 16]. The current study and the NCIC BR-21 trial concluded that unselected EGFR mutations were not significantly associated with survival. The BR-21 molecular analysis was criticized for unexpectedly high rate of uncommon mutations, however updated analysis limited to exon 19 and 21 mutations did not change initially reported study conclusion [21]. Exon 20 mutations were not analyzed in our study as they represent a small minority of EGFR mutations [35], although may include known resistance mutation T790M [41]. We found that the type of EGFR mutation associated with survival. Patients with deletions in EGFR exon 19 had superior PFS and OS compared with those without these mutations and compared with patients with exon 21 mutations. In prior studies, EGFR mutations were associated with a better prognosis independent of the type of therapy, indicating that EGFR mutations represent a more indolent biological course [42, 43]. Recently, two studies reported that patients with EGFR exon 19 deletions had a longer survival than patients with EGFR L858R point mutations [44, 45]. Future and larger studies will have to verify whether differences in prognosis and prediction exist between the subtypes of EGFR mutations.
The current study demonstrated that FISH analysis for EGFR gene copy number in combination with IHC assessment for EGFR protein is a useful paradigm for selection of advanced NSCLC patients to gefitinib. Validation of our results is currently undergoing in prospective studies with enriched populations. The proposed selection paradigm is on the basis of two already established laboratorial platforms; both of them are easily applicable to the formalin-fixed, paraffin-embedded tissue material, routinely available for diagnosis. The good clinical outcome in patients with advanced NSCLC patients who are FISH and/or IHC+ for EGFR expression, should also encourage studies of EGFR TKIs in early disease settings, for example as adjuvant therapy after surgery in enriched study populations.
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National Cancer Institute (NCI) grants 2P30-CA 46934, P01-CA 58187 and Southwest Oncology Group grants CA32102 and CA38926. Cappuzzo was supported by the grants from the Department of Oncology, Bellaria Hospital, Bologna, Italy and the Italian Association for Cancer Research (grant # 2881). Dziadziuszko is on leave from Medical University of Gdansk, Poland and supported by International Association for the Study of Lung Cancer.
Received for publication August 28, 2006. Revision received November 9, 2006. Accepted for publication January 5, 2007.
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