Ann Thorac Surg 2008;85:216-223. doi:10.1016/j.athoracsur.2007.07.046
© 2008 The Society of Thoracic Surgeons
Original Articles: General Thoracic
Epidermal Growth Factor Receptor Signaling in Adenocarcinomas With Bronchioloalveolar Components
Inderpal S. Sarkaria, MDa,c,*,
Maureen F. Zakowski, MDb,d,
Duykhanh Pham, MDa,c,
Michael Hezel, BAa,
Michael I. Ebright, MDc,
Shaokun Chuai, MAe,
Ennapadam S. Venkatraman, PhDe,
Mark G. Kris, MDb,f,
Valerie W. Rusch, MDb,c,
Bhuvanesh Singh, MDa,c
a Laboratory of Epithelial Cancer Biology, Memorial Sloan-Kettering Cancer Center, New York, New York
b Thoracic Oncology Service, Memorial Sloan-Kettering Cancer Center, New York, New York
c Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York
d Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York
e Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York
f Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York
Accepted for publication July 11, 2007.
* Address correspondence to Dr Sarkaria c/o Dr Singh, Memorial Sloan-Kettering Cancer Center, Division of Head & Neck Surgery, 1275 York Ave, New York, NY 10021 (Email: singhb{at}mskcc.org).
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Abstract
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Background: Epidermal growth factor receptor (EGFR) has gained importance in non–small cell lung cancer given impressive responses to agents targeting this molecule, particularly in bronchioloalveolar carcinoma (BAC) and adenocarcinomas, mixed subtype, with BAC components (adeno/BAC). This study assesses EGFR signaling in these tumors.
Methods: One hundred fifty tumors were classified as BAC or adeno/BAC. Tumor marker expression was determined by immunohistochemistry. Correlations with expression were examined for all tumors (BAC and adeno/BAC), and by BAC and adeno/BAC subset analyses.
Results: Positive immunophenotype was observed in 40.6% of tumors for EGFR, 51.3% for p-AKT, 58.7% for p-ERK, and 28.0% for PTEN, with increased overexpression of EGFR (p = 0.025) and p-AKT (p < 0.0001) in adeno/BAC. Epidermal growth factor receptor immunophenotype was greater in never-smokers (p = 0.008) and correlated with improved overall survival (p = 0.018). On subset analysis, EGFR correlated with improved overall survival (p = 0.05) and disease-free interval (p = 0.044) only in adeno/BAC. Epidermal growth factor receptor independently predicted improved disease-free interval in adeno/BAC (p = 0.03; hazard ratio, 0.47; 95% confidence interval, 0.23 to 0.94).
Conclusions: Overexpression of EGFR in lung adenocarcinomas with components of BAC histology correlate with never-smoker status and improved overall survival and disease-free interval. Epidermal growth factor receptor immunophenotype may be a useful predictor of clinical outcomes in this tumor subset.
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Introduction
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Lung cancer represents the leading cause of cancer mortality in the United States, with more than 174,000 new cases and 160,000 deaths in 2006. Greater than 90% of patients present with regional or distant spread of disease with 5-year survival rates less than 20%, underscoring the need for development of novel markers and therapeutics to improve longevity in these patients [1].
Interest has arisen around the biologic and clinical associations between the epidermal growth factor receptor (EGFR) and bronchioloalveolar carcinomas (BAC) and adenocarcinomas, mixed subtype, with BAC components (adeno/BAC). As defined by the World Health Organization, BAC displays a lepidic pattern of growth along the alveolar epithelium with no evidence of invasion, whereas adeno/BAC presents as a spectrum of lesions with varying combinations of invasive adenocarcinoma intermixed with areas of the noninvasive BAC growth pattern [2]. These non–small cell lung cancers (NSCLC) occur in a higher percentage of female and nonsmoking patients, and are increasing in incidence [3].
Tyrosine kinase inhibitor therapies targeting EGFR have shown tremendous clinical responses in NSCLC [4]. Epidermal growth factor receptor mutations in the tyrosine kinase–binding domain are thought to be targets of these therapies, and correlate strongly with observed clinical responses [5, 6]. Additionally, the majority of responses have been in tumors containing the BAC histologic subtype (predominantly adeno/BAC) arising in patients with a history of never-smoking [4, 7]. This association has led some authors to support the presence of the BAC growth pattern as a clinical indicator for starting tyrosine kinase inhibitor therapies in never-smokers [4]. Accordingly, investigations surrounding the biology and signaling of EGFR in NSCLC have gained momentum in the past few years, with the goals of identifying appropriate markers of clinical and pathologic behavior and targets for novel therapeutics.
Epidermal growth factor receptor is a key mediator of oncogenesis in NSCLC with activation inducing tumor proliferation and growth, angiogenesis, inhibition of apoptosis, invasion, and metastasis [8]. The effects of EGFR in NSCLC are mediated through two principal mechanisms: RAS (rat sarcoma virus) dependent Raf (murine leukemia viral oncogene homolog)/MEK (mitogen activated protein kinase kinase)/ERK (extracellular signal related kinase) signaling, and activation of the AKT (murine thymoma viral oncogene homolog) serine/threonine kinase through PI3K (phosphatidylinositol 3' kinase) signaling [8, 9].
Given the aforementioned strong clinical correlations, the purpose of this study was to clarify biologic patterns of EGFR-mediated signaling through immunohistochemical (IHC) analyses in noninvasive BAC and adeno/BAC. We assessed EGFR and components of PI3K signaling, including phosphorylated (activated) AKT and its suppressor, PTEN (phosphatase and tensin homolog). We also analyzed expression of phosphorylated/activated ERK, a downstream mediator of EGFR through RAS/RAF/MEK signaling. Finally, we tested expression patterns for clinically significant clinicopathologic correlations in these tumors, including overall survival (OS) and disease-free interval (DFI).
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Material and Methods
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This study was approved by our institutional review board for retrospective review and analysis with a waiver of individual consent.
Tumor Selection and Classification
Sections from 150 BAC and adeno/BAC tumors consecutively resected at Memorial Sloan-Kettering Cancer Center from January 1988 to December 2002 were reviewed. Sections were reviewed again and tumor classification confirmed by M.F.Z. as BAC if there was no evidence of invasive adenocarcinoma within the specimen, or adeno/BAC for mixed adenocarcinomas containing variable degrees of the BAC growth pattern. As in previous studies, adeno/BAC tumors were further classified into those containing only focal areas of invasive adenocarcinoma (<10% of overall tumor), and those with a predominantly invasive pattern (>85% of overall tumor) [10, 11].
Clinical Data Acquisition
Clinical data were obtained by review of medical records. All patients were followed up at Memorial Sloan-Kettering Cancer Center. Dates of death were confirmed by the Social Security Death Index. Smoking history was divided into never-smokers (<100 cigarettes over lifetime), former smokers (quit >1 month before resection), and current smokers. Tumors were staged by joint review (V.W.R. and I.S.S) according to the fifth edition of the American Joint Committee on Cancer (AJCC) Staging Handbook [12]. Tumors presenting with a pneumonic pattern of disease were classified as stage Px. The Martini-Melamed/AJCC criteria were used to define synchronous tumors from intrapulmonary metastases, as previously described in detail [10–13]. Relapse was defined as locoregional if along a staple line or the pleura, or distant if within bone, brain, or the adrenal glands. New cancers were defined as new lesions within the lungs at sites distinct from the original tumor.
Tissue Microarray Construction
Construction of the array and our scoring methodology have previously been described in thorough detail [11]. Briefly, areas of tumor-adjacent benign lung, BAC, and invasive adenocarcinoma were marked for all tumor specimens. Triplicate 0.6-mm biopsy cores were arrayed onto master paraffin blocks (Beecher Instruments, Inc, Sun Prairie, WI). For each tumor, all areas of histologic variation were represented on the tissue array with three cores per variation. Presence of tumor in cores and their histologic characterization was confirmed by M.F.Z.
Immunohistochemistry and Grading
Immunohistochemistry was performed using commercially available monoclonal antibodies optimized and routinely used for clinical specimen analysis by our institution’s IHC core facility, and as described in other studies [14, 15]. These included antibodies against EGFR (3IG7 clone) and PTEN (clone 26H9), and phosphorylation-specific monoclonal antibodies against the activated isoforms p-AKT (clone 587F11, Ser473 specific) and p-ERK (clone E10, phospho-p42/44 thr202/tyr204 specific; anti-EGFR, Zymed Laboratories, South San Francisco, CA; anti-PTEN, anti-p-AKT, and anti-p-ERK, Cell Signaling Technologies, Beverly, MA). Microarray sections were cut at 4 to 5 µm, placed on Superfrost/Plus microscope slides (Fisher Scientific International, Hampton, NH), and baked at 60°C for 60 minutes. Antigen retrieval was performed by treatment in either a citric acid buffer, pH 6.0, for 30 minutes at 97°C (PTEN, p-AKT, and p-ERK), or in 0.05% pepsin for 30 minutes at 37°C (EGFR). Quenching was performed in 3% hydrogen peroxide in distilled water for 5 minutes. Blocking was performed in 0.05% bovine serum albumin (BSA) diluted in phosphate-buffered saline (PBS) for 1 minute, and then by incubation in a humidity chamber for 10 minutes with the appropriate animal serum at a 1:20 dilution in 2% BSA/PBS. Slides were incubated in a humidity chamber with primary antibody overnight at 4°C. Antibodies were used at concentrations of 1:100 for anti-EGFR, 1:50 for anti-PTEN and anti-p-AKT, and 1:500 for anti-p-ERK. Slides were incubated with secondary antibody at a 1:500 dilution in 1% BSA/PBS for 60 minutes, and with peroxidase-conjugated streptavidin at a dilution of 1:500 in 1% BSA/PBS for 45 to 60 minutes at room temperature in a humidity chamber. Diaminobenzidine was used as the chromogen and hematoxylin as the nuclear counterstain.
Immunohistochemistry expression scoring was adapted from previously described methodologies [15]. Level of expression was scored for EGFR, p-AKT, and p-ERK in each tissue core with an intensity grade of 0 (absent), 1 (moderate), or 2 (high) by two investigators (M.F.Z. and I.S.S.) blinded to the clinical data, with interobserver and intraobserver concordance rates greater than 90% for all markers. Specimens were positive for EGFR marker immunophenotype if at least 20% of cancer cells had an IHC grade of at least 1, and for p-AKT and p-ERK if cytoplasmic staining intensity was at least 2 or at least 1, respectively. Expression of PTEN was positive if the level of staining intensity was equal to or greater than that of the lung endothelium. Representative stains are shown in Figure 1. Tissues expressing the study antigens were used as positive controls (EGFR and PTEN, breast adenocarcinoma; p-AKT, papillary thyroid carcinoma; p-ERK, endometrial carcinoma). Tumor-adjacent benign tissue cores were used as internal controls of tumor-specific staining.
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Fig 1. Representative examples of marker expression in tumor cohort. (EGFR = epidermal growth factor receptor; p-AKT = phosphorylated murine thymoma viral oncogene homolog; PTEN = phosphatase and tensin homolog; p-ERK = phosphorylated extracellular signal related kinase.)
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Each specimen was given an averaged IHC grade based on staining of all cores for a given specimen, a methodology previously described in detail and validated by our group [11, 16].
Statistical Analysis
Correlations between marker expression and clinical factors were determined using 
2 and Fisher’s exact test. Univariate correlation of coexpression between markers was tested by the Pearson correlation coefficient and multivariate analyses by logistic regression. Curves for OS and DFI were generated by the Kaplan–Meier method, and univariate and multivariate comparison of prognostic indicators was performed using log-rank testing and Cox proportional hazards modeling, including sex, smoking status, pattern of tumor presentation, and tumor histology as covariates. Marker coexpression, OS, and DFI analyses were conducted across the entire tumor cohort (BAC and adeno/BAC), as well as individually. Further analysis of marker interrelationships was performed using multidimensional scaling. In our multidimensional scaling model, dissimilarities between variables are calculated as one minus the Pearson correlation squared and are plotted on a two-dimensional configuration of points minimizing a "stress" function, providing a mathematical association or "distance" between two given variables [15]. All statistical analyses were performed by the study biostatisticians (S.C. and E.S.V.) using SAS statistical software (SAS Institute, Inc, Cary, NC), and approved by the biostatistical joint review committee.
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Results
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Demographics are summarized in Table 1. One tumor specimen did not have available clinical data, and was excluded in all clinical correlations. Median and mean follow-up was 61.4 months (range, 0.2 to 145.9 months) and 56.4 ± 3.1 months (± standard error of the mean).
Rates of overexpression and correlations are summarized in Table 2. The incidence of EGFR overexpression was significantly greater in never-smokers. Epidermal growth factor receptor overexpression increased with progressively more invasive histologic phenotypes, with 28% seen in noninvasive BAC versus 48% in adeno/BAC specimens with less than 15% invasive histologic phenotype. This rate increased further to 53% in adeno/BAC specimens with greater than 85% invasive histologic phenotype (p = 0.05 across all groups). Both EGFR and p-AKT expression were higher in invasive adeno/BAC. Expression of p-ERK correlated with pneumonic clinical presentation.
Univariate analyses of marker coexpression are summarized in Table 3. Levels of PTEN and p-AKT expression were positively correlated when analyzed across all tumors. Subset analysis showed the correlation between PTEN and p-AKT mainly reflected cases of BAC. A significant correlation between EGFR and p-ERK expression was also found in noninvasive BAC. These associations were maintained on multivariate logistic regression (p-AKT regressed on PTEN, odds ratio, 0.087; 95% confidence interval, 0.018 to 0.0407; p = 0.002; p-ERK regressed on EGFR, odds ratio, 0.062; 95% confidence interval, 0.007 to 0.522; p = 0.01). These correlations were not statistically significant in invasive adeno/BAC tumors. Two-dimensional multidimensional scaling analysis shows a pattern paralleling known signaling relationships between these markers (Fig 2).
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Fig 2. (A) Multidimensional scaling of study markers in bronchioloalveolar carcinoma. (B) Simplified schematic of known epidermal growth factor receptor (EGFR) signaling through phosphatidylinositol 3' kinase (PI3K)-dependent and ras-dependent pathways.
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Curves for OS and DFI are depicted in Figure 3. Epidermal growth factor receptor immunophenotype was associated with improved OS in our tumor cohort as a whole. This association was maintained in invasive adeno/BAC tumors. Epidermal growth factor receptor expression was associated with a longer DFI in adeno/BAC, but not in BAC tumors. On multivariate modeling, EGFR status remained an independent predictor of improved DFI in adeno/BAC (Table 4).
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Fig 3. Overall survival and disease-free interval analyses by epidermal growth factor receptor status. The cumulative number of patients at risk (N at Risk) by year is represented by positive (pos) and negative (neg) immunophenotype status. Crosshatch marks represent censured data points. (A, B) OS and DFI for all tumors (median follow-up 73.0 and 31.5 months, respectively); (C, D) OS and DFI for BAC (median follow-up 49.1 and 25.7 months, respectively); (E, F) OS and DFI for adeno/BAC (median follow-up 81.5 and 45.3 months, respectively). (OS = overall survival; DFI = disease free interval; EGFR = epidermal growth factor receptor; BAC = bronchioloalveolar carcinoma; adeno/BAC = adenocarcinoma, mixed subtype, with bronchioloalveolar components.)
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Table 4 Multivariate Analysis of Disease-Free Interval With Tumor Stage as a Covariate in Adenocarcinoma, Mixed Subtype, With Bronchioloalveolar Components
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Comment
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Anti-EGFR tyrosine kinase inhibitor therapies exhibit a clinical or pathologic response in approximately 9% to 18% of lung cancer patients [17–20]. A history of never-smoking, the BAC and adeno/BAC subtype of adenocarcinoma, and female sex predict therapeutic responses to treatment with these drugs [4]. More recently, investigators have identified mutations within the EGFR molecule present in approximately 90% of responders to tyrosine kinase inhibitors [5, 6]. A significant proportion of mutations are found in BAC and adeno/BAC tumors from patients who never smoked tobacco [7].
Despite these associations, few studies have examined EGFR expression and signaling in tumors containing BAC histologic phenotype. Erman and colleagues [21] found IHC detected overexpression of EGFR, p-AKT, and p-ERK in 60%, 87%, and 87% of BAC specimens, respectively. However, this observational study was limited by small size (n = 15) and lack of clinical outcomes testing. Hirsch and associates [22] found an 80% rate of EGFR overexpression by IHC in BAC, but excluded adeno/BAC. A more recent trial found increased EGFR copy number (as detected by fluorescent in-situ hybridization in 32% of specimens) associated with improved survival in gefitinib-treated BAC patients [23]. This study was not designed to assess EGFR signaling. Our current study design examines EGFR expression and signaling in a uniform cohort of BAC and adeno/BAC tumors alone.
All markers were overexpressed in our cohort (Table 2). Increasing degrees of EGFR and p-AKT overexpression from noninvasive BAC to invasive adeno/BAC support previous reports suggesting a paradigm of tumor progression with escalating genetic dysregulation driving malignant phenotypes in the spectrum from atypical adenomatous hyperplasia, to noninvasive BAC and increasingly invasive forms of adeno/BAC, and ultimately to pure adenocarcinomas [11, 24].
This progression may be further supported by observed loss of strong positive intermarker expression correlations in the progression from noninvasive BAC to invasive adeno/BAC (Table 3). Multidimensional scaling analysis supports these interrelationships, and resembles EGFR signaling reported in other tumor types (Fig 2) [15]. These observations suggest that, with the progression from noninvasive BAC to invasive variants of adeno/BAC, the contribution of EGFR to RAS-mediated and PI3K-mediated expression is diminished, with additional levels of genetic dysregulation allowing inputs from other oncogenic molecules to play greater roles in activation of these pathways.
These findings shed insight into RAS and PI3K activation in these tumors, with respect to EGFR as a putative common mediator of pathway dysregulation [9]. In the case of PI3K signaling, positive, rather than inverse, correlation between p-AKT and PTEN expression discredits loss of PTEN activity as the impetus for AKT dysregulation. In fact, mutational loss or gain of function of PI3K-mediated signaling is thought to be a rare occurrence in BAC [25]. Overexpression of PTEN in these cancers may reflect activity of negative regulatory mechanisms in response to overexpression of AKT. However, given the lack of correlation between EGFR and expression of PTEN or p-AKT observed in our study, it is difficult to draw conclusions regarding contributions of EGFR signaling to this pathway, and suggests the presence of inputs from other mechanisms. Alternatively, EGFR expression in our cohort was significantly related to RAS signaling, a pathway also with low rates of mutation in BAC [26]. These results support a primary role for EGFR-mediated RAS activity in noninvasive BAC, again with increasing inputs from other mediators with the progression to more invasive adeno/BAC lesions.
Although other studies support p-ERK overexpression as a marker of aggressive NSCLCs, conclusions regarding pneumonic variants of BAC are limited by the small number of these patients in our study (Table 2) [27].
Positive EGFR immunophenotype was increased in tumors from never-smokers (Table 2). This is distinct from NSCLC as a whole in which increased EGFR overexpression is associated with tumors from smokers, with increased levels of p-AKT seen in never-smokers [14, 28]. Nonetheless, the association between EGFR and never-smoker status in this uniform cohort supports a molecular link to improved outcomes reported with tyrosine kinase inhibitor therapies in never-smoker patients with BAC or adeno/BAC, and echoes the increased rates of EGFR mutation in tumors from never-smokers responding to these agents [4, 7].
Epidermal growth factor receptor immunophenotype correlated with improved OS in our cohort as a whole, and with OS and DFI in our invasive lesions (adeno/BAC alone; Fig 3). Epidermal growth factor receptor overexpression remained an independent predictor of improved DFI in the invasive adeno/BAC variants (Table 4). This suggests EGFR expression in surgically resected specimens may be a marker of tumors disposed to better outcomes, even before the additional advantage gained by the initiation of medical therapies.
Overall, the claim that IHC detected EGFR overexpression as a clinical prognostic factor in NSCLC has been disputed. Although some have found EGFR overexpression to be associated with advanced tumors and worse survival, others have not [22, 29–31]. Only one study showed a tendency toward better survival with positive EGFR status [32]. In a 16-study meta-analysis of EGFR expression in NSCLC, no significant correlation between EGFR and survival was demonstrated [33]. However, in subset analysis of studies detecting EGFR by IHC, positive immunophenotype was associated with worse survival. The highly uniform makeup of our tumor cohort may explain the disagreement seen between this study and those with mixed populations of NSCLC.
Certain caveats must be considered in interpreting our results. Interstudy discrepancies in marker overexpression may be influenced by cohort histologic makeup, specific primary antibodies used and processing techniques, and criteria for scoring expression. Also, our study population represents a cohort of predominantly early stage lesions treated by surgery, potentially introducing bias in interpretation of these results compared with other studies. Apart from these limitations, we believe this study presents significant and stimulating preliminary information regarding EGFR signaling in this highly uniform tumor cohort, warranting further work to delineate the prognostic and therapeutic significance of these markers in BAC and adeno/BAC.
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Acknowledgments
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This research was supported by a grant from the T.J. Martell Foundation for Leukemia, Cancer, and AIDS research.
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Abstract
Introduction
