Affiliations of authors: University of Colorado Health Sciences Center and University of Colorado Cancer Center, Aurora, CO (FC, FRH, LB, JH, SW, WAF, PAB, MVG); Bellaria Hospital, Bologna, Italy (FC, SB, EM, LC); CINECA Interuniversity Consortium, Bologna, Italy (ER, ID); Scientific Institute University Hospital San Raffaele, Milan, Italy (GLC, VG, CD); Response Genetic, Inc., Los Angeles, CA (KD); Policlinico Monteluce, Perugia, Italy (VL, AS, MT)
Correspondence to: Fred R. Hirsch, MD, PhD, University of Colorado Cancer Center, Department of Medicine and Pathology, 12801 E. 17th Ave., P.O. Box 6511, Mail Stop 8111, Aurora, CO 80010 (e-mail: fred.hirsch{at}uchsc.edu).
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ABSTRACT |
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INTRODUCTION |
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Given the biologic importance of the EGFR molecular network in carcinomas, several molecules have been synthesized that inhibit the EGFR tyrosine kinase domain (11,12). These inhibitors include gefitinib (Iressa; AstraZeneca, Macclesfield, UK) and erlotinib (Tarceva; OSI Pharmaceuticals Inc., Melville, NY), both of which are orally active, selective EGFR tyrosine kinase inhibitors that produce objective response rates of 12%27% in previously treated or untreated advanced NSCLC (1316). Recently, the Canadian trial BR.21 reported a survival benefit for erlotinib versus placebo as a second- or third-line therapy that was not confined to objective responders or to a single sex or histology (17).
Ways to identify NSCLC patients who are most likely to respond to EGFR tyrosine kinase inhibitors are being investigated. Interestingly, data from retrospective studies suggest that the level of EGFR protein expression is not associated with gefitinib response (1820). Activating mutations cause ligand-independent activity of receptor tyrosine kinases, and recent reports show that specific missense and deletion mutations in the tyrosine kinase domain of the EGFR gene (2123) are associated with EGFR tyrosine kinase inhibitor sensitivity and with female sex, adenocarcinoma histology, and never-smoking statusall clinical characteristics that are known to be related to tyrosine kinase inhibitor sensitivity (1316,24). Although these EGFR mutations can account for almost all objective responses obtained with tyrosine kinase inhibitors, the clinical benefit observed with these drugs and the survival benefit identified in the BR.21 trial cannot be explained only by the presence of mutations.
Among other important players in EGFR tyrosine kinase inhibitor sensitivity, the activation status of the Akt protein has been highlighted in preclinical and clinical studies (25,26). Akt is a serine/threonine kinase that acts downstream of EGFR to regulate many cellular processes, including cell survival, proliferation, and growth, and it is activated by phosphorylation at amino acids Thr308 and Ser473 (27). Sordella et al. (25) showed that gefitinib-sensitizing EGFR mutations activate antiapoptotic pathways involving Akt in lung cancer cell lines, and we have previously shown (26) that the activation status of Akt is associated with gefitinib sensitivity of NSCLC patients, in terms of response and time to progression, but not in terms of survival. The lack of association with survival could be explained by the presence of a subset of phosphorylated (P)-Akt-positive patients who are resistant to gefitinib therapy as a consequence of Akt activation by a nonEGFR-dependent mechanism (2830).
Gene amplification is another molecular mechanism responsible for oncogene overexpression, and this phenomenon has been associated with mutations in the EGFR gene in glioblastoma (31) and in the HER2 gene in breast cancer (32). The NSCLC cell line NCI-H3255 has been reported to carry the EGFRL858R mutation, which is found in patients who respond to gefitinib, and has gene amplifications as well, and is reported to be sensitive to gefitinib in vitro (33). However, the role of EGFR genomic gain in predicting gefitinib sensitivity in patients and its association with mutation remains to be clarified.
Here we investigated the status of EGFR at the genomic, mRNA, and protein levels using fluorescence in situ hybridization (FISH), DNA sequencing, quantitative reverse-transcriptionpolymerase chain reaction, and immunohistochemistry in 102 advanced NSCLC patients treated with gefitinib. We compared the results with P-Akt status and with clinical features and outcome.
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SUBJECTS AND METHODS |
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Patients included in this study were accrued from a prospective study of gefitinib (26) and the Expanded Access Study of gefitinib conducted at Bellaria Hospital (Bologna), Scientific Institute University Hospital San Raffaele (Milan), and Policlinico Monteluce (Perugia). Complete clinical information and tissue blocks were available from 80 of 106 patients enrolled in the Akt clinical trial (26) and from an additional 22 patients in the Expanded Access Study who were treated consecutively at the end of the Akt study and followed up in the same way as patients in the Akt trial. These studies were approved by the Bellaria Hospital institutional ethical review board, and written informed consent was obtained from each patient before enrollment. In the subgroup of patients participating in the Expanded Access Study of gefitinib, institutional review board approval was obtained according to Good Clinical Practice, and specific written informed consent was obtained from each patient (Expanded Access Study consent form, Italian version). Eligibility for both studies included histologically confirmed NSCLC with measurable, locally advanced or metastatic disease, progressing or relapsing after chemotherapy or with medical contraindications for chemotherapy. Patients had performance status ranging from grade 0 to 2. Performance status was defined according to Eastern Cooperative Oncology Group (34) and considered grade 0 when the patient was fully active and able to perform all predisease activities without restriction, grade 1 when the patient was restricted in physically strenuous activity but ambulatory and able to perform work of a light or sedentary nature, and grade 2 when the patient was ambulatory and capable of all self-care but unable to perform any work activities. Patients received gefitinib (250 mg/day) and were evaluated for response according to the Response Evaluation Criteria in Solid Tumors criteria (35). Tumor response was assessed by computed tomography scan after 2 months, with a confirmatory evaluation to be repeated in responders and in patients with stable disease at least 4 weeks after the initial determination of response. Time to disease progression was calculated from the date of initiation of gefitinib treatment to the date of detection of progressive disease or to the date of last contact. Survival was calculated from the date of therapy initiation to the date of death or to the date of last contact.
Tissue Preparation; EGFR Gene, mRNA, and Protein Analyses
Tumor specimens were obtained before any cancer therapy and embedded in paraffin. Serial sections (4 µm) containing representative malignant cells were stained with hematoxylin and eosin and classified based on the World Health Organization criteria (36). Gene copy number per cell was investigated by FISH using the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe (Vysis, Abbott Laboratories, IL), according to a published protocol (10). Sections were incubated at 56 °C overnight, deparaffinized by washing in CitriSolv (Fisher Scientific, Pittsburgh, PA), and dehydrated in 100% ethanol. After incubation in 2x saline sodium citrate buffer (2x SSC; pH 7.0) at 75 °C for 1525 minutes, sections were digested with proteinase K (0.25 mg/mL in 2x SSC; pH 7.0) at 37 °C for 1525 minutes, rinsed in 2x SSC (pH 7.0) at room temperature for 5 minutes, and dehydrated using ethanol in a series of increasing concentrations (70%, 85%, 100%). The EGFR/CEP 7 probe set was applied per the manufacturer's instructions onto the selected area based on the presence of tumor foci on each slide, and the hybridization area was covered with a glass coverslip and sealed with rubber cement. The slides were incubated at 80 °C for 810 minutes for codenaturation of chromosomal and probe DNA and were then placed in a humidified chamber at 37 °C for 2024 hours to allow hybridization to occur. Posthybridization washes were performed in 1.5 M urea and 0.1x SSC (pH 7.07.5) at 45 °C for 30 minutes and in 2x SSC for 2 minutes at room temperature. After the samples were dehydrated in ethanol as above, 4',6'-diamidino-2-phenylindole (DAPI; 0.15 mg/mL in Vectashield mounting medium, Vector Laboratories, Burlingame, CA) was applied for chromatin counterstaining. FISH analysis was performed independently by two authors (F. Cappuzzo and M. Varella-Garcia) who were blinded to the patients' clinical characteristics and all other molecular variables.
Patients were classified into six FISH strata with ascending number of copies of the EGFR gene per cell according to the frequency of tumor cells with specific number of copies of the EGFR gene and chromosome 7 centromere: 1) disomy (2 copies in >90% of cells); 2) low trisomy (
2 copies in
40% of cells, 3 copies in 10%40% of the cells,
4 copies in <10% of cells); 3) high trisomy (
2 copies in
40% of cells, 3 copies in
40% of cells,
4 copies in <10% of cells); 4) low polysomy (
4 copies in 10%40% of cells); 5) high polysomy (
4 copies in
40% of cells); and 6) gene amplification (defined by presence of tight EGFR gene clusters and a ratio of EGFR gene to chromosome of
2 or
15 copies of EGFR per cell in
10% of analyzed cells).
EGFR protein expression was evaluated by immunohistochemistry using methods and assessment criteria described elsewhere (10) with the mouse antihuman EGFR, clone 31G7 monoclonal antibody (Zymed Laboratories, San Francisco, CA). P-Akt was also detected by immunohistochemistry using the rabbit antimouse P-Akt (Ser 473) polyclonal antibody (Cell Signaling Technology, Beverly, MA), according to the manufacturer's protocol. P-Akt expression and EGFR expression were scored based on intensity and fraction of positive cells. The intensity score was defined as follows: 0 = no appreciable staining in the tumor cells, 1 = barely detectable staining in the cytoplasm and/or nucleus compared with the stromal elements, 2 = readily appreciable brown staining distinctly marking the tumor cell cytoplasm and/or nucleus, 3 = dark brown staining in tumor cells obscuring the cytoplasm and/or nucleus, or 4 = very strong staining of nucleus and/or cytoplasm. The score was based on the fraction of positive cells (0%100%). The total score was calculated by multiplying the intensity score and the fraction score producing a total range of 0400. For statistical analyses, scores of 0200 were considered negative/low expression, and scores of 201400 were considered positive/high expression. This cutoff level was based on consistency with previous studies from our group, in which we found a statistical correlation between increased EGFR protein expression and increased gene copy number (10). Immunohistochemistry assays were scored jointly by two authors (W.A. Franklin and F.R. Hirsch); blinded to clinical, FISH, and EGFR mutation results; if discrepancies occurred, a consensus score was made by the two readers after discussion of the slide.
RNA was isolated, cDNA transcribed, and quantitative real-time polymerase chain reactions performed as described previously (37). Microdissection of tumor cells was performed by manual or by laser capture technique using the PALM instrument (PALM Microlaser Technologies AG, Bernried, Germany), according to the manufacturer's guidelines. Primers and probes were as follows: forward EGFR primer: 5'-TCCGTCTCTTGCCGGGAAT-3'; reverse EGFR primer: 5'-GGCTCACCCTCCAGAACCTT-3'; EGFR Taqman probe: 5'-ACGCATTCCCTGCCTCGGCTG-3' (GenBank accession number NM_005228). DNA for mutation analysis was also isolated from the same tumors. Fifty nanograms of genomic DNA was amplified for EGFR exons 18, 19, and 21 by touchdown heminested polymerase chain reaction (38,39) and sequenced in both sense and antisense directions. Exons 18, 19, and 21 were examined because they harbor 98% of the 56 EGFR mutations in NSCLC reported to date (2123).
Statistical Analysis
Differences between and among groups were compared using Fisher's exact test or Pearson's chi-square test for qualitative variables and using Student's t test or analysis of variance for continuous variables. Normality of the distribution was assessed using the KolmogorovSmirnov test (40). Time to progression, overall survival, and 95% confidence intervals (CIs) were calculated and evaluated by the KaplanMeier method (41); different groups were compared using the log-rank test. Association of risk factors associated with survival was evaluated using Cox proportional hazards regression modeling with a step-down procedure (42). Only those variables with significant results in univariate analysis were included in the multivariable analysis. The criterion for variable removal was the likelihood ratio statistic, based on the maximum partial likelihood estimates (default P value of .10 for removal from the model). The study design guarantees independence of the observations. The proportional hazard assumption was tested by log-survival function analysis and found to hold. All statistical tests were two-sided, and statistical significance was defined as P<.05. All analyses were performed using the statistical package SPSS version 11.5 (SPSS Italia srl, Bologna, Italy).
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RESULTS |
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The clinical outcome based on sex, stage, histology, performance status, and smoking status, most of which was reported in a previous publication (25), is shown in Table 1. For the entire group, the objective response rate was 14%, the progression rate was 60%, the median time to progression was 2.9 months, the median survival was 9.4 months, and 1-year survival was 40.7%. Female sex (mean difference = 22.6%, 95% CI = 6.6 to 38.6; P = .004) and never-smoking status (mean difference = 30.8%, 95% CI = 5.3 to 56.3; P = .006) were statistically significantly associated with better response, and female sex (mean difference = 3.0 months, 95% CI = 4.5 to 10.5 months; P = .03), adenocarcinoma and bronchioloalveolar histology (mean difference = 5.0 months, 95% CI = 2.8 to 7.2 months; P = .03), and performance status 01 (mean difference = 7.4 months, 95% CI = 5.6 to 9.1 months; P = .004) were statistically significantly associated with longer survival.
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First we assessed EGFR gene copy number by FISH. Disomy for the EGFR gene was present in 35.3% of case patients, low trisomy in 16.7%, high trisomy in 2%, low polysomy in 13.7%, high polysomy in 19.6%, and gene amplification in 12.7% (Table 2; Fig. 1, AD). Patients with gene amplification and high polysomy had relatively high response rates (54% and 25%), long time to progression (9.1 and 6.3 months), and high 1-year survival rates (48.8% and 62.3%). Patients with high trisomy also had a high response rate (100%), a long time to progression (14 months), and a high 1-year survival (100%), but this group included only two patients. In contrast, among the patients with tumors in the disomy, low trisomy, and low polysomy categories, none were responders, more than 70% had progressive disease, time to progression was 3.5 months or less, and median survival was 7 months or less. For further analyses, patients with high gene copy numbers (gene amplification or high polysomy) were combined because of similar outcome and designated FISH+, and patients in all other categories (disomy, low trisomy, high trisomy, and low polysomy) were categorized as FISH. FISH+ patients represented 32.4% of the total group, and FISH patients represented 67.6% of the patients.
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EGFR Protein Expression and Clinical Outcome
EGFR protein expression was evaluated by immunohistochemistry in 98 patients (Fig. 1, EH), and the outcome of patients according to protein score is shown in Table 2 and Fig. 2, CD. Patients with the lowest scores (099) had no response, and only one had stable disease. These patients had a short time to progression (median 2.1 months) and short median survival (4.5 months), and 27% had 1-year survival. Patients with scores of 100199 also had a poor outcome, with a 65% rate of progressive disease, short time to progression (median 2.3 months), and poor survival (only 35% of the patients alive at 1 year). Because their outcomes were similarly poor, the 40 patients (41%) with scores less than 100 and in the range 100199 were combined (EGFR IHC). Patients with EGFR immunohistochemistry scores of 200299 and of 300399 had much better outcomes than patients in the EGFR IHC group, and because they had similar response rates, progression times, and survival, they were also grouped together (EGFR IHC+). EGFR IHC+ patients, compared with IHC patients, had statistically significantly higher objective response rate (21% versus 5%; P = .03), lower progression rate (44.8% versus 80%; P<.001), longer time to progression (5.2 versus 2.3 months; P = .001), and longer survival (11.5 versus 5.0 months; P = .01). Protein status was not associated with clinical characteristics (Table 3) but was statistically significantly correlated with gene copy numbers (Pearson r = .28, P = .006).
EGFR Mutation Analysis and Clinical Outcome
Mutation analysis for EGFR exons 18, 19, and 21 was performed in 89 case patients (60 microdissected and 29 nonmicrodissected specimens). EGFR mutations were found in 15 patients (EGFR mutation positive = 17%), 12 from microdissected and three from nonmicrodissected specimens (P = .30), and consisted of missense mutations in exon 21 (n = 8) or small in-frame deletions in codons 746753 in exon 19 (n = 7) (Fig. 3). All of these mutations have previously been described (1113), with the exception of the missense mutation in exon 21 (valine 851 to isoleucine, V851I), which occurred in a male patient experiencing progressive disease. The presence of EGFR mutations was associated with never-smoking history (P = .007). The associations with sex and histology were not statistically significant (P = .10 for both), although mutations were more frequent in women and in patients with adenocarcinoma (Table 3).
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To define which variables were predictive for survival, those factors that were statistically significant in the univariate analysis (sex, histology, performance status, FISH, and protein status) were included in a multivariable model. Mutation and smoking status were not included because they were not associated with survival (P = .09 and P = .20, respectively) in univariate analyses. Poor performance status (PS 2) remained statistically significantly associated with increased risk of death (hazard ratio [HR] = 3.27, 95% CI = 1.49 to 7.17; P = .003), whereas adenocarcinoma/bronchioloalveolar histologies (HR = 0.58, 95% CI = 0.35 to 0.96; P = .035) and FISH status (HR = 0.44, 95% CI = 0.23 to 0.82; P = .01) were statistically significantly associated with better survival. Protein status (HR = 0.60, 95% CI = 0.36 to 1.01; P = .056) and sex (HR = 1.43, 95% CI = 0.79 to 2.6; P = .20) were not statistically significantly associated with survival. To demonstrate that EGFR adds to the predictive power of the other variables, we fit the multivariable model with and without EGFR and compared the two models by likelihood ratio method. The 2 log likelihood for the first model without EGFR was 505.16. The 2 log likelihood for the second model with EGFR was 500.20. The difference between the two models is 4.96 (P<.05). Statistical significance is based on the chi-square test with 1 degree of freedom.
Association Between EGFR and P-Akt
Evaluation of the P-Akt protein was successful in 98 patients (Table 4). P-Aktpositive status was significantly associated with better response rate (21% versus 0%, mean difference = 20.6%, 95% CI = 11.0 to 30.2; P = .004), disease control rate (50% versus 22%, mean difference = 28.1%, 95% CI = 9.5 to 46.7; P = .008), longer time to progression (4.2 versus 2.1 months, mean difference = 2.1 months, 95% CI = 0.7 to 3.4 months; P = .01), but not with survival (11.4 versus 9.4 months, mean difference = 2.0 months, 95% CI = 1.3 to 5.3 months; P = .20). P-Aktpositive status was also statistically significantly associated with EGFR gene gain (FISH+ Pearson r = .30; P = .01) and high level of protein expression (EGFR IHC+ Pearson r = .27; P = .01), but not with EGFR mutation (P = .08).
Combining FISH and P-Akt data (Table 5), we observed that double-positive patients (EGFR FISH+/P-Akt+) had a statistically significantly higher response rate (41% versus 3%, mean difference = 38.5%, 95% CI = 20.1 to 56.8; P<.001) and disease control rate (72% versus 28%, mean difference = 44.9%, 95% CI = 26.6 to 65.3; P<.001), longer time to progression (9.0 versus 2.5 months, mean difference = 6.5 months, 95% CI = 3.3 to 9.8 months; P<.001) and survival (18.7 versus 9.4 months, mean difference = 9.3 months, 95% CI = 4.7 to 13.9 months; P = .04) compared with EGFR FISH patients and/or P-Akt patients. Similar findings were observed when EGFR immunohistochemistry and mutation data were combined with P-Akt data. Compared with EGFR and/or P-Akt patients, EGFR IHC+/P-Akt+ patients had a statistically significantly better response rate (29% versus 4%, mean difference = 25.8%, 95% CI = 10.9 to 40.4; P<.001), disease control rate (66% versus 23%, mean difference = 43.1%, 95% CI = 23.9 to 60.6; P<.001), longer time to progression (6.2 versus 2.3 months, mean difference = 3.9 months, 95% CI = 1.5 to 6.3 months; P = .001), and longer survival (14.9 versus 8.3 months, mean difference = 6.6 months, 95% CI = 4.0 to 9.2 months; P = .03). EGFR mutation+/P-Akt+ patients had a statistically significantly better response rate (67% versus 6%, mean difference = 61.2%, 95% CI = 34.0 to 88.4; P<.001), disease control rate (75% versus 32%, mean difference = 43.5%, 95% CI = 16.8 to 70.2; P = .008), longer time to progression (11.2 versus 2.6 months, mean difference = 8.6 months, 95% CI = 3.3 to 14.0 months; P = .004), and longer survival (20.8 versus 9.3 months, mean difference = 11.5 months, 95% CI = 1.1 to 24.2 months; P = .044) than EGFR mutation and/or P-Akt patients.
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Unfavorable outcomes were also observed in the group of patients negative for EGFR but positive for P-Akt (Table 5). Compared with the double-positive group, the EGFR FISH/P-Akt+ group had a statistically significantly worse response rate (5% versus 41%, mean difference = 36.1%, 95% CI = 16.8 to 55.4; P<.001), disease control rate (32% versus 72%, mean difference 40.8%, 95% CI = 3.7 to 9.1 months; P = .001) and a nonstatistically significantly shorter survival (8.4 versus 18.7 months, mean difference = 10.3 months, 95% CI = 7.2 to 13.4 months; P = .083). Similar findings were observed when EGFR was evaluated by immunohistochemistry or for mutations. In both cases, the EGFR/P-Akt+ group had a statistically significantly worse response rate (P = .034 and P<.001, respectively, for protein and mutation), disease control rate (P = .002 and P = .025), time to progression (P = .010 and P = .009) and had a nonstatistically significantly worse survival (P = .080 and P = .070), compared with the double-positive group.
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DISCUSSION |
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Some limitations to the study must be taken into consideration. Our finding is so far based on a single retrospective study with a relatively small number of patients, and before any clinical recommendation can be made, our data need to be verified in a larger cohort of patients and prospectively. The EGFR status was determined on tumor tissue at time of primary diagnosis, and possible changes after chemotherapy were not determined in this study. Finally, both the classifications of the EGFR gene copy status and protein status represent a posthoc aggregation based on our previous reported studies (10). However, although our group and others are exploring other methods to express the results of the EGFR expression, our classifications seems to be associated with a meaningful clinical outcome after gefitinib therapy.
The question could be raised whether increased EGFR copy number per se has a positive impact on prognosis, independent of the treatment. The converse appears to be the case; we have previously reported that NSCLC patients with resected tumors carrying high EGFR gene copy number have a tendency to a shorter survival (10). Thus, similar to the findings in breast cancer for HER2 and trastuzumab (Herceptin; Genentech/Roche), increased EGFR gene copy number in NSCLC seems to be a poor prognostic feature but a good predictor for sensitivity to EGFR inhibitors.
The presence of EGFR gene mutations was also related to better response to gefitinib and time to progression, but the difference in survival did not reach statistical significance. An interesting finding was the association between EGFR mutations and increased gene copy number, a phenomenon that was recently described in the human lung cancer cell line H3255 (32) and that is probably relevant to gefitinib sensitivity. In fact, among the eight patients with EGFR mutations who responded to gefitinib therapy, seven were also FISH+, and among the six nonresponding patients with EGFR mutations, four presented a disomic pattern. This observation suggests that the impact of genomic gain is critical for EGFR mutations to predict gefitinib sensitivity.
Another important finding was the virtual absence of EGFR mutations in patients with stable disease. Among the 21 patients with stable disease who were assessed for EGFR mutations, only one patient had an EGFR mutation. We defined stable disease as neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, as confirmed by two consecutive observations no less than 4 weeks apart. The small number of mutations in patients with stable disease is of clinical relevance because data from the BR.21 trial (17) show that the survival benefit of gefitinib is not confined to responding patients. It is possible that survival improvement in the gefitinib-treated patients, as a whole, is due to the presence of a group of patients with an intermediate benefit from the treatment, such as those with stable disease, who would be excluded from tyrosine kinase inhibitor treatment if mutation analysis were established as the test of choice for patient selection. Moreover, although previous studies suggested that EGFR mutations are present in almost all responding patients (2123), in this study we observed that 40% of patients with EGFR mutations had progressive disease. These somewhat discrepant results could be explained by the fact that this is the first study conducted in a large and unselected cohort of gefitinib-treated patients, in whom clinical results are similar to those obtained in large clinical trials with gefitinib (13,14).
In this study, gefitinib sensitivity was associated with high EGFR protein expression; outcomes in patients with low EGFR expression scores (<200) were as poor as those in patients with low gene copy numbers or lacking mutations, which is different from what has been observed in previous studies (1820). The lack of standardization in staining procedures and guidelines for interpretation of the EGFR assessment may be the major reason for the conflicting results across studies. The cutoff level used to classify a patient as "positive" and "negative" also varies from one study to another. In an earlier study (18), we used the Zymed monoclonal antibody (Zymed Lab, San Francisco, CA) and scored the specimens according to the Herceptest criteria; tumors were classified as negative when the immunohistochemical staining score was 0 to 1+ and positive when the score was 2+ to 3+. Parra et al. (20), using the DAKO EnVision visualization system (DAKO Corp, Glostrup, Denmark), scored staining intensity as negative to faint (0/1+) or medium to strong (2+/3+) and classified tumors with less than 20% immunoreactive cells as negative/low expressors and those with at least 20% immunoreactive cells as high expressors. In specimens from the IDEAL trials (19), the pharmDx kit (DakoCytomation, Glostrup, Denmark) was used, and EGFR expression was measured for both membrane and cytoplasmic staining using a scoring system with values ranging from 0 to 3. In this study, we used the same antibody as we did in our previous study (18), but a different scoring system. The final score of EGFR expression was made by multiplication of the percentage of positive cells (0%100%) and the dominant pattern of intensity staining intensity (1 = negative or trace; 2 = weak; 3 = moderate; 4 = intense), creating a score ranging from 0 to 400.
The sampling size and selection of tissue material for immunohistochemical staining might also contribute to explain differences in results across the studies. For instance, tumors from only 43 and 50 patients were evaluated by Cappuzzo et al. (18) and Parra et al. (20), respectively. In the retrospective immunohistochemical analysis of tumor tissue from the IDEAL trials, less than 40% of the total population of patients were studied (19), whereas in our study more than 90% of patients had tissue available for immunohistochemical staining. Although the authors of the IDEAL trials have reported that the subgroup of patients who underwent EGFR analysis was representative of the whole study population in terms of sex, age, and histology, it is possible that the larger tumors, which have more tissue available for immunohistochemical staining analysis, represent distinct biology from that of small tumors, from which tumor tissue may not have been available. The impact on the outcome by using different antibodies and scoring systems for immunohistochemical staining assessment of EGFR should be addressed in future comparative investigations. To our knowledge, such data are not currently available either for NSCLC or for other tumors.
In this study, we also found an association between activated Akt pathway (e.g., expression of phosphorylated Akt) and gefitinib sensitivity, an association that has also been described and discussed by others (25,26). The combinatorial analysis of EGFR and P-Akt status indicated that, independent of the method of EGFR assessment, when EGFR status was positive, the presence of Akt phosphorylation was statistically significantly related to better response, disease control rate, time to progression, and survival. Importantly, better outcome was observed not only when the subset of EGFR+/P-Akt+ patients was compared with all the other groups combined but also when this subset was compared with patients EGFR positive but P-Akt negative. These findings support the hypothesis that, when the gefitinib target is present but the antiapoptotic pathway is not activated, the patient is not sensitive to the inhibitory effects of gefitinib, as suggested by our previous study (26) and as demonstrated in preclinical models (28,29). As expected, the EGFR+/P-Akt+ group also had a statistically significantly better outcome compared with the EGFR-negative and P-Aktpositive group, confirming preclinical data indicating that aberrant, EGFR-independent Akt activation may lead to gefitinib resistance (29,30). These data indicate that P-Aktpositive status is relevant in EGFR-positive patients for the identification of a subgroup of patients particularly sensitive to the drug. In EGFR-negative patients, P-Aktpositive status may identify a group of patients with a very low chance of benefiting from gefitinib treatment.
Information regarding the relationship between EGFR protein expression and Akt pathway activation would greatly advance the understanding of the mechanisms of gefitinib sensitivity. We compared EGFR protein and P-Akt expression in a subgroup of patients and, in general, we found expression of EGFR and P-Akt proteins in the same cell populations (data not shown), suggesting that the observed P-Akt was a result of EGFR activity. However, in some cases we found discrepancies in the expression (i.e., some cells expressed EGFR and not P-Akt and vice versa.). Reasons for discrepancies could be biological; for instance, Akt in EGFR-negative cell populations could be activated by other pathways. However, the discrepancies could also be explained by technical reasons, because the sections compared were not always in immediate proximity to each other. Future prospective studies should be undertaken to verify whether the same cells express both EGFR protein and activated Akt or whether the Akt activation for drug activity might also occur through other paracrine pathway mechanisms.
In conclusion, results from this study demonstrate that gefitinib is most effective in advanced NSCLC patients with high EGFR gene copy number, protein expression, or EGFR mutations. Because only high EGFR gene copy number was associated with prolonged survival in the multivariable analysis, and because FISH is a readily available clinical test, the EGFR FISH analysis represents an ideal test for selecting candidate NSCLC patients for gefitinib therapy. Because patients who had either high EGFR gene copy number or protein expression and P-Akt expression had a better response, disease control rate, time to progression, and survival, analysis of the activating status of the Akt protein may also be relevant for proper patient selection. Prospective studies are ongoing to validate these results.
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NOTES |
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Within the last two years, F.R. Hirsch has participated in advisory board meetings for AstraZeneca, Eli Lilly, and Ligand Pharmaceuticals, and P.A. Bunn has been a consultant for Bristol-Myers Squibb, ImClone, AstraZeneca, Genentech/OSI, Pfizer, and Roche.
Supported by NCI Cancer Center Shared Grant 2P30-CA46934, Specialized Program of Research Excellence P01-CA58187, and NCI Early Detection Network U01-CA 85070. FC was a visiting professor at the University of Colorado Health Sciences Center sponsored by the Department of Medical Oncology of the Bellaria Hospital, Bologna, Italy, and was in part supported by the Italian Association for Cancer Research (AIRC) grant number 2881.
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Manuscript received October 14, 2004; revised February 7, 2005; accepted March 10, 2005.
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