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© 2002 American Society for Clinical Oncology High-Throughput Tissue Microarray Analysis Used to Evaluate Biology and Prognostic Significance of the E-Cadherin Pathway in Non–Small-Cell Lung CancerByFrom the Departments of Pathology and Preventive Medicine, and Division of Medical Oncology, University of Colorado Cancer Center, Denver, CO; Department of Pathology, Johns Hopkins Medical Center, Baltimore, MD, and Department of Oncology, University Hospital of Tromsø, Tromsø, Norway. Address reprint requests to Roy M. Bremnes, MD, PhD, Department of Oncology, University Hospital of Tromsø, N-9038 Tromsø, Norway; email: roy.bremnes{at}rito.no
PURPOSE: E-cadherin (E-cad) and its associated intracellular molecules, catenins, are critical for intercellular epithelial adhesion and are often expressed in non–small-cell lung carcinomas (NSCLCs). We constructed tissue microarrays (TMAs) to investigate the expression of cadherins and catenins and their prognostic significance in NSCLC.
PATIENTS AND METHODS: Tumor tissue samples from 193 patients with stages I to III NSCLC were obtained from the University of Colorado Cancer Center and Johns Hopkins Medical Institutions. Viable tumor was sampled in triplicate for the TMAs, and slides were stained by immunohistochemistry with antibodies against E-cad, N-cadherin, alpha (
RESULTS: Absent or severely reduced membranous expression for E-cad, CONCLUSION: Reduced expression of E-cad and catenins is associated with tumor cell dedifferentiation, local invasion, regional metastasis, and reduced survival in NSCLC. E-cad is an independent prognostic factor for NSCLC survival.
AMONG PATIENTS TREATED for stages I to III non–small-cell lung cancer (NSCLC) and considered tumor-free after surgery, approximately 65% will relapse within 2 years after surgery and subsequently die of metastatic spread.1,2 Hitherto, detailed histopathologic examinations and known clinical prognostic factors have been of limited help in predicting lung cancer outcome. A better understanding of changes in tumor cell biology that results in a more aggressive neoplastic phenotype may help identify patients with resectable lung cancer at risk of recurrent disease and lead to the development of more effective therapeutic modalities.
The main characteristics of malignancy are invasive growth of tumor cells and the tendency to metastasize. One of the crucial steps in the invasive growth process is the detachment of intercellular junctions of tumor cells. Cadherins, transmembrane glycoproteins whose major function is calcium-dependent cell-to-cell adhesion, play an important role in this process.3,4 In epithelial cells, intercellular adhesion is achieved as the extracellular E-cadherin (E-cad) domain interacts with E-cad on neighboring cells through a dimeric zipper-like structure, thus binding the cells together (Fig 1). E-cad is linked intracellularly to the cytoskeleton via catenins that comprise at least four molecules classified according to their molecular weight: alpha (
The full adhesive function of E-cad depends on the integrity of the entire cadherin-catenin-actin network. Any significant change in expression or structure of one of the involved components leads to adherens junction disassembly and consequently nonadhesive invasive and metastatic cells.11,12 Perturbation of the E-cad pathway in lung cancer and its association with dedifferentiation, metastasis, and reduced survival have been reported previously, but mainly in smaller clinical studies.13-20 Herein, we report results of the first study in which tissue microarrays (TMAs) were used for large-scale investigation of the biologic and prognostic value of E-cad pathway markers in NSCLC tumors. TMA technology makes it possible to quickly and comprehensively evaluate coexpression of proteins in signaling pathways.
Patients and Clinical Samples We used anonymized primary tumor tissue samples from patients diagnosed with NSCLC pathologic stage I to III at the University of Colorado Cancer Center (UCCC) and the Johns Hopkins Medical Institutions (JHMI) in the period from 1993 through 1999. The study protocol was approved by the Colorado and the JHMI institutional review boards. In cases in which a lung cancer resection was not performed (n = 11), histopathologic examinations and staging were based on lymph node tissue from mediastinoscopy. One hundred ninety-three patients had complete medical records, had been followed by the tumor registries for survival time and outcome, and had adequate paraffin-embedded fixed tissue blocks at these institutions. These patients had a median follow-up of 51 months (range, 18 to 100 months). Demographic and clinical data were collected retrospectively. None of the patients received radiotherapy or chemotherapy before surgery. Formalin-fixed and paraffin-embedded tumor specimens were obtained from the archives of the departments of pathology at UCCC and JHMI; the control specimens were from the UCCC. The tumors were staged according to the International Union Against Cancer’s tumor-node-metastasis (TNM) classification and histologically subtyped and graded according to the World Health Organization guidelines.21 There were 95 squamous cell carcinomas (SCCs), 73 adenocarcinomas, 10 bronchioloalveolar carcinomas, and 15 large-cell carcinomas (LCCs). All tumors and control tissues were reviewed by two pathologists (R.V. and W.A.F.).
Microarray Construction
Immunohistochemistry Antibodies applied to sectioned arrays are specified in Table 2. The two included anti–E-cad antibodies react with the cytoplasmic (monoclonal, clone 36) and the extracellular domain (polyclonal, clone H-108) of the molecule. Before they were used on the arrays, the antibodies were titered against normal controls and the concentrations determined to give optimal sensitivity and specificity in the control tissue were used for the TMAs (Table 2). All antibodies had been subjected to in-house validation by the manufacturer for immunohistochemical analysis on paraffin-embedded material, except N-cadherin,  -catenin, and p120. The latter antibodies had been validated for immunofluorescence or had been used for immunohistochemistry in paraffin-embedded tissues.23-25 Antigen retrieval was performed in citrate buffer using a Biocare Medical (Walnut Creek, CA) decloaking chamber. Peroxide blocking was preformed with 3% peroxide in absolute methanol. Blocking was performed with Powerblock (Biogenics, San Ramon, CA) or avidin/biotin block. After incubation of primary antibodies for 1 hour at 37°C (except p27, 1 hour at room temperature), the secondary antibody (Dako Biotinylated Multi-Link antimouse, goat, and rabbit immunoglobulin with 40% human serum) was applied for 30 minutes at room temperature. This was followed by application of streptavidin horseradish peroxidase enzyme complex and diaminobenzidine chromogen. The slides were then counterstained in hematoxylin and covered with a coverslip.
Scoring of Immunohistochemistry By light microscopy, the tissue sections were scored semiquantitatively for membranous and cytoplasmic staining. Labeling scores were determined by multiplying the percentage of positive tumor cells per slide (0% to 100%) by the dominant staining intensity (0 = negative, 1 = trace, 2 = weak, 3 = intermediate, and 4 = strong). Resulting scores ranged from 0 to 400. Specimens with overall scores of 5 to 100, 101 to 200, 201 to 300, and 301 to 400 were classified as trace, low-level, intermediate-level, and high-level expression, respectively. Scores for nuclear staining followed a three-step classification (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong). Overall scores of 5 to 100, 101 to 200, 201 to 300 were classified as low-level, intermediate-level, and high-level expression, respectively. Specimens that exhibited complete absence of staining or faint staining in less than 5% of the cells were classified as negative. All samples were evaluated and scored (by R.M.B and R.V.) randomly without knowledge of the patients’ histories. In case of disagreement, the slides were reviewed again and a consensus was reached. To evaluate the interindividual variability with regard to immunohistochemistry scoring, 60 specimens were evaluated for each antibody by four independent investigators (R.M.B., R.V., F.R.H., and W.A.F.).
Statistical Methods
Clinicopathologic Variables Demographic, clinical, and histopathologic variables are shown in Table 3. The median age was 65 years (range, 34 to 86 years), and the majority of the patients were male (59%). Data on smoking history were missing for 45 patients. Among the other 148 patients, 45% were present smokers, 48% were ex-smokers (abstinence > 1 year), and 7% were nonsmokers. Mean pack years was 58 (range, 1 to 150 pack years). Ninety-four percent of the patients underwent thoracotomy; lobectomies were performed in the majority of cases. Surgical margins were positive for tumor involvement in 8%. Mediastinoscopy with lymph node resection was performed in the nonresectable patients. Histopathologic examinations revealed that 52% of the patients had pathologic stage I disease. SCC was the dominant histologic group (49%). Of all tumors, the majority was poorly differentiated (52%), and vascular invasion by tumor cells was present in 16%.
Patterns in Expression of Cadherins, Catenins, p27, and APC In normal and tumor cells of the lung, expression of cadherin and catenin was primarily membranous (Fig 3B), while APC and p27 expression was predominantly cytoplasmic and nuclear, respectively. For E-cad, membranous staining for both antibodies (monoclonal, polyclonal) was essentially identical, whereas nuclear expression was observed mainly with the monoclonal antibody (Fig 3D). Data on the distribution of marker expression in normal lung tissues and in 16 other normal tissues are presented in Table 1. Although E-cad,  -, ß-, and -catenin, and p120 were moderately to highly expressed in the membrane of normal lung tissue cells, their expression in tumor cells was absent or severely reduced (trace to low grade) in 10% (Fig 3C), 17%, 8%, 31%, and 61% of the cases, respectively. Membranous E-cad expression was recognized as absent or severely reduced only if this was the case for both antibodies. Furthermore, the membranous expression was significantly more heterogeneous in tumors than in normal tissues. The scores for membranous N-cadherin expression were 70% lower than those for E-cad, and ß-catenin showed 14%, 26%, and 75% higher membranous expression scores than -catenin, -catenin, and p120, respectively (Table 4). When all tumors were assessed, correlations between membranous E-cad expression and membranous -catenin (P < .001), ß-catenin (P < .001), -catenin (P < .001), and p120 (P < .001) expression were significant. Nuclear expression was observed only for E-cad (79% of tumors), ß-catenin (57%), -catenin (26%), and p27 (77%). When all tumors were examined for nuclear expression, E-cad correlated with ß-catenin (P < .001) and p27 (P = .005) expression. In mitotic cells in the lung cancer tissues, we observed migration of all nuclear E-cad protein to the centriolar poles and along the mitotic spindle apparatus (Fig 4). This phenomenon was best observed in cases with intermediate levels of staining and was obscured when staining was dense. Staining for APC was generally localized along cell membranes and was weak but distinct (maximum score, 200). Seven percent of the tumors had minimal or no labeling (scores < 5). There was no evident relationship between APC expression and expression of other markers, nor between APC expression and clinical outcome.
When tumor immunohistochemistry scores from each observer were compared for interobserver reliability, the intraclass correlation coefficients were 0.98 for membranous/cytoplasmic staining and 0.95 for nuclear staining, indicating excellent reproducibility of the scores among the four investigators.
Correlation Between Marker Expression, Pathologic Features, and Stage of Disease
Dedifferentiation of SCC correlated strongly with reduced membranous expression of E-cad (P = .009), ß-catenin (P = .017), and
Reduced expression of E-cad (P = .041) and
Cadherin/Catenins and Clinical Outcome
The survival analyses according to membranous E-cad expression in subsets of patients with SCC and adenocarcinoma are given in Fig 5. In SCC patients, high tumor expression of E-cad yielded a 2-year survival rate of 78%, compared with 14% in patients whose tumors showed low or no expression (Fig 5B, P = .0008), whereas in adenocarcinoma patients, the 2-year survival rate was 75% in those with tumors showing high E-cad expression and 40% when expression was low or absent (Fig 5C, P = .07). ß-Catenin membranous expression also had a significant prognostic value in SCC patients (P = .0006), whereas survival of adenocarcinoma patients were not associated with ß-catenin expression status (P = .93) (Fig 6). In SCC patients, 2-year survival was 72% to 80% when ß-catenin expression was moderate to strong compared with 29% for patients whose tumors had low or no expression. For -catenin and -catenin, there were no differences in prognostic significance between the SCC and adenocarcinoma patients. Since variables found to have prognostic influence by univariate analysis may covariate, all statistically significant variables from the univariate analysis were included in the multiple regression analysis to identify independent prognostic factors. Data from the multivariate analyses are presented in Table 6. Membranous E-cad expression was found to have an independent prognostic value, together with clinical variables such as pN status, pT status, pathologic surgical margins, and age.
There is increasing evidence that modulation of the E-cad–catenin cell-cell adhesion complex is an important step in the initiation and progression of human cancers.26 Recently, several clinical studies have suggested that dedifferentiation, metastasis, and reduced survival in NSCLC are results of reduced expression of E-cad and catenins.13-20 However, the number of tumors and markers examined, follow-up time, and correlation with clinical data have been limited in these studies. We present a large-scale clinical study that efficiently used a high-throughput TMA to evaluate the prognostic role of the E-cad–catenin adhesion complex in patients with stage I to III NSCLC. In our opinion, the tissue array is an important innovation in prognostic marker research. We were able to obtain more than 100 slides from each TMA we created. Since we could place up to 130 cores in each array, using up to three cores for each tumor specimen, the entire cohort of 193 tumors could be accommodated on five slides. Each slide required about 200 µL of primary antibody, and the entire tumor set could thus be efficiently prepared and analyzed with only 1 mL of dilute antibody. We found that the 1.5-mm cores used to create the arrays were easy to work with, included enough tumor tissue that histologic relationships were easily evaluated, and focused attention on limited regions of tumor, thus ensuring high reproducibility of scoring. The use of TMAs for immunophenotyping of malignant tumors has recently been validated by Hoos et al,27 and their data showed an excellent concordance for staining of three antibodies between TMAs with triplicate cores per tumor and the full sections. We found that reduced membranous E-cad expression was related to LCC histology, dedifferentiation, local invasion, regional metastasis, and significantly reduced survival. Although the reported associations between tumor cell differentiation and E-cad expression in NSCLC have been inconsistent,14,17,28-30 we found a strong association as (1) undifferentiated LCC tumors had the weakest membranous E-cad expression and (2) reduced expression correlated with dedifferentiation when all tumors were analyzed. In subset analysis according to histologic subtype, the observed correlation with tumor cell differentiation in the SCC group only was in agreement with the study of 52 lung carcinomas by Böhm et al.31 They reported that E-cad expression levels were associated with tumor cell differentiation in SCC but not in adenocarcinomas. Provided an accurate pathologic TNM classification, we were able to demonstrate an inverse correlation between E-cad expression and pathologic stage. Our finding corroborated data from most previous studies,13,14,30 although this association could not be detected in the smaller lung cancer studies.17,28 The association between reduced E-cad expression and shortened survival reported by Sulzer et al14 is supported by the present work and extended by the finding that the survival advantage was conferred predominantly in patients with SCC. In these patients, high E-cad expression yielded a five-fold higher long-term survival when compared with low or no expression. The subtype-specific association between E-cad expression and survival corresponded to the observed association between this marker and tumor cell differentiation. These findings may indicate that different cellular mechanisms are responsible for the progression of adenocarcinomas versus SCC of the lung.
Since each catenin, independently, plays a critical role in the regulation of cadherin-mediated adhesion, E-cad immunoreactivity alone will not always imply the presence or absence of a functionally normal cadherin-catenin complex.11,32 Thus, to evaluate the impact of this complex on tumor invasion and metastasis, it is necessary to assess the expression of the catenins in addition to E-cad in tumor cells. We found that reduced expression of two or more of the catenins was associated with histologic subtype, dedifferentiation, local invasion, regional metastasis, and prognosis. The histologic subtype LCC was associated with reduced expression for all catenins, whereas
In some studies,13,15 reduced membranous
Our finding that membranous Nuclear expression of E-cad was not anticipated because it is a transmembrane protein. There have been isolated reports of nuclear E-cad expression in Merkel cell carcinomas34 but no such reports in lung carcinomas. Nuclear staining was observed only in sections stained with the E-cad antibody that reacts with the cytoplasmic domain of the molecule. This suggests that E-cad may be cleaved at the cell membrane35 with the subsequent translocation of the cleaved cytoplasmic fragment to the nucleus. A similar mechanism has recently been described for several transmembrane proteins and referred to as "regulated intramembranous proteolysis."36 The migration of this fragment to the centriole and polar parts of the mitotic spindle in dividing cells has previously not been reported and suggests that the cleaved fragment may have a role in chromosomal segregation. Additional molecular studies will be required to thoroughly explain this morphologic finding. Nuclear expression of ß-catenin has frequently been reported in solid tumors,37,38 as this catenin also plays a role in signal transduction via the Wnt pathway. By activation of this pathway, the level of free cytosolic ß-catenin increases, and the molecule translocates to the cell nucleus and binds directly to the transcription factors LEF1 and Tcf, leading to gene activation, apoptosis inhibition, and increased cellular proliferation and migration.39 Nuclear expression of ß-catenin, present in the majority of our NSCLC tumors (57%), was observed less frequently in the tumors investigated by Pirinen et al.18 This discrepancy may be caused, at least in part, by the 10-fold higher dilution (1/1,000) of the ß-catenin antibody in the Finnish study. In conclusion, membranous E-cad expression is an independent prognostic factor for NSCLC survival. The finding that reduced E-cad or ß-catenin expression relates to dedifferentiation and shortened survival primarily in SCC may indicate that progression of adenocarcinomas may depend more on other cellular factors than components of the E-cad–catenin complex. This study supports a functional relationship between E-cad and the catenins. Moreover, the poor prognosis in patients with reduced expression of these molecules is considered to be due to adherence junction disassembly and, consequently, nonadhesive invasive and metastatic cells. However, our data need to be confirmed in future prospective studies. Our study further demonstrates that TMAs are beneficial in large-scale screening for potential prognostic markers and should be implemented in clinical trials. Because TMAs assess the end point of protein synthesis and expression, this method may supplement and, in cases where protein turnover is low, have an advantage over other methods that quantify gene expression, such as oligonucleotide arrays.
Supported by research grants from the National Cancer Institute (SPORE in lung cancer [P50-CA58187 and P50-CA58184], Early Detection Research Network [UO1-CA15070]), Bethesda, MD; Norwegian Cancer Society, Oslo, Norway (to R.M.B.); and International Association for the Study of Lung Cancer/Cancer Research Foundation of America (to F.R.H.). We thank Paul Bunn, MD, for ideas and constructive remarks in preparation of the manuscript.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
