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Soluble Adhesion Molecules E-Cadherin, Intercellular Adhesion Molecule-1, and E-Selectin as Lung Cancer Biomarkers
CHEST
Original Research LUNG CANCER
Soluble Adhesion Molecules E-Cadherin, Intercellular Adhesion Molecule-1, and E-Selectin as Lung Cancer Biomarkers Athena Gogali, MD; Konstantinos Charalabopoulos, MD; Iris Zampira, BSc; Athanasios K. Konstantinidis, MD, PhD; Fanny Tachmazoglou, BSc; George Daskalopoulos, MD; Stavros H. Constantopoulos, MD, FCCP; and Yotanna Dalavanga, MD
Background: Altered levels of circulating adhesion molecules found in several carcinomas, including lung cancer, reflect local loss of diffusion barriers and tumor volume and can be potentially used as biomarkers. In the present study, we investigated the role of soluble E-cadherin (sE-cad), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble E-selectin (sE-sel) as biomarkers in lung cancer. Methods: Sixty-two patients with recently diagnosed lung cancer, 42 with small cell lung cancer (SCLC), and 20 with non-small cell lung cancer (NSCLC), as well as 29 healthy volunteers were enrolled. Blood samples were collected at the time of diagnosis and measurement of soluble adhesion molecules in the serum samples was performed by enzyme-linked immunoassay using monoclonal antibodies against E-cadherin, E-selectin, and ICAM-1. Results: Serum levels of sE-cad, sE-sel, and sICAM-1 in both SCLC and NSCLC were significantly elevated compared with control subjects (P , .001). In addition, patients with SCLC or NSCLC with distant metastasis had a marked increase of sE-Cad (P , .001), but no such correlation with sE-sel and sICAM-1 was found. Conclusions: Our findings suggest that sE-cad, sE-sel, and sICAM-1 have an adjunctive diagnostic role in lung cancer. Furthermore, sE-cad may also have a prognostic role and could be a useful biomarker in the prediction of lung cancer outcome. CHEST 2010; 138(5):1173–1179 Abbreviations: NSCLC 5 non-small cell lung cancer; SCLC 5 small cell lung cancer; sE-cad 5 soluble E-cadherin; sE-sel 5 soluble E-selectin; sICAM-1 5 soluble intercellular adhesion molecule-1
is mounting evidence relating the basic charThere acteristics of malignancy, such as uncontrolled proliferation, disorganization of cellular differentiation, tumor cell invasion, and metastasis, with changes
Manuscript received January 19, 2010; revision accepted April 20, 2010. Affiliations: From the Department of Medicine, Pulmonary Section (Drs Gogali, Konstantinidis, Daskalopoulos, and Constantopoulos), Laboratories of Physiology (Dr Charalabopoulos), and Anatomy (Ms Zampira and Dr Dalavanga), and the Neurosurgical Institute (Ms Tachmazoglou), Medical School, University of Ioannina, Ioannina, Greece. Correspondence to: Stavros H. Constantopoulos, MD, FCCP, Department of Medicine, Pulmonary Section, Medical School of the University of Ioannina, Ioannina 45500, Greece; e-mail: [email protected] © 2010 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-0157 www.chestpubs.org
in the structure and function of adhesion molecules in the primary site or the metastatic organ target.1-3 Reduced, absent, or disorganized expression of adhesion molecules has been observed in a variety of human carcinomas, including lung, gastric, head and neck, bladder, prostate, colorectal, and breast cancer.4 Cleavage of membrane adhesion proteins by various proteases is the major mechanism resulting in the release of soluble adhesion molecules. Circulating adhesion molecules can derive either from the cancer cells themselves or the associated stroma, and their presence in the circulation reflects the local loss of diffusion barriers, such as cell junction and the basement membrane, as well as the tumor volume.5 Soluble adhesion molecules function in a different way from the intact molecule of origin, usually as decoy receptors for the respective ligands blocking the normal function of the cells.5 CHEST / 138 / 5 / NOVEMBER, 2010
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Lung cancer is the leading cause of death from cancer, with a 5-year survival as low as 13%.6 As a major factor of lung cancer mortality is late diagnosis, a large number of studies have sought to discover serum biomarkers that would serve for initial screening as well as staging, prognosis, prediction of relapse, and therapeutic monitoring. Several markers have been used, including squamous cell carcinoma antigen, carcinoembryonic antigen, cytokeratin 19, and neuron-specific enolase7,8; however, none of them has proved highly sensitive and specific so as to be used routinely. Soluble adhesion molecules have been the subject of several studies that describe their role in various types of cancer,9-16 but existing data on lung cancer are rather limited, especially in the field of prognosis. Major adhesion molecules that are involved in lung cancer spread include E-cadherin, intercellular adhesion molecule (ICAM)-1, and E-selectin. E-cadherin is a prime adhesion molecule on epithelial cells mediating cell adhesion in homotypic fashion.17 The cytoplasmic domain of E-cadherin interacts with three proteins, called catenins (a, b, and g), that are crucial for cadherin function.18 E-cadherin participates in the development and architectural maintenance of epithelial tissues and has signaling capabilities.19 E-cadherin is dysregulated in lung cancer and reduced E-cadherin levels have been found in non-small cell lung cancer (NSLC)20 and small cell lung cancer (SCLC).21 We have previously shown that patients with NSCLC and metastatic disease have increased circulating levels of soluble E-cadherin (sE-cad) compared with controls.22 ICAM-1 (CD54) belongs to the immunoglobulin superfamily and is the ligand for the b2 integrins lymphocyte function-associated antigen-1 and CD11b/CD18. ICAM-1 is expressed on a variety of hematopoietic and nonhematopoietic cells, including B and T cells, macrophages, dendritic cells, fibroblasts, keratinocytes, endothelial cells, and some epithelial tissues.23 In normal lung tissue ICAM-1 is expressed mainly by bronchial and alveolar epithelial cells.24 ICAM-1 expression is upregulated by inflammatory cytokines, such as interferon-g and tumor necrosis factor-a, phorbolic esters, and lipopolysaccharide.25,26 The stable adhesion between ICAM-1 on leukocytes and endothelium is an essential step in the migration of leukocytes to sites of inflammation.27 ICAM-1 has been found to enhance the metastatic ability of malignant tumors28 and is strongly expressed in SCLC and in all histologic types of NSCLC.29 In addition, increased serum levels of soluble ICAM-1 (sICAM-1) were found in patients with NSCLC compared with controls.30 E-selectin (CD62E) is expressed exclusively by cytokine-activated endothelial cells and recognizes complex sialylated carbohydrate groups related to
Lewis X or Lewis A family found on polymorphonuclear leukocytes, monocytes, and some previously activated effector and memory T cells.31 E-selectin is important for weak tethering and rolling of leukocytes at sites of inflammation.31 Increased expression of E-selectin has been found on endothelial cells of venules and capillaries in the tumor stroma of NSCLC and serum levels of soluble E-selectin (sE-sel) were higher in patients with squamous lung cancer compared with normal donors.32 Given the limited available data on the role of sE-cad, sICAM-1, and sE-sel in lung cancer, especially SCLC type, we sought to investigate the role of these three markers as adjunctive tools in diagnosis and metastatic spread of lung cancer by determining their evaluation of serum levels in patients with newly diagnosed NSLC and SCLC as well as healthy smokers. Materials and Methods Sixty-two patients with recently diagnosed lung cancer were enrolled in the present study. The study population was selected in a cross-sectional manner and included 20 patients with NSCLC (adenocarcinoma: nine patients, squamous cell carcinoma: eight patients, and large cell carcinoma: three patients) and 42 with SCLC. Patients were either referred to our outpatient chest clinic or were treated as inpatients. All subjects underwent clinical examination; plain chest radiograph; CT scan of the chest, upper abdomen, and brain; fiberoptic bronchoscopy; and bone scan. Blood samples were collected from the patients at the time of diagnosis, before any kind of treatment (surgery, radiation, or chemotherapy). Twenty-nine healthy, age-matched smokers were used as a control group. The samples were centrifuged and sera were kept at -80°C until use. The measurement of soluble adhesion molecules in the serum samples was performed using commercially available enzyme-linked immunoassay kits with specific monoclonal antibodies against E-cadherin, E-selectin, and ICAM-1 (R&D Systems; Abingdon, England). Results were statistically analyzed using t test and analysis of variance test. Statistical analysis was carried out using SPSS software, version 16.0 for Windows (SPSS Inc; Chicago, IL), and a statistical level of 0.05 was considered as statistically significant. The Ioannina University Hospital Ethical Committee approved the study and written informed consent was obtained from all participants.
Results As shown in Table 1, when compared with healthy subjects, patients with NSCLC or SCLC presented no differences in age, sex, or smoking status. Staging in the NSCLC group revealed that 50% of the patients (10/20) had distant metastasis; similarly, in the SCLC group 50% of the patients (21/42) had distant metastatic disease. Thus, we divided patients with SCLC into two groups; the first group included patients with limited disease (without distant metastasis), and in the second group patients had extensive disease (presence of distant metastasis). Patients with NSCLC
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Table 1—Demographic Data and Serum Levels of Soluble Adhesion Molecules in Healthy Subjects and Patients With Non-Small Cell Cancer or Small Cell Lung Cancer Controls (n 5 29)
Data Age Smoking status, smoker:nonsmoker Sex, M (F) sE-cad sE-sel sICAM-1
63.3 6 9 29:0 27 (2) 1,015 6 125 458 6 89 55.8 6 19
NSCLC (n 5 20)
SCLC (n 5 42)
P Value
62.2 6 11.2 19:1 17 (3) 3,455 6 1,055.0 1,984.55 6 854.3 993.85 6 79.1
64.1 6 10.2 39:3 39 (3) 3,285.7 6 672.1 1,868.3 6 638.0 941.2 6 111.9
.79 .26 .61 , .001 , .001 , .001
Values represent mean 6 SD. F 5 female; M 5 male; NSCLC 5 non-small cell lung cancer; SCLC 5 small cell lung cancer; sE-cad 5 soluble E-cadherin; sE-sel 5 soluble E-selectin; sICAM-1 5 soluble intercellular adhesion molecule-1.
were divided in two groups; the first group included patients without distant metastasis (stage I-III), and the second group included patients with distant metastasis, pleural dissemination, or metastasis to the contralateral lung (stage IV).33 Serum Levels of Soluble Adhesion Molecules in Patients and Controls Serum levels of sE-cad in both SCLC and NSCLC patients were found significantly increased compared with controls (P , .001) (Table 1). In particular, levels of sE-cad in the control group ranged between 655 ng/mL and 1,753 ng/mL (mean 1,015 6 125 ng/mL), in the SCLC group between 1,876 ng/mL and 4,265 ng/mL (mean 3,428.3 6 1,198.8 ng/mL), and in the NSCLC group between 1,942 ng/mL and 4,877 ng/mL (mean 3,455 6 1,082.4 mg/mL). Serum levels of sE-sel in patients of both groups (SCLC and NSCLC) were also found significantly elevated compared with the control group (P , .001) (Table 1). In particular, levels in control subjects ranged between 40.2 ng/mL and 71 ng/mL (mean 55.8 6 19 ng/mL), in the SCLC group between 1,037 ng/mL and 3,962 ng/mL (mean 1,868 6 638 ng/mL), and in the NSCLC group between 510 ng/mL and 4,263 mg/mL (mean 1,984 6 876.48 ng/mL). Serum levels of ICAM-1 in patients with SCLC and NSCLC were found significantly increased compared with controls (P , .001) (Table 1). In the control group, serum levels ranged between 340 ng/mL and 572 ng/mL (mean 458 6 89 ng/mL), in the SCLC group between 789 ng/mL and 1,163 ng/mL (mean 945.45 6 105.17 ng/mL) and in the NSCLC group between 865 ng/mL and 1,098 ng/mL (mean 964 6 81 ng/mL). Comparison of levels of all three adhesion molecules between NSCLC and SCLC groups did not reveal any statistically significant differences (data not shown). Correlation Between Serum Levels of Soluble Adhesion Molecules and Distant Metastasis Increasing serum levels of sE-cad were correlated with distant metastasis in patients with SCLC or www.chestpubs.org
NSCLC ( Fig 1 ). Thus, mean levels of sE-cad in patients with SCLC with metastatic disease (extensive disease; 3,871 6 77.7 ng/mL) were significantly increased ( P , .001) compared with patients with SCLC with no metastasis (limited disease; 3,035 6 586.9 ng/mL). Similarly, mean levels of sE-cad in patients with NSCLC with metastatic disease (stage IV; 4,579.5 6 279.3 ng/mL) were significantly elevated (P , .001) compared with patients with NSCLC with no metastasis (stage I-III; 2,460 6 388.2 ng/mL) (Fig 1). In contrast, no correlation was found between levels of sE-sel and sICAM-1 and presence of distant metastasis in both NSCLC and SCLC groups (Figs 2, 3).
Figure 1. Serum concentrations of soluble E-cadherin (sE-cad) and correlation with presence of distant metastasis. A, Patients with small cell lung cancer (SCLC). B, Patients with non-small cell lung cancer (NSCLC). 瀭 5 P , .001, using analysis of variance. CHEST / 138 / 5 / NOVEMBER, 2010
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Discussion In the present study we found that serum levels of sE-cad, sE-sel, and sICAM-1 were significantly elevated in patients with SCLC and NSCLC when compared with healthy individuals, making them potential adjunctive tools for diagnosis of lung cancer. Furthermore, levels of sE-cad were significantly increased in patients with SCLC and NSCLC with metastatic disease compared with those with no metastasis. Patients with SCLC and NSCLC in our study had significantly higher levels of sE-cad. Our findings are in agreement with those of Cioffi et al,34 who found increased sE-cad in patients with lung cancer. In their study, the diagnostic sensitivity of sE-cad at a specificity level of 90% was calculated and was 66.6%, 47.6%, and 43.7% in patients affected with squamous cell carcinoma, small cell carcinoma, and adenocarcinoma, respectively. Notably, a recent study showed a correlation between sE-cad levels and response to chemotherapy in patients with NSCLC, as E-cadherin levels declined significantly after treatment in patients who responded, suggesting an additional role of E-cadherin in disease monitoring.35 In many instances, epithelial tumors lose E-cadherin-mediated adhesions as they progress toward malignancy36 and decreased local expression
Figure 2. Serum concentrations of soluble E-selectin (sE-sel) in patients with and without distant metastasis. A, Patients with SCLC. B, Patients with NSCLC. 瀭 5 P , .001, using analysis of variance. See Figure 1 legend for expansion of abbreviations.
of E-cadherin by tumor cells has been found in both NSCLC and SCLC.20,21 Mechanisms for loss of E-cadherin-mediated adhesions include inactivating mutations, epigenetic silencing, proteolytic cleavage, endocytosis, proteasomal degradation, and increased expression of nonepithelial cadherins.36,37 sE-cad is generated by cleavage of the extracellular domain of the anchored protein (120 kDa) resulting in the release of fragments of 80 kDa.38 Increased levels of its soluble form in the serum may reflect serious dysfunction of the cell surface molecule. Soluble cadherin fragments may retain specific biologic activities in the tumor environment favoring aggressiveness and metastasis by antagonizing the intact molecule or serving as anchor for migration when bound to extracellular matrix.38 Increased amounts of sE-cad have been found in the serum or urine in various cancers, including gastric,11 bladder,12 prostate,13 ovarian,14 and colorectal cancer,15 as well as in melanoma.16 Although no clear correlation with TMN staging was found, it was evident in the above studies that high levels of serum E-cadherin correlate with tumor grade and extensive disease. In our study, increased levels of sE-cad were correlated with distant metastasis in patients with SCLC or NSCLC. To our knowledge, this is the first study to report a correlation between sE-cad and distant metastasis in both types of lung cancer. In the present study, patients with NSCLC had significantly higher levels of serum sICAM-1 consistent with previous reports.30,39-41 The presence of ICAM-1 on lung cancer cells indicates their participation in inflammatory processes accompanying neoplasia as part of the immune response to the tumor, although the exact role of ICAM-1 in lung cancer is not yet clear. Thus, a previous study demonstrated that cytokine-induced expression of ICAM-1 increases the vulnerability of tumor cells to lysis by monocytes and T lymphocytes.42 In sharp contrast, in vitro data from another study showed that upregulation of human umbilical vein endothelial ICAM-1 promotes increased adhesion of SCLC cells to endothelium, thus enhancing the metastatic potential.43 We found that patients with SCLC had also significantly elevated levels of sICAM-1 compared with controls. To the best of our knowledge, this is the first comprehensive study showing that sICAM-1 levels are elevated in patients with SCLC. The only previous study evaluating levels of sICAM-1 between patients with SCLC and normal individuals included only three patients with SCLC and concluded that serum sICAM-1 levels were significantly elevated in patients with SCLC.40 In our study, serum levels of sICAM-1 did not show any correlation with the presence of distant metastasis. This is in contrast to other investigators who found higher levels of ICAM-1 in patients
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Figure 3. Serum concentrations of sICAM-1 in patients with and without distant metastasis. A, Patients with SCLC. B, Patients with NSCLC. 瀭 5 P , .001, using analysis of variance. sICAM-1 5 soluble intercellular adhesion molecule-1. See Figure 1 legend for expansion of other abbreviations.
with NSCLC with extensive disease.40,41 In the studies by Taguchi et al40 and Kamiyoshihara et al,41 soluble ICAM-1 levels correlated with clinical stage and tumor progression, whereas in the study by Grothey et al,30 increasing levels predicted a short-term fatal outcome but overall did not correlate with the prognosis. Further studies are needed to determine the prognostic role of serum ICAM-1 in NSCLC and SCLC. Serum levels of sE-sel in our study were significantly increased in patients with NSCLC or SCLC as compared with healthy controls. E-selectin is not normally expressed by lung endothelium, but its expression can be induced after activation by inflammatory cytokines (IL-1, tumor necrosis factor-a) or endotoxin.44,45 It mediates leukocyte attachment to inflammatory sites through the antigen Lewis on leukocytes surface and it has been postulated that E-selectin mediates adhesion of cancer cells to endothelium in the same way.46,47 The expression of E-selectin on endothelial cells of neoplastic tissue of the lungs indicates vascular involvement in the process.48 The role of sE-sel in lung cancer has been studied only by Roselli et al,32 who found that sE-sel levels were strongly associated only with squamous lung cancer at late stages. Therefore, our study is the first one to our knowledge to report elevated levels of E-sel in patients with SCLC. In the study by www.chestpubs.org
Roselli et al,32 elevated levels of soluble E-sel were independently related to the presence of distant metastasis in patients with squamous lung cancer. However, we found no correlation between sE-sel and presence of distant metastasis either in SCLC or in NSCLC. A strong point of our study is that it is the first to comprehensively evaluate the three major adhesion molecules in the same group of patients, highlighting the importance of sE-cad as a marker of extensive disease. Given that existing literature regarding SCLC and soluble adhesion molecules is rather limited, we have demonstrated that levels of soluble adhesion molecules are altered in SCLC, too. More importantly, our data show that markedly elevated levels of E-cadherin are highly suggestive of distant metastasis both in SCLC and NSCLC. There are two possible biases in our study. The first one is inherent to the cross-sectional nature of the study. Because we have not prospectively followed our patients, because recruitment took place when the initial diagnosis was made, we were unable to determine the role of sE-cad, sICAM-1, and sE-sel as markers for disease relapse and overall survival. The second bias is that we have recruited patients referred to or treated in our pulmonary department. Thus, the clinical profile might have been different if patients from other departments (eg, thoracic surgery, oncology, and so forth) had been included. Our staging protocol did not include PET scan for SCLC cases; thus, a small underestimation of occult adrenal metastasis may have occurred. We must stress, though, that PET scanning has been widely studied as a component of the staging evaluation for NSCLC, but fewer data are available on its role in patients with SCLC,49,50 and in combination with its high cost, it is not yet part of routine SCLC staging. In fact, our results could potentially lead to a new indication for PET scanning, as markedly increased levels of E-cadherin create suspicion of diffuse disease. We believe that the results of the current study are of significant clinical relevance. We demonstrated a potential role of the soluble adhesion molecules studied in the detection of lung cancer, which could be helpful in cases with inconclusive results of standard diagnostic methods. More importantly, serum levels of E-cadherin were markedly increased in cases with generalized disease, giving E-cadherin a possible prognostic value, because high concentrations reveal a more malignant phenotype. Thus, we speculate that therapeutic approach could become more effective in the future, as patients with markedly increased levels of sE-cad might be considered as belonging to a high-risk patient group. For example, remarkably high levels of sE-cad in a patient with operable disease might lead to adjuvant chemotherapy, as micrometastases not detectable by conventional imaging CHEST / 138 / 5 / NOVEMBER, 2010
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might be present. Finally, we believe that our study serves as a pilot study that opens novel research fields. For example, could soluble adhesion molecules help in the early detection of suspected lung cancer when bronchoscopy and other established diagnostic workup are negative. In addition, it would be of great interest to determine potential differences of soluble adhesion molecule levels among various TNM stages. Of course, such a study would require a larger number of patients with NSCLC compared with the current study. In conclusion, we report that serum levels of sE-cad, sICAM-1, and sE-sel were significantly elevated in patients with SCLC and NSCLC compared with healthy controls, suggesting that all three soluble adhesion molecules may have an adjunctive diagnostic role in lung cancer, especially in controversial cases, although they cannot substitute for tissue diagnosis. More importantly, sE-cad levels were also correlated with presence of distant metastasis in patients with NSCLC and SCLC, suggesting that E-cadherin may have a possible prognostic value. Additional prospective studies with larger numbers of patients are needed to evaluate the role of these three soluble adhesion molecules as markers for diagnosis, prognosis, and response to therapy in lung cancer. Acknowledgments Author contributions: Dr Gogali: contributed to the design and coordination of the study, performed the bronchoscopies and patient enrollment in the study, prepared the manuscript, and read and approved the final manuscript. Dr Charalabopoulos: contributed to the design and coordination of the study and read and approved the final manuscript. Ms Zampira: contributed to the statistical analysis and read and approved the final manuscript. Dr Konstantinidis: contributed to preparing the manuscript and read and approved the final manuscript. Ms Tachmazoglou: contributed to performing the ELISA tests and read and approved the final manuscript. Dr Daskalopoulos: contributed to performing the bronchoscopies and patient enrollment in the study and read and approved the final manuscript. Dr Constantopoulos: contributed to the design and coordination of the study, revised the article for important intellectual content, and read and approved the final manuscript. Dr Dalavanga: contributed to the design and coordination of the study, revised the article for important intellectual content, and read and approved the final manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.
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25. Pohlman TH, Stanness KA, Beatty PG, Ochs HD, Harlan JM. An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin 1, and tumor necrosis factoralpha increases neutrophil adherence by a CDw18-dependent mechanism. J Immunol. 1986;136(12):4548-4553. 26. Pober JS, Gimbrone MA Jr, Lapierre LA, et al. Overlapping patterns of antigenic modulation by interleukin-1, tumor necrosis factor and immune interferon. J Immunol. 1986; 137(6):1893-1896. 27. Sligh JE Jr, Ballantyne CM, Rich SS, et al. Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1. Proc Natl Acad Sci U S A. 1993;90(18):8529-8533. 28. Miele ME, Bennett CF, Miller BE, Welch DR. Enhanced metastatic ability of TNF-alpha-treated malignant melanoma cells is reduced by intercellular adhesion molecule-1 (ICAM-1, CD54) antisense oligonucleotides. Exp Cell Res. 1994;214(1):231-241. 29. Azuma A, Yagita H, Matsuda H, Okumura K, Niitani H. Induction of intercellular adhesion molecule 1 on small cell lung carcinoma cell lines by gamma-interferon enhances spontaneous and bispecific anti-CD3 x antitumor antibodydirected lymphokine activated killer cell cytotoxicity. Cancer Res. 1992;52(18):4890-4894. 30. Grothey A, Heistermann P, Philippou S, Voigtmann R. Serum levels of soluble intercellular adhesion molecule-1 (ICAM-1, CD54) in patients with non-small-cell lung cancer: correlation with histological expression of ICAM-1 and tumour stage. Br J Cancer. 1998;77(5):801-807. 31. Bevilacqua M, Butcher E, Furie B, et al. Selectins: a family of adhesion receptors. Cell. 1981;67(2):233. 32. Roselli M, Mineo TC, Martini F, et al. Soluble selectin levels in patients with lung cancer. Int J Biol Markers. 2002;17(1): 56-62. 33. Goldstraw P, Crowley J, Chansky K, et al; International Association for the Study of Lung Cancer International Staging Committee; Participating Institutions. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol. 2007;2(8):706-714. 34. Cioffi M, Gazzerro P, Di Finizio B, et al. Serum-soluble E-cadherin fragments in lung cancer. Tumori. 1999;85(1): 32-34. 35. Reckamp KL, Gardner BK, Figlin RA, et al. Tumor response to combination celecoxib and erlotinib therapy in non-small cell lung cancer is associated with a low baseline matrix metalloproteinase-9 and a decline in serum-soluble E-cadherin. J Thorac Oncol. 2008;3(2):117-124.
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36. Van Aken E, De Wever O, Correia da Rocha AS, Mareel M. Defective E-cadherin/catenin complexes in human cancer. Virchows Arch. 2001;439(6):725-751. 37. Cavallaro U. N-cadherin as an invasion promoter: a novel target for antitumor therapy? Curr Opin Investig Drugs. 2004;5(12):1274-1278. 38. Noë V, Fingleton B, Jacobs K, et al. Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci. 2001;114(pt 1):111-118. 39. Osaki T, Mitsudomi T, Yoshida Y, et al. Increased levels of serum intercellular adhesion molecule-1 (ICAM-1) in patients with non-small cell lung cancer. Surg Oncol. 1996;5(3):107-113. 40. Taguchi O, Gabazza EC, Kobayashi T, Yoshida M, Yasui H, Kobayashi H. Circulating intercellular adhesion molecule-1 in patients with lung cancer. Intern Med. 1997;36(1):14-18. 41. Kamiyoshihara M, Kawashima O, Otani Y, Morishita Y. Clinical significance of the preoperative serum-soluble intercellular adhesion molecule-1 in non-small cell lung cancer. J Cardiovasc Surg (Torino). 2002;43(5):729-734. 42. Webb DS, Mostowski HS, Gerrard TL. Cytokine-induced enhancement of ICAM-1 expression results in increased vulnerability of tumor cells to monocyte-mediated lysis. J Immunol. 1991;146(10):3682-3686. 43. Finzel AH, Reininger AJ, Bode PA, Wurzinger LJ. ICAM-1 supports adhesion of human small-cell lung carcinoma to endothelial cells. Clin Exp Metastasis. 2004;21(3):185-189. 44. Nelson RM, Venot A, Bevilacqua MP, Linhardt RJ, Stamenkovic I. Carbohydrate-protein interactions in vascular biology. Annu Rev Cell Dev Biol. 1995;11:601-631. 45. Bevilacqua MP, Nelson RM. Selectins. J Clin Invest. 1993; 91(2):379-387. 46. Varki A. Selectin ligands. Proc Natl Acad Sci U S A. 1994; 91(16):7390-7397. 47. Furukawa Y, Tara M, Ohmori K, Kannagi R. Variant type of sialyl Lewis X antigen expressed on adult T cell leukemia cells is associated with skin involvement. Cancer Res. 1994;54(24):6533-6538. 48. Staal-van den Brekel J, Wouters E. Expression of E-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 in non-small-cell lung carcinoma. Virchows Arch. 1996;428(1):21-27. 49. Fischer BM, Mortensen J, Langer SW, et al. A prospective study of PET/CT in initial staging of small-cell lung cancer: comparison with CT, bone scintigraphy and bone marrow analysis. Ann Oncol. 2007;18(2):338-345. 50. Vinjamuri M, Craig M, Campbell-Fontaine A, Almubarak M, Gupta N, Rogers JS. Can positron emission tomography be used as a staging tool for small-cell lung cancer? Clin Lung Cancer. 2008;9(1):30-34.
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Original Research LUNG CANCER
Soluble Adhesion Molecules E-Cadherin, Intercellular Adhesion Molecule-1, and E-Selectin as Lung Cancer Biomarkers Athena Gogali, MD; Konstantinos Charalabopoulos, MD; Iris Zampira, BSc; Athanasios K. Konstantinidis, MD, PhD; Fanny Tachmazoglou, BSc; George Daskalopoulos, MD; Stavros H. Constantopoulos, MD, FCCP; and Yotanna Dalavanga, MD
Background: Altered levels of circulating adhesion molecules found in several carcinomas, including lung cancer, reflect local loss of diffusion barriers and tumor volume and can be potentially used as biomarkers. In the present study, we investigated the role of soluble E-cadherin (sE-cad), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble E-selectin (sE-sel) as biomarkers in lung cancer. Methods: Sixty-two patients with recently diagnosed lung cancer, 42 with small cell lung cancer (SCLC), and 20 with non-small cell lung cancer (NSCLC), as well as 29 healthy volunteers were enrolled. Blood samples were collected at the time of diagnosis and measurement of soluble adhesion molecules in the serum samples was performed by enzyme-linked immunoassay using monoclonal antibodies against E-cadherin, E-selectin, and ICAM-1. Results: Serum levels of sE-cad, sE-sel, and sICAM-1 in both SCLC and NSCLC were significantly elevated compared with control subjects (P , .001). In addition, patients with SCLC or NSCLC with distant metastasis had a marked increase of sE-Cad (P , .001), but no such correlation with sE-sel and sICAM-1 was found. Conclusions: Our findings suggest that sE-cad, sE-sel, and sICAM-1 have an adjunctive diagnostic role in lung cancer. Furthermore, sE-cad may also have a prognostic role and could be a useful biomarker in the prediction of lung cancer outcome. CHEST 2010; 138(5):1173–1179 Abbreviations: NSCLC 5 non-small cell lung cancer; SCLC 5 small cell lung cancer; sE-cad 5 soluble E-cadherin; sE-sel 5 soluble E-selectin; sICAM-1 5 soluble intercellular adhesion molecule-1
is mounting evidence relating the basic charThere acteristics of malignancy, such as uncontrolled proliferation, disorganization of cellular differentiation, tumor cell invasion, and metastasis, with changes
Manuscript received January 19, 2010; revision accepted April 20, 2010. Affiliations: From the Department of Medicine, Pulmonary Section (Drs Gogali, Konstantinidis, Daskalopoulos, and Constantopoulos), Laboratories of Physiology (Dr Charalabopoulos), and Anatomy (Ms Zampira and Dr Dalavanga), and the Neurosurgical Institute (Ms Tachmazoglou), Medical School, University of Ioannina, Ioannina, Greece. Correspondence to: Stavros H. Constantopoulos, MD, FCCP, Department of Medicine, Pulmonary Section, Medical School of the University of Ioannina, Ioannina 45500, Greece; e-mail: [email protected] © 2010 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-0157 www.chestpubs.org
in the structure and function of adhesion molecules in the primary site or the metastatic organ target.1-3 Reduced, absent, or disorganized expression of adhesion molecules has been observed in a variety of human carcinomas, including lung, gastric, head and neck, bladder, prostate, colorectal, and breast cancer.4 Cleavage of membrane adhesion proteins by various proteases is the major mechanism resulting in the release of soluble adhesion molecules. Circulating adhesion molecules can derive either from the cancer cells themselves or the associated stroma, and their presence in the circulation reflects the local loss of diffusion barriers, such as cell junction and the basement membrane, as well as the tumor volume.5 Soluble adhesion molecules function in a different way from the intact molecule of origin, usually as decoy receptors for the respective ligands blocking the normal function of the cells.5 CHEST / 138 / 5 / NOVEMBER, 2010
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Lung cancer is the leading cause of death from cancer, with a 5-year survival as low as 13%.6 As a major factor of lung cancer mortality is late diagnosis, a large number of studies have sought to discover serum biomarkers that would serve for initial screening as well as staging, prognosis, prediction of relapse, and therapeutic monitoring. Several markers have been used, including squamous cell carcinoma antigen, carcinoembryonic antigen, cytokeratin 19, and neuron-specific enolase7,8; however, none of them has proved highly sensitive and specific so as to be used routinely. Soluble adhesion molecules have been the subject of several studies that describe their role in various types of cancer,9-16 but existing data on lung cancer are rather limited, especially in the field of prognosis. Major adhesion molecules that are involved in lung cancer spread include E-cadherin, intercellular adhesion molecule (ICAM)-1, and E-selectin. E-cadherin is a prime adhesion molecule on epithelial cells mediating cell adhesion in homotypic fashion.17 The cytoplasmic domain of E-cadherin interacts with three proteins, called catenins (a, b, and g), that are crucial for cadherin function.18 E-cadherin participates in the development and architectural maintenance of epithelial tissues and has signaling capabilities.19 E-cadherin is dysregulated in lung cancer and reduced E-cadherin levels have been found in non-small cell lung cancer (NSLC)20 and small cell lung cancer (SCLC).21 We have previously shown that patients with NSCLC and metastatic disease have increased circulating levels of soluble E-cadherin (sE-cad) compared with controls.22 ICAM-1 (CD54) belongs to the immunoglobulin superfamily and is the ligand for the b2 integrins lymphocyte function-associated antigen-1 and CD11b/CD18. ICAM-1 is expressed on a variety of hematopoietic and nonhematopoietic cells, including B and T cells, macrophages, dendritic cells, fibroblasts, keratinocytes, endothelial cells, and some epithelial tissues.23 In normal lung tissue ICAM-1 is expressed mainly by bronchial and alveolar epithelial cells.24 ICAM-1 expression is upregulated by inflammatory cytokines, such as interferon-g and tumor necrosis factor-a, phorbolic esters, and lipopolysaccharide.25,26 The stable adhesion between ICAM-1 on leukocytes and endothelium is an essential step in the migration of leukocytes to sites of inflammation.27 ICAM-1 has been found to enhance the metastatic ability of malignant tumors28 and is strongly expressed in SCLC and in all histologic types of NSCLC.29 In addition, increased serum levels of soluble ICAM-1 (sICAM-1) were found in patients with NSCLC compared with controls.30 E-selectin (CD62E) is expressed exclusively by cytokine-activated endothelial cells and recognizes complex sialylated carbohydrate groups related to
Lewis X or Lewis A family found on polymorphonuclear leukocytes, monocytes, and some previously activated effector and memory T cells.31 E-selectin is important for weak tethering and rolling of leukocytes at sites of inflammation.31 Increased expression of E-selectin has been found on endothelial cells of venules and capillaries in the tumor stroma of NSCLC and serum levels of soluble E-selectin (sE-sel) were higher in patients with squamous lung cancer compared with normal donors.32 Given the limited available data on the role of sE-cad, sICAM-1, and sE-sel in lung cancer, especially SCLC type, we sought to investigate the role of these three markers as adjunctive tools in diagnosis and metastatic spread of lung cancer by determining their evaluation of serum levels in patients with newly diagnosed NSLC and SCLC as well as healthy smokers. Materials and Methods Sixty-two patients with recently diagnosed lung cancer were enrolled in the present study. The study population was selected in a cross-sectional manner and included 20 patients with NSCLC (adenocarcinoma: nine patients, squamous cell carcinoma: eight patients, and large cell carcinoma: three patients) and 42 with SCLC. Patients were either referred to our outpatient chest clinic or were treated as inpatients. All subjects underwent clinical examination; plain chest radiograph; CT scan of the chest, upper abdomen, and brain; fiberoptic bronchoscopy; and bone scan. Blood samples were collected from the patients at the time of diagnosis, before any kind of treatment (surgery, radiation, or chemotherapy). Twenty-nine healthy, age-matched smokers were used as a control group. The samples were centrifuged and sera were kept at -80°C until use. The measurement of soluble adhesion molecules in the serum samples was performed using commercially available enzyme-linked immunoassay kits with specific monoclonal antibodies against E-cadherin, E-selectin, and ICAM-1 (R&D Systems; Abingdon, England). Results were statistically analyzed using t test and analysis of variance test. Statistical analysis was carried out using SPSS software, version 16.0 for Windows (SPSS Inc; Chicago, IL), and a statistical level of 0.05 was considered as statistically significant. The Ioannina University Hospital Ethical Committee approved the study and written informed consent was obtained from all participants.
Results As shown in Table 1, when compared with healthy subjects, patients with NSCLC or SCLC presented no differences in age, sex, or smoking status. Staging in the NSCLC group revealed that 50% of the patients (10/20) had distant metastasis; similarly, in the SCLC group 50% of the patients (21/42) had distant metastatic disease. Thus, we divided patients with SCLC into two groups; the first group included patients with limited disease (without distant metastasis), and in the second group patients had extensive disease (presence of distant metastasis). Patients with NSCLC
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Table 1—Demographic Data and Serum Levels of Soluble Adhesion Molecules in Healthy Subjects and Patients With Non-Small Cell Cancer or Small Cell Lung Cancer Controls (n 5 29)
Data Age Smoking status, smoker:nonsmoker Sex, M (F) sE-cad sE-sel sICAM-1
63.3 6 9 29:0 27 (2) 1,015 6 125 458 6 89 55.8 6 19
NSCLC (n 5 20)
SCLC (n 5 42)
P Value
62.2 6 11.2 19:1 17 (3) 3,455 6 1,055.0 1,984.55 6 854.3 993.85 6 79.1
64.1 6 10.2 39:3 39 (3) 3,285.7 6 672.1 1,868.3 6 638.0 941.2 6 111.9
.79 .26 .61 , .001 , .001 , .001
Values represent mean 6 SD. F 5 female; M 5 male; NSCLC 5 non-small cell lung cancer; SCLC 5 small cell lung cancer; sE-cad 5 soluble E-cadherin; sE-sel 5 soluble E-selectin; sICAM-1 5 soluble intercellular adhesion molecule-1.
were divided in two groups; the first group included patients without distant metastasis (stage I-III), and the second group included patients with distant metastasis, pleural dissemination, or metastasis to the contralateral lung (stage IV).33 Serum Levels of Soluble Adhesion Molecules in Patients and Controls Serum levels of sE-cad in both SCLC and NSCLC patients were found significantly increased compared with controls (P , .001) (Table 1). In particular, levels of sE-cad in the control group ranged between 655 ng/mL and 1,753 ng/mL (mean 1,015 6 125 ng/mL), in the SCLC group between 1,876 ng/mL and 4,265 ng/mL (mean 3,428.3 6 1,198.8 ng/mL), and in the NSCLC group between 1,942 ng/mL and 4,877 ng/mL (mean 3,455 6 1,082.4 mg/mL). Serum levels of sE-sel in patients of both groups (SCLC and NSCLC) were also found significantly elevated compared with the control group (P , .001) (Table 1). In particular, levels in control subjects ranged between 40.2 ng/mL and 71 ng/mL (mean 55.8 6 19 ng/mL), in the SCLC group between 1,037 ng/mL and 3,962 ng/mL (mean 1,868 6 638 ng/mL), and in the NSCLC group between 510 ng/mL and 4,263 mg/mL (mean 1,984 6 876.48 ng/mL). Serum levels of ICAM-1 in patients with SCLC and NSCLC were found significantly increased compared with controls (P , .001) (Table 1). In the control group, serum levels ranged between 340 ng/mL and 572 ng/mL (mean 458 6 89 ng/mL), in the SCLC group between 789 ng/mL and 1,163 ng/mL (mean 945.45 6 105.17 ng/mL) and in the NSCLC group between 865 ng/mL and 1,098 ng/mL (mean 964 6 81 ng/mL). Comparison of levels of all three adhesion molecules between NSCLC and SCLC groups did not reveal any statistically significant differences (data not shown). Correlation Between Serum Levels of Soluble Adhesion Molecules and Distant Metastasis Increasing serum levels of sE-cad were correlated with distant metastasis in patients with SCLC or www.chestpubs.org
NSCLC ( Fig 1 ). Thus, mean levels of sE-cad in patients with SCLC with metastatic disease (extensive disease; 3,871 6 77.7 ng/mL) were significantly increased ( P , .001) compared with patients with SCLC with no metastasis (limited disease; 3,035 6 586.9 ng/mL). Similarly, mean levels of sE-cad in patients with NSCLC with metastatic disease (stage IV; 4,579.5 6 279.3 ng/mL) were significantly elevated (P , .001) compared with patients with NSCLC with no metastasis (stage I-III; 2,460 6 388.2 ng/mL) (Fig 1). In contrast, no correlation was found between levels of sE-sel and sICAM-1 and presence of distant metastasis in both NSCLC and SCLC groups (Figs 2, 3).
Figure 1. Serum concentrations of soluble E-cadherin (sE-cad) and correlation with presence of distant metastasis. A, Patients with small cell lung cancer (SCLC). B, Patients with non-small cell lung cancer (NSCLC). 瀭 5 P , .001, using analysis of variance. CHEST / 138 / 5 / NOVEMBER, 2010
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Discussion In the present study we found that serum levels of sE-cad, sE-sel, and sICAM-1 were significantly elevated in patients with SCLC and NSCLC when compared with healthy individuals, making them potential adjunctive tools for diagnosis of lung cancer. Furthermore, levels of sE-cad were significantly increased in patients with SCLC and NSCLC with metastatic disease compared with those with no metastasis. Patients with SCLC and NSCLC in our study had significantly higher levels of sE-cad. Our findings are in agreement with those of Cioffi et al,34 who found increased sE-cad in patients with lung cancer. In their study, the diagnostic sensitivity of sE-cad at a specificity level of 90% was calculated and was 66.6%, 47.6%, and 43.7% in patients affected with squamous cell carcinoma, small cell carcinoma, and adenocarcinoma, respectively. Notably, a recent study showed a correlation between sE-cad levels and response to chemotherapy in patients with NSCLC, as E-cadherin levels declined significantly after treatment in patients who responded, suggesting an additional role of E-cadherin in disease monitoring.35 In many instances, epithelial tumors lose E-cadherin-mediated adhesions as they progress toward malignancy36 and decreased local expression
Figure 2. Serum concentrations of soluble E-selectin (sE-sel) in patients with and without distant metastasis. A, Patients with SCLC. B, Patients with NSCLC. 瀭 5 P , .001, using analysis of variance. See Figure 1 legend for expansion of abbreviations.
of E-cadherin by tumor cells has been found in both NSCLC and SCLC.20,21 Mechanisms for loss of E-cadherin-mediated adhesions include inactivating mutations, epigenetic silencing, proteolytic cleavage, endocytosis, proteasomal degradation, and increased expression of nonepithelial cadherins.36,37 sE-cad is generated by cleavage of the extracellular domain of the anchored protein (120 kDa) resulting in the release of fragments of 80 kDa.38 Increased levels of its soluble form in the serum may reflect serious dysfunction of the cell surface molecule. Soluble cadherin fragments may retain specific biologic activities in the tumor environment favoring aggressiveness and metastasis by antagonizing the intact molecule or serving as anchor for migration when bound to extracellular matrix.38 Increased amounts of sE-cad have been found in the serum or urine in various cancers, including gastric,11 bladder,12 prostate,13 ovarian,14 and colorectal cancer,15 as well as in melanoma.16 Although no clear correlation with TMN staging was found, it was evident in the above studies that high levels of serum E-cadherin correlate with tumor grade and extensive disease. In our study, increased levels of sE-cad were correlated with distant metastasis in patients with SCLC or NSCLC. To our knowledge, this is the first study to report a correlation between sE-cad and distant metastasis in both types of lung cancer. In the present study, patients with NSCLC had significantly higher levels of serum sICAM-1 consistent with previous reports.30,39-41 The presence of ICAM-1 on lung cancer cells indicates their participation in inflammatory processes accompanying neoplasia as part of the immune response to the tumor, although the exact role of ICAM-1 in lung cancer is not yet clear. Thus, a previous study demonstrated that cytokine-induced expression of ICAM-1 increases the vulnerability of tumor cells to lysis by monocytes and T lymphocytes.42 In sharp contrast, in vitro data from another study showed that upregulation of human umbilical vein endothelial ICAM-1 promotes increased adhesion of SCLC cells to endothelium, thus enhancing the metastatic potential.43 We found that patients with SCLC had also significantly elevated levels of sICAM-1 compared with controls. To the best of our knowledge, this is the first comprehensive study showing that sICAM-1 levels are elevated in patients with SCLC. The only previous study evaluating levels of sICAM-1 between patients with SCLC and normal individuals included only three patients with SCLC and concluded that serum sICAM-1 levels were significantly elevated in patients with SCLC.40 In our study, serum levels of sICAM-1 did not show any correlation with the presence of distant metastasis. This is in contrast to other investigators who found higher levels of ICAM-1 in patients
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Figure 3. Serum concentrations of sICAM-1 in patients with and without distant metastasis. A, Patients with SCLC. B, Patients with NSCLC. 瀭 5 P , .001, using analysis of variance. sICAM-1 5 soluble intercellular adhesion molecule-1. See Figure 1 legend for expansion of other abbreviations.
with NSCLC with extensive disease.40,41 In the studies by Taguchi et al40 and Kamiyoshihara et al,41 soluble ICAM-1 levels correlated with clinical stage and tumor progression, whereas in the study by Grothey et al,30 increasing levels predicted a short-term fatal outcome but overall did not correlate with the prognosis. Further studies are needed to determine the prognostic role of serum ICAM-1 in NSCLC and SCLC. Serum levels of sE-sel in our study were significantly increased in patients with NSCLC or SCLC as compared with healthy controls. E-selectin is not normally expressed by lung endothelium, but its expression can be induced after activation by inflammatory cytokines (IL-1, tumor necrosis factor-a) or endotoxin.44,45 It mediates leukocyte attachment to inflammatory sites through the antigen Lewis on leukocytes surface and it has been postulated that E-selectin mediates adhesion of cancer cells to endothelium in the same way.46,47 The expression of E-selectin on endothelial cells of neoplastic tissue of the lungs indicates vascular involvement in the process.48 The role of sE-sel in lung cancer has been studied only by Roselli et al,32 who found that sE-sel levels were strongly associated only with squamous lung cancer at late stages. Therefore, our study is the first one to our knowledge to report elevated levels of E-sel in patients with SCLC. In the study by www.chestpubs.org
Roselli et al,32 elevated levels of soluble E-sel were independently related to the presence of distant metastasis in patients with squamous lung cancer. However, we found no correlation between sE-sel and presence of distant metastasis either in SCLC or in NSCLC. A strong point of our study is that it is the first to comprehensively evaluate the three major adhesion molecules in the same group of patients, highlighting the importance of sE-cad as a marker of extensive disease. Given that existing literature regarding SCLC and soluble adhesion molecules is rather limited, we have demonstrated that levels of soluble adhesion molecules are altered in SCLC, too. More importantly, our data show that markedly elevated levels of E-cadherin are highly suggestive of distant metastasis both in SCLC and NSCLC. There are two possible biases in our study. The first one is inherent to the cross-sectional nature of the study. Because we have not prospectively followed our patients, because recruitment took place when the initial diagnosis was made, we were unable to determine the role of sE-cad, sICAM-1, and sE-sel as markers for disease relapse and overall survival. The second bias is that we have recruited patients referred to or treated in our pulmonary department. Thus, the clinical profile might have been different if patients from other departments (eg, thoracic surgery, oncology, and so forth) had been included. Our staging protocol did not include PET scan for SCLC cases; thus, a small underestimation of occult adrenal metastasis may have occurred. We must stress, though, that PET scanning has been widely studied as a component of the staging evaluation for NSCLC, but fewer data are available on its role in patients with SCLC,49,50 and in combination with its high cost, it is not yet part of routine SCLC staging. In fact, our results could potentially lead to a new indication for PET scanning, as markedly increased levels of E-cadherin create suspicion of diffuse disease. We believe that the results of the current study are of significant clinical relevance. We demonstrated a potential role of the soluble adhesion molecules studied in the detection of lung cancer, which could be helpful in cases with inconclusive results of standard diagnostic methods. More importantly, serum levels of E-cadherin were markedly increased in cases with generalized disease, giving E-cadherin a possible prognostic value, because high concentrations reveal a more malignant phenotype. Thus, we speculate that therapeutic approach could become more effective in the future, as patients with markedly increased levels of sE-cad might be considered as belonging to a high-risk patient group. For example, remarkably high levels of sE-cad in a patient with operable disease might lead to adjuvant chemotherapy, as micrometastases not detectable by conventional imaging CHEST / 138 / 5 / NOVEMBER, 2010
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might be present. Finally, we believe that our study serves as a pilot study that opens novel research fields. For example, could soluble adhesion molecules help in the early detection of suspected lung cancer when bronchoscopy and other established diagnostic workup are negative. In addition, it would be of great interest to determine potential differences of soluble adhesion molecule levels among various TNM stages. Of course, such a study would require a larger number of patients with NSCLC compared with the current study. In conclusion, we report that serum levels of sE-cad, sICAM-1, and sE-sel were significantly elevated in patients with SCLC and NSCLC compared with healthy controls, suggesting that all three soluble adhesion molecules may have an adjunctive diagnostic role in lung cancer, especially in controversial cases, although they cannot substitute for tissue diagnosis. More importantly, sE-cad levels were also correlated with presence of distant metastasis in patients with NSCLC and SCLC, suggesting that E-cadherin may have a possible prognostic value. Additional prospective studies with larger numbers of patients are needed to evaluate the role of these three soluble adhesion molecules as markers for diagnosis, prognosis, and response to therapy in lung cancer. Acknowledgments Author contributions: Dr Gogali: contributed to the design and coordination of the study, performed the bronchoscopies and patient enrollment in the study, prepared the manuscript, and read and approved the final manuscript. Dr Charalabopoulos: contributed to the design and coordination of the study and read and approved the final manuscript. Ms Zampira: contributed to the statistical analysis and read and approved the final manuscript. Dr Konstantinidis: contributed to preparing the manuscript and read and approved the final manuscript. Ms Tachmazoglou: contributed to performing the ELISA tests and read and approved the final manuscript. Dr Daskalopoulos: contributed to performing the bronchoscopies and patient enrollment in the study and read and approved the final manuscript. Dr Constantopoulos: contributed to the design and coordination of the study, revised the article for important intellectual content, and read and approved the final manuscript. Dr Dalavanga: contributed to the design and coordination of the study, revised the article for important intellectual content, and read and approved the final manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.
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