Affiliations of authors: Department of Oncologic Sciences, Bellaria-Maggiore Hospital, Bologna, Italy (FC, EM, SB, CL, AC, SD, LL, CC, LC); Division of Radiochemotherapy, Scientific Institute University Hospital San Raffaele-Milano, Italy (GLC, VG, AS, CTP); CINECAInteruniversity Consortium, Bologna, Italy (ER); Policlinico Monteluce, Division of Medical Oncology, Perugia, Italy (VL, GB, MT)
Correspondence to: Federico Cappuzzo, MD, Bellaria Hospital, Division of Medical Oncology, Via Altura 3, 40139-Bologna, Italy (e-mail: federico.cappuzzo{at}ausl.bo.it)
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ABSTRACT |
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INTRODUCTION |
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One potential family of therapeutic targets is the epidermal growth factor receptor (EGFR) superfamily. These cell membrane receptors have been implicated in the development and progression of cancer through their effects on cell cycle progression, apoptosis, angiogenesis, and metastasis (36). The EGFR superfamily includes four distinct receptors: EGFR/ErbB-1, HER2/ErbB-2, HER3/ErbB-3, and HER4/ErbB-4. Although specific soluble ligands that bind to the extracellular domains of EGFR, HER3, and HER4 have been identified, no ligand has been identified for the HER2 receptor. Several ligands can bind to EGFR, including EGF and transforming growth factor (TGF-
). After the ligand binds the receptor, the receptor dimerizeseither as a homodimer or as a heterodimer with other members of the EGFR family, but preferentially with HER2and undergoes autophosphorylation at specific tyrosine residues within the intracellular domain. These autophosphorylation events in turn activate downstream signaling pathways, including the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3'-kinase (PI3K)Akt pathway. Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs. The MAPKs ERK1 and ERK2 subsequently regulate gene transcription and have been linked to cell proliferation, survival, and transformation in laboratory studies (7). Akt also plays a critical role in controlling the balance between cell survival and apoptosis (8). Phosphorylation of Akt is required for its activation; once activated, Akt inactivates proapoptotic proteins, including the Bcl-2 family member Bad and caspase-9, and cell cycleregulatory molecules (910). Akt function is also important for cell survival when cells are exposed to different apoptotic stimuli, such as growth factor withdrawal, ultraviolet radiation, and DNA damage (1115). Given its association with cell survival, it is not surprising that Akt has been found to be overexpressed in several cancers, including lung (15), pancreatic (16), thyroid (17), and ovarian (1819) cancers. Recently, Akt phosphorylation has been observed in more than 60% of patients with NSCLC (20).
Drugs interfering with EGFR pathways are thought to be of potential therapeutic benefit in tumors expressing or overexpressing EGFR, and during the last decade, several molecules have been synthesized to inhibit the tyrosine kinase domain of EGFR (21,22). Among the more promising new drugs is ZD-1839 (gefitinib or Iressa; AstraZeneca, London, U.K.), an orally active, selective EGFR tyrosine kinase inhibitor with antitumor activity against a variety of human cancer cell lines expressing EGFR (23). After objective responses were observed in 11% of study participants in phase I trails (2427), two large phase II trials (IDEAL 1 and 2) (28,29) were conducted. The results of the IDEAL trials confirmed that gefitinib is active in approximately 10% of patients with NSCLC in whom standard therapy failed (28,29). Although these results were considered encouraging because they were obtained from patients in whom standard therapies were largely ineffective, only a small percentage of patients responded to single-agent gefitinib. Preclinical data have suggested that EGFR expression is not the critical factor in determining whether a patient will respond to gefitinib (30), although tumors that overexpress HER2 are highly sensitive to gefitinib (31). In our previous trial (32), conducted on 63 patients with NSCLC who gave their consent for EGFR and HER2 analysis, we examined the association between efficacy and tolerability of gefitinib and EGFR and HER2 expression. In that small study (32), in which HER2 status was determined with immunohistochemistry, there was no association between HER2 expression and gefitinib activity.
Although the degree of EGFR expression does not affect response to gefitinib, clinical responses to EGFR-blocking agents suggest that, in some patients with NSCLC, the EGFR signaling pathway may be the critical pathway for tumor growth. It is possible that patients who respond to EGFR-blocking agents such as gefitinib are those patients in whom tumor cell survival is maintained by activated EGFR-dependent pathways. To test this hypothesis and to determine if it is possible to identify which patients may respond to gefitinib therapy, we evaluated the association between the phosphorylation status of MAPK and Akt in patients with advanced NSCLC before starting gefitinib therapy and evaluated their response to gefitinib therapy. This trial was designed to evaluate possible associations between the phosphorylation status of Akt and of MAPK and gefitinib activity in terms of response rate and time to progression.
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PATIENTS AND METHODS |
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Study enrollment began in February 2001, and the last patients were enrolled in August 2003. In the period, February 2001June 2002, because of the ongoing HER2 study (32), patients were asked to give their consent for inclusion in the HER2 study (N = 55), the Akt/MAPK study (N = 22) (the results of which are described in this article), or both studies (N = 8).
Patients included in the present study had histologically confirmed, measurable, locally advanced or metastatic NSCLC and had progressed or relapsed after standard therapy. Patients who had not received any previous chemotherapy were included in the study if they were considered not eligible for systemic therapy because of age, poor performance status, or any medical condition contraindicating chemotherapy. To be eligible for the study, patients had to be aged older than 18 years, have an ECOG performance status of 2 or less, have a white blood cell count of at least 3.5 x 109/L, have a platelet count of at least 100 x 109/L, have a hemoglobin level of at least 9 g/dL, have an absolute granulocyte count of more than 2.0 x 109/L, have a bilirubin level that was less than 1.5-fold the upper limit of normal, have a prothrombin time or activated partial thromboplastin time of less than 1.5 times that of control, have an alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels of less than threefold the upper limits of normal (this level was increased to fivefold for patients with known hepatic metastases), and have a calculated creatinine clearance rate of more than 45 mL/min. Patients were ineligible for the study if they had evidence of prior or concurrent malignancy, with the exception of in situ carcinoma of the cervix, adequately treated basal cell carcinoma of the skin, or no evidence of recurrence of a malignancy treated 5 or more years ago.
Before patients were included in the trial, all patients were asked about their smoking history and classified as nonsmoker (never smoker), former smoker (stopped smoking more than 6 months before enrollment onto the trial), or current smoker (stopped smoking less than 6 months before enrollment onto the trial or still an active smoker). Lung cancer histology was defined according to the World Health Organization pathology classification (33). Written informed consent was obtained from each patient entering the study. The study was approved by the appropriate ethics review boards and followed the recommendations of the Declaration of Helsinki for biomedical research involving human subjects and the guidelines for good clinical practice.
Study Design and Treatment
In this study, 106 consecutive patients with NSCLC who progressed or relapsed after standard therapy failed received gefitinib daily at a dose of 250 mg. Patients received the drug until their disease progressed (n = 77), they experienced unacceptable toxicity (n = 1), or they refused to comply further (n = 1). The drug was provided by AstraZeneca on a compassionate-use basis.
Before the time of entry on the trial, a baseline evaluation was performed for each patient that included a complete history and physical examination, a complete blood cell count and serum chemistry analysis, urinalysis, an electrocardiogram, chest x-ray, and a total-body computed tomography (CT) scan. Other imaging modalities, such as magnetic resonance imaging and bone scintigraphy, were performed according to specific clinical indications. All baseline imaging procedures were performed within 4 weeks before study entry. Biochemical screening, which was performed every 4 weeks, included assessments of serum levels of creatinine, electrolytes, alkaline phosphatase, bilirubin, AST, ALT, calcium, magnesium, and total protein. Patients were evaluated for response according to the RECIST (response evaluation criteria in solid tumors) criteria (36). Tumor response was assessed by CT scan every 2 months, with a confirmatory evaluation that was repeated in patients who had not progressed to therapy at least 4 weeks after the initial determination of response.
Immunohistochemical Staining
Tumor specimens obtained at the time of primary diagnosis or at the time of study entry were collected for P-Akt and P-MAPK immunohistochemistry. Paraffin-embedded tissue sections were stained with antibodies against phospho-Akt (P-Akt; Cell Signaling Technology, Beverly, MA) and phospho-MAPK (P-MAPK; Cell Signaling Technology), according to the manufacturer's recommended protocol. Briefly, 4-µmthick tissue sections were placed on glass slides and deparaffinized. The tissue sections were incubated in 1x Microstain Unmasker Buffer (pH 8) (Ventana, Tucson, AZ) for 40 minutes at 98 °C to unmask the antigens. The reaction was quenched with 1% hydrogen peroxide. Nonspecific binding sites were blocked with 5% goat serum in phosphate-buffered saline (PBS) for 1 hour at room temperature. The sections were then incubated with P-Akt (Ser 473) rabbit polyclonal antibody (1 : 50 diluted in phosphate buffer) or P-p44/42 Map Kinase (Thr202/Tyr204) polyclonal antibody (1 : 100 diluted in phosphate buffer) overnight at 4 °C. The sections were developed by using a two-step method involving a large-volume biotinylated goat polyvalent antibody and a large-volume streptavidin peroxidase reagent (Lab Vision, Fremont, CA). The negative controls were incubated with nonimmune solution in place of primary antibody.
Immunohistochemically stained sections were interpreted independently by two pathologists who were blinded to all patient information. The intensity of staining seen in different areas of the same slide was analyzed according to criteria described previously for p53 (34). Immunohistochemical analyses were centralized, and all were performed at the Bellaria Hospital Pathology Department. Immunohistochemical results were blinded, and the referring physician was informed of the results only at the end of the trial or when the patient was withdrawn from the study.
At present, there are no validated scoring systems for interpreting immunohistochemical staining for P-MAPK or P-Akt. We used a system for interpreting P-MAPK staining that was based on staining intensity. If none of the tumor cells stained, the intensity was coded as 0; if more than 10% of the tumor cells stained weakly, the intensity was coded as 1+; if more than 10% of the tumor cells stained moderately, the intensity was coded as 2+; and if more than 10% of the tumor cells stained strongly, the intensity was coded as 3+. Specimens having a score of 0 or 1+ were considered negative, whereas a score of 2+ or 3+ was considered positive. We used a system for interpreting P-Akt staining that was based on subcellular staining localization. Activation of Akt by phosphorylation results in its translocation from the cytoplasm to the nucleus (35). Thus, staining was considered positive if nuclear staining was present and negative if nuclear staining was absent.
Statistical Analysis
Intent-to-treat analyses were performed on data from all patients who entered the study and received treatment. The trial was designed to detect a difference in response rate between the patients whose tumors were P-Akt positive and patients whose tumors were P-Akt negative. A sample size of 94 patients, with 47 patients in each group, was considered adequate to detect a difference of 25% (5%30%) between the two groups, with a two-sided significance level () of 5% and a power of 90%. Sample size was calculated using SPSS version 11.5.1 (SPSS Italia srl, Bologna, Italy).
Statistical analyses were performed with respect to clinical characteristics and response to therapy. For both proteins, differences between the two groups (positive and negative) were compared by Fisher's exact test or chi-square test for nominal variables and the MannWhitney test or Student's t test for continuous variables. Normality of the distribution of continuous variables was assessed with the KolmogorovSmirnov test. Time to progression, overall survival, and 95% confidence intervals (CIs) were evaluated with the KaplanMeier method (37), and differences between the two groups were evaluated with the log-rank test. Risk factors associated with time to progression were evaluated using Cox's proportional hazards regression model with a step-down procedure (38). Proportional hazard assumptions were checked and satisfied. Only those variables with statistically significant results in univariate analysis were included in the multivariable analysis. The criterion for removing a variable was the likelihood ratio statistic, which was based on the maximum partial likelihood estimate (default P value of .10 for removal from the model). Data analysis was planned for 3 months after the enrollment of the last patient. This time was selected because standard therapies had failed in our cohort, and the patients therefore had a poor prognosis. The degree of agreement between the two pathologists involved in the study (EM and SD) was assessed by a weighted statistic (39). All statistical analyses were performed using SPSS version 11.5.1 (SPSS Italia srl).
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RESULTS |
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From February 2001 through August 2003, 106 consecutive patients from three Italian institutions fulfilled the selection criteria and received gefitinib daily at a dose of 250 mg. The majority of patients (76 patients) were enrolled after June 2002. Tumor tissue specimens were not suitable for immunohistochemistry from four patients: sections from two patients could not be stained for P-Akt and P-MAPK, sections from one patient could not be stained for P-Akt, and sections from one patient could not be stained for P-MAPK. Thus, 103 patients contributed immunohistochemistry results for either P-Akt or P-MAPK. Patient characteristics are listed in Table 1. The majority of the patients were male (63.1%), with a median age of 63 years (range = 2583 years) and with a good ECOG (Eastern Cooperative Oncology Group) performance status (86.4% had a performance status of 0 or 1). Among the patient cohort, 55.3% had adenocarcinoma, 12.6% had bronchioloalveolar carcinoma, 20.4% had squamous-cell carcinoma, 9.8% had undifferentiated carcinoma, and 1.9% had large-cell carcinoma. Seven patients (6.8%) received gefitinib as first-line therapy: One patient was aged older than 80 years, and six patients had comorbidities contraindicating chemotherapy. The remaining 96 patients had all received chemotherapy before gefitinib, and of these patients, 74.7% had received chemotherapy that included a platinum salt. At the time of study entry, the majority of patients were current (50.5%) or former (31.1%) smokers.
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We next evaluated the association between P-Akt and P-MAPK status and clinical variables. P-Akt status was statistically significantly associated with being female (P<.001), never-smoking history (P = .004), and a bronchioloalveolar carcinoma histology (P = .034). No association was found between P-Akt status and other tumor histologies. P-MAPK status was not statistically significantly related to any clinical variable.
Response to Therapy
One hundred patients were evaluable for response. Six patients were considered not evaluable for response because of the absence of any measurable lesion (one patient) or because the patient was lost to follow-up after the baseline visit (five patients). Gefitinib was administered orally, with a median treatment duration of 3.6 months (range = 0.623.5 months). One (1%) patient had a complete response, 13 (13%) patients had a partial response, and 26 (26%) patients had stable disease, for an overall response rate of 14% (95% CI = 7.9% to 22.4%). The overall response rate, including complete responses and partial responses, was 26.1% for patients with P-Aktpositive tumors and 3.9% for patients with P-Aktnegative tumors (mean difference = 22.2%, 95% CI = 14.0 to 30.4; P = .003). The disease control rate, which includes complete responses, partial responses, and stable disease, was 60.9% for patients with P-Aktpositive tumors and 23.5% for patients with P-Aktnegative tumors (mean difference = 37.4%, 95% CI = 27.9 to 46.9; P<.001). There was no difference in terms of response rate and disease control rate among patients with P-MAPKnegative tumors and patients with P-MAPKpositive tumors (response rates = 12% versus 22.7%, respectively; P = .298; disease control rate = 38.7% versus 50%, respectively; P = .461) (Table 2). Eighteen patients had tumors that were positive for both P-Akt and P-MAPK. In this subgroup of patients, the response rate was 29.4% and disease control rate was 58.8%, values that were not statistically significantly different from those observed for patients with tumors positive for P-Akt and negative for P-MAPK (response rate = 24.1%, disease control rate = 62.1%) but statistically significantly better than those observed for patients whose tumors were negative for both P-Akt and P-MAPK (response rate = 4.4%; P = .014; and disease control rate = 24.4%; P = .011) (Table 3). No comparisons were made with six patients whose tumors were negative for P-Akt but positive for P-MAPK because of the small sample size.
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Final analysis was performed in November 2003, after more than 3 months since the enrollment of the last patient. With a median follow-up time of 6.6 months (range = 0.624.1 months), the median time to progression for the entire cohort was 3.4 months (range = 0.624.1 months), and median overall survival time was 9.4 months (range = 0.624.1 months). At the time of the final analysis, 33 patients with P-Aktpositive tumors and 44 patients with P-Aktnegative tumors had radiologically confirmed disease progression, whereas 27 patients with P-Aktpositive tumors and 41 patients with P-Aktnegative tumors had symptomatic progression (onset of new symptoms or worsening of existing disease-related symptoms). Median time to progression was statistically significantly longer for patients with P-Aktpositive tumors than for patients with P-Aktnegative tumors (5.5 versus 2.8 months, mean difference = 2.7 months, 95% CI = 1.5 to 3.9 months; P = .004) (Fig. 1). Time to progression did not differ on the basis of P-MAPK status (3.2 months for patients with P-MAPKnegative tumors versus 4.8 months for patients with P-MAPKnegative tumors; P = .29). Median time to progression was 6.2 months for patients with tumors that were positive for both P-Akt and P-MAPK, which was not statistically significantly different from the median time to progression of patients with tumors that were negative for P-Akt and P-MAPK (2.8 months; P = .05) or from that of patients with tumors that were positive for P-Akt and negative for P-MAPK (0/1+) (5.3 months; P = .63).
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DISCUSSION |
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On the basis of previous findings, we hypothesized that response to gefitinib would occur only in patients whose tumors were dependent on sustained activation of the EGFR signaling pathways, and only in those in whom these pathways were suppressed by the drug. In this study, we evaluated the two major EGFR signaling pathways (PI3KAkt and Ras/Raf/MAPK). Only the PI3KAkt pathway was statistically significantly associated with gefitinib activity. Compared with patients whose tumors were negative for P-Akt, patients whose tumors were positive for P-Akt had a better response rate (26.1% versus 3.9%; P = .003) and disease control rate (60.9% versus 23.5%; P<.001). No difference in response and disease control rate was observed between patients with P-MAPKnegative and P-MAPKpositive tumors. Patients whose tumors were positive for both P-Akt and P-MAPK had better response and disease control rates only in comparison with patients whose tumors were negative for both markers (P = .01) and not in comparison with patients whose tumors were positive for P-Akt but negative for P-MAPK. These findings are consistent with preclinical data suggesting that the PI3KAkt pathway plays a critical role in the antitumor effects of gefitinib and that inhibition of the MAPK pathway is not sufficient to mediate response to the drug (40).
In two large phase II studies evaluating gefitinib, only being female and having an adenocarcinoma histology were associated with a clinical response (28,29). More recently, Shah et al. (41) also reported that, in a multivariable analysis of 140 patients, those patients who had never smoked cigarettes and those with bronchioloalveolar carcinoma histology were more likely to respond to gefitinib. These findings are in agreement with those of our study, in which P-Akt status was statistically significantly associated with being female (P<.001), never smoking history (P = .004), and bronchioloalveolar carcinoma histology (P = .034). Results from analyses of the time-to-progression data support the central role of Akt activation in gefitinib activity. Compared with patients whose tumors were negative for P-Akt, patients whose tumors were positive for P-Akt had a statistically significantly longer time to progression (5.5 versus 2.8 months; P = .004). We found no difference in the time to progression among patients grouped on the basis of P-MAPK status (P = .29), although we noted slight, albeit not statistically significant, evidence of a longer time to progression among patients whose tumors were positive for both markers compared with patients whose tumors were negative for both markers (P = .05).
To further investigate the difference in time to progression observed among patients whose tumors were positive or negative for P-Akt, we performed a planned multivariable analysis, and only those variables that were statistically significant in the univariate analysis (sex, performance status, smoking history, and P-Akt status) were included in the model. To ensure that only relevant factors were retained in the multivariable model, the backward regression technique was used at the 10% significance level. In the multivariable analysis, P-Akt status was statistically significantly associated with a reduced risk of disease progression (HR = 0.58, 95% CI = 0.35 to 0.94). Importantly, after being adjusted for P-Akt status, performance status and smoking history remained statistically significantly associated with an increased risk of disease progression (HR = 2.65 [95% CI = 1.33 to 5.27] and 1.75 [95% CI = 1.08 to 2.85], respectively), and female sex was immediately removed at the first step of the backward elimination. These data indicate that P-Akt status, performance status, and smoking history are independent factors for disease progression but that being female is not when Akt status is considered. These findings do not represent a variation in respect to the IDEAL trial results because in those studies (28,29), sex was related to response rate and not to time to progression. Moreover, the impact of P-Akt was not evaluated in the multivariable model used in the IDEAL trials.
Although response rate, disease control rate, and time to progression were better for 51 patients whose tumors were positive for P-Akt than for 52 patients whose tumors were negative, two patients among the latter group had major responses and 10 patients had stable disease. This finding may be explained by the fact that the tumor specimens used for the immunohistochemical assays were generally obtained at the time of primary diagnosis and not immediately before starting the trial. Preclinical data showed that NSCLC tumors may become more dependent on the EGFR signaling pathway, and therefore more sensitive to EGFR blocking strategies, as they are exposed to different chemotherapies (42,43). This finding may also explain the negative results of the INTACT trials, in which standard chemotherapy plus gefitinib was compared with standard chemotherapy alone in previously untreated patients with NSCLC (44,45). In our study, only 6.6% of individuals received gefitinib as first-line therapy, and in five of those patients, the PI3KAkt pathway was not activated (i.e., their tumors were P-Akt negative). Another important finding is that almost 40% of patients with P-Aktpositive tumors did not benefit from gefitinib therapy. Recent data indicate that sensitivity to gefitinib therapy requires intact EGFR-stimulated Akt signaling activity and that loss of PTEN, a phosphatase that negatively regulates Akt by dephosphorylating it, can lead to aberrant Akt activation and, finally, to gefitinib resistance (40,46,47). In this study, PTEN status was not determined, but a retrospective analysis of PTEN status in all patients included in this trial is ongoing.
No difference in survival was found among any group of patients separated on the basis of Akt or MAPK phosphorylation status, but survival was not the primary end point of the study, and at the time of this analysis, median follow-up was too short and the number of censored cases too high to detect any difference. Analysis of survival curves does show a trend that favors patients whose tumors are positive for P-Akt, and it is possible that with longer follow-up a difference in survival outcome could be detected.
Recent reports (48,49) showed that specific missense and deletion mutations in the tyrosine kinase domain of the EGFR gene are statistically significantly associated with gefitinib sensitivity. However, although objective responses were reported in up to 18%, and symptomatic improvement in 40%, of the unselected gefitinib-treated NSCLC patients (28,29), the low frequency of these mutations in unselected U.S. patients (49) suggests that other mechanisms could be involved in the response to gefitinib, such as Akt activation. Further studies should evaluate the association between Akt activation and EGFR gene status.
Finally, this trial showed that Akt is activated in approximately 50% of patients with NSCLC. Our findings are in agreement with the results of Brognard et al. (15), who showed that Akt is constitutively active in many cases of NSCLC, and our findings confirm the recent report by Lee et al. (20), who showed that tumors in patients with NSCLC frequently express phosphorylated Akt. Despite the observation that many patients with NSCLC express activated Akt, the relationship between Akt activation and lung carcinogenesis remains unclear.
In conclusion, our findings suggest that gefitinib therapy is more active in patients with tumors that are positive for P-Akt than in those with tumors that are negative for P-Akt. Further prospective studies are needed to evaluate the role of other associated markers such as PTEN and to assess the impact of previous therapies on Akt signaling pathway.
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NOTES |
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Manuscript received January 8, 2004; revised May 28, 2004; accepted June 4, 2004.
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