Human Cancer Biology Analysis of ErbB Receptors in Pulmonary Carcinoid Tumors

Purpose: This study aimed to investigate the expression of the ErbB family of receptor tyrosine kinases in pulmonary typical carcinoid and atypical carcinoid tumors and to understand the role of epidermal growth factor receptor (EGFR) signaling in pulmonary carcinoid tumor proliferation. Experimental Design: Surgically resected typical carcinoid (n = 24) and atypical carcinoid (n = 7) tumor tissues were analyzed by immunohistochemical staining for EGFR, ErbB2, ErbB3, and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene and the KRAS gene was carried out. Biochemical analysis of lung carcinoid cell lines was used to investigate EGFR signal transduction and response to erlotinib inhibition. Results: The analysis showed that 45.8% of typical carcinoid and 28.6% of atypical carcinoid tumors express EGFR, 100% of the tumors lack expression of ErbB2, and 100% have moderate to intense staining for ErbB3 and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene revealed the absence of tyrosine kinase domain mutations in these tumors. Instead, 80.6% tumors harbored a synonymous single nucleotide polymorphism in exon 20. Because EGFR and KRAS mutations tend not to be present at the same time, we sequenced the KRAS gene from pulmonary carcinoid tumor DNA and found that 100% were wild-type. Using a lung carcinoid cell line that expresses EGFR, we found that erlotinib reduced proliferation by inhibiting EGFR signal transduction. Conclusions: Our findings suggest clinical potential for the use of EGFR inhibitors in the treatment of patients with pulmonary carcinoid tumors, particularly for patients with EGFR-positive pulmonary carcinoid tumors not amenable to surgical resection. Lung cancer is the leading cause of cancer-related death in the United States (1). Pulmonary carcinoid tumors are malignant neoplasms comprising neuroendocrine cells that account for 2% to 5% of all lung cancers (2). Pulmonary typical carcinoid, which is considered a low-grade tumor, has scarce mitotic figures (<2 per 10 high-powered fields) and absence of necrosis in histologic specimens (3). Pulmonary atypical carcinoid, a higher-grade tumor, has 2 to 10 mitotic figures per 10 high-powered fields, and/or areas of focal necrosis (3). Typical carcinoid tumors usually present at an average age of 45 years, whereas atypical carcinoid tumors present a decade later. Pulmonary carcinoid tumors can develop without any known risk factors for lung cancer, and in particular less than half of patients are cigarette smokers. Epidemiologic data show that pulmonary typical carcinoid tumors are about four times more frequent than atypical carcinoid tumors, and that women are at higher risk than men of developing these tumors. The 5-year survival for pulmonary typical carcinoid tumors averages 88% and ranges from 25% to 56% for atypical carcinoid tumors (4–6). Surgical resection of the tumor in the affected lung is the standard treatment, but complete surgical excision can be difficult or unattainable depending upon the location of the tumor in the chest and whether it has metastasized. Chemotherapy and radiation therapy have limited success in treating pulmonary carcinoid tumors (7–9). Widely metastatic pulmonary carcinoid tumors can result in carcinoid heart disease or the carcinoid syndrome (10). At the time of diagnosis, up to 30% of patients will have the carcinoid syndrome with symptoms due to excessive production of serotonin from the tumor burden (11). The outcome for patients with widely metastatic disease is poor. A better understanding of the biology of these tumors could lead to the development of novel therapeutic agents to improve patient outcomes. Altered growth factor signaling through cell surface receptors is thought to contribute to tumorigenesis through abnormal cell proliferation. The ErbB family of receptor tyrosine kinases consists of the epidermal growth factor receptor Authors' Affiliations: Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care and Internal Medicine, Department of Medicine, Departments of Laboratory Medicine and Pathology, and Surgery, Mayo Clinic College of Medicine, Rochester, Minnesota Received 10/2/08; revised 2/6/09; accepted 2/9/09; published online 5/15/09. Grant support: Mayo Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Charles F. Thomas Jr., Thoracic Diseases Research Unit, 826 Stabile Building, Mayo Clinic College of Medicine, Rochester, MN 55905. Phone: 507-284-2494; Fax: 507-284-4521; E-mail: thomas.charles@ mayo.edu. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2549 3315 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Research. on April 3, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from (EGFR/ErbB1/Her1), ErbB2 (Her2/neu), ErbB3 (Her3) and ErbB4 (Her4; ref. 12). These receptor tyrosine kinases mediate cellular responses to growth factors through their intracellular domain and interact with downstream pathways important for development, differentiation, and proliferation. The EGFR gene is overexpressed and amplified in non–small cell lung cancer (NSCLC), and the presence of somatic mutations in the tyrosine kinase domain of EGFR confers sensitivity to the agents erlotinib and gefitinib (13, 14). ErbB2 (Her2/neu) overexpression in breast and ovarian cancer is associated with poor clinical outcome (15–18). Treatment with trastuzumab (Herceptin) to specifically target ErbB2 (Her2/neu) in breast cancer has resulted in improved patient outcome for this cancer type (19). ErbB3 expression has been found in varying degrees in cancers of the lung, breast, ovary, prostate, and elsewhere (20). ErbB3 is the only member of this receptor tyrosine kinase family that does not have intrinsic kinase activity (21, 22). ErbB3 therefore must heterodimerize with another ErbB receptor for signal transduction. ErbB4 is required for a number of cellular responses, such as development, differentiation, and proliferation. ErbB4 is expressed mainly in developing neural tissues and myocardium (23, 24). The role of ErbB4 in tumorigenesis is complex and not completely understood. In breast cancer ErbB4 expression is low to moderate and its expression correlates with a favorable prognosis (25). ErbB4 expression in ependymoma is high and correlated with worse patient survival (26). In a study of 80 patients with NSCLC who underwent surgery, tumors that expressed ErbB4 had decreased survival compared with tumors that did not express ErbB4 (27). A positive correlation was also found with lymph node metastasis and ErbB4 expression in the tumors in this series. Overexpression of ErbB4 in a NSCLC cell line resulted in increased cell proliferation in comparison with ErbB4-negative cells (27). In the present study, we define the expression pattern for the ErbB family of receptor tyrosine kinases from archival tumor specimens of 31 patients with pulmonary carcinoid tumors, 24 with typical carcinoid, and 7 with atypical carcinoid, for the first time. Additionally we sequenced exons 18 to 21 of the EGFR gene and the KRAS to determine whether activating mutations are present in these rare tumors. Finally using H727 lung carcinoid cells that express EGFR, we found that erlotinib reduced proliferation of this tumor cell line by inhibiting EGFR signal transduction. Our findings might have clinical application by inhibiting EGFR signaling for patients with EGFR-positive pulmonary carcinoid tumors. Materials and Methods Immunohistochemistry. These studies were approved by the Mayo Clinic Institutional Review Board. The Mayo Clinic Lung Cancer Specimen Registry was searched for tissue of carcinoid tumors from January 1, 2001 to January 31, 2006. Sufficient-quality frozen tissue and the corresponding archived paraffin-embedded tissue were available in 31 patients. All tumors were reviewed and confirmed as typical and atypical carcinoid tumors based on the 2004 WHO classification by an expert lung pathologist experienced in neuroendocrine lung cancers (3). Immunohistochemical stains were done on representative 4-μm formalin-fixed, paraffin-embedded tissue sections from the lung tumors using antibodies to chromogranin (Millipore; clone LK2H10; 1:500 dilution), EGFR (Dako; EGFR pharmDx), phospho-EGFR (Dako), ErbB2/Her2Neu (Dako; HercepTest), ErbB3/Her3 (Santa Cruz Biotechnology; polyclonal, 1:100), and ErbB4/Her4 (Santa Cruz Biotechnology; polyclonal, 1:100). For chromogranin, ErbB3/Her3, and ErbB4/Her4, epitope retrieval was done in a heated 1 mmol/L EDTA pH 8.0 solution for 30 min. Antigen-antibody reactions were visualized using a polymer-based detection system (Dako) using diaminobenzidine as the chromogen. For antibodies to EGFR and ErbB2/ Her2Neu, the procedure was done according to the manufacturer's instructions for EGFR pharmDx and HercepTest for the Dako Autostainer. Appropriate positive and negative controls were employed for all conditions. Stains were scored 0 to 4+ based on the percentage of positive tumor cells as follows: 0, <5%; 1+, 5% to 10%; 2+, 11% to 50%; 3+, 50% to 80%; 4+, >80%. Cell culture. H727, H720, UMC-11, and HCC827 were purchased from the American Type Culture Collection and were grown at 37°C in 5% CO2 in RPMI-1640 media with 1% antibiotics (penicillin/ streptomycin) and 10% fetal bovine serum. EGFR and KRAS gene sequencing. DNAwas extracted from all of the frozen carcinoid tumor specimens using standard procedures. PCR amplification and sequencing of EGFR exon 18 to 21 was done using the identical conditions and PCR primers described by Lynch and colleagues (13). The PCR primers used were (a) exon 18: 5′-CAA-ATG-AGC-TGGCAA-GTG-CCG-TGTC-3′, and 5′-GAG-TTT-CCC-AAA-CAC-TCA-GTGAAAC-3′; (b) exon 19: 5′-GCA-ATA-TCA-GCC-TTA-GGT-GCG-GCTC3′, and 5′-CAT-AGA-AAG-TGA-ACA-TTT-AGG-ATG-TG-3′; (c) exon 20: 5′-CCA-TGA-GTA-CGT-ATT-TTG-AAA-CTC-3′, and 5′-CAT-ATC-CCCATG-GCA-AAC-TCT-TGC-3′; and (d) exon 21: 5′-CTA-ACG-TTC-GCCAGC-CAT-AAG-TCC-3′, and 5′-GCT-GCG-AGC-TCA-CCC-AGA-ATGTCT-GG-3′. For KRAS the following PCR primers were used: 5′GTACTG-GTG-GAG-TAT-TTG-AT-3′ and 5′-TGA-AAA-TGG-TCA-GAG-AAACC-3′. Amplification was done using 0.2 μmol/L primers and 100 ng DNA with the following conditions: 94°C for 5 min, then 30 cycles of 94°C for 1 min, 58°C for 1 min (55°C for KRAS), 72°C for 1 min. PCR amplicons were resolved by electrophoresis on 2% agarose gels stained with ethidium bromide. The PCR amplicons were cut from gel, gene-cleaned, and sequenced with the forward and reverse