Tumor Biol. DOI 10.1007/s13277-013-1401-z
RESEARCH ARTICLE
Clinical significance of serum epithelial cell adhesion molecule (EPCAM) and vascular cell adhesion molecule-1 (VCAM-1) levels in patients with epithelial ovarian cancer Faruk Tas & Senem Karabulut & Murat Serilmez & Rumeysa Ciftci & Derya Duranyildiz
Received: 19 August 2013 / Accepted: 6 November 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract Cellular adhesion molecules might be good markers in some types of malignant tumors, useful information in diagnosis and prognosis. The objective of this study was to evaluate the serum levels of epithelial cell adhesion molecule (EPCAM) and vascular cell adhesion molecule-1 (VCAM-1) in epithelial ovarian cancer (EOC) patients. Fifty patients were enrolled into the study. Serum EPCAM and VCAM-1 levels were determined by the solid-phase sandwich ELISA method. Age- and sex-matched 30 healthy controls were included in the analysis. The median age of the patients was 56.5 years, range 22 to 83 years. Majority of the patients had advanced disease (stages III–IV) (90 %). The baseline serum EPCAM levels of the EOC patients were significantly higher than in those in the control group (p =0.03). However, there was no significant difference in the serum VCAM-1 level between EOC patients and controls (p = 0.24). Metastatic patients had higher serum VCAM-1 levels compared with the non-metastatic patients (p =0.03). Moreover, no other clinical variables including response to chemotherapy were found to be correlated with both serum assays (p >0.05). No correlation was found between serum EPCAM and VCAM-1 levels in EOC patients (r s =0.105, p =0.362). Neither serum EPCAM level nor serum VCAM-1 level had significant adverse effect on survival. In conclusion, the higher baseline serum levels of VCAM-1 were associated with metastatic disease, and serum EPCAM level was found to be a diagnostic marker in EOC patients. However, both serum assays had no prognostic roles on outcome. Keywords EPCAM . VCAM-1 . Serum . Ovarian cancer F. Tas (*) : S. Karabulut : M. Serilmez : R. Ciftci : D. Duranyildiz Institute of Oncology, University of Istanbul, Capa, 34390 Istanbul, Turkey e-mail: [email protected]
Introduction Epithelial ovarian cancer (EOC) is the fourth most frequent cause of death from cancer and the most fatal gynecologic malignancy in women. Because EOC typically cause few specific symptoms, more than two thirds of patients are diagnosed with advanced disease, 5-year survival rates are less than 30 %. Therefore, early detection of EOC has great promise to improve clinical outcome. Similar to serum CA 125, several EOC-related serum protein biomarkers lack sufficient sensitivity and specificity for detection of early stage disease. Thus, the need to develop a diagnostic assay in a large EOC diagnosed population still exists. Cellular adhesion molecules (CAMs) mediate a great variety of cellular interactions in normal tissue physiology and process of carcinogenesis [1]. In normal, CAMs participate in tissue growth and maintenance, blood coagulation, wound healing, and inflammation. Moreover, they are involved in invasion and metastasis in malign neoplastic transformation. In oncology practice, CAMs might be good markers in some types of malignant tumors, useful information in diagnosis and prognosis [1]. Epithelial cell adhesion molecule (EPCAM) is a transmembrane, epithelial-specific intercellular cell adhesion molecule [2–11]. Although its function is still largely unknown, it is involved also in cellular signaling, cell migration, proliferation, and differentiation in addition to cell adhesion. In human, EPCAM is expressed exclusively in epithelia and epithelialderived neoplasms. Therefore, it can be used as a histologically diagnostic and differentiative marker for epithelial tumors from non-epithelial tumors. EPCAM expression is not found in mesenchymal, muscular, neuroendocrine, lymphoid, and melanoma-originated tumor. EPCAM is detected by immunohistochemistry (IHC) in histological tumor samples [2]. More specifically, EPCAM is detected at the basolateral cell
Tumor Biol.
membrane of normal human tissues [2]. EPCAM is more intensely positive in tumor cells than in the normal tissue. High EPCAM expression has been reported in various epithelial tumors such as, colorectal, pancreas, stomach, breast, prostate, lung cancer, and ovarian cancer [2]. In other types of cancer including ovarian cancer, the role of EPCAM as diagnostic is not clear, and contradictory results have been reported (2–11). Although a high expression level of EPCAM has been reported in ovarian cancer, the clinical implication has not been determined fully [2–11]. So far, the diagnostic, prognostic, and predictive marker roles of EPCAM are unclear in ovarian cancer. Vascular cell adhesion molecule (VCAM) is a member of the immunoglobulin gene superfamily, which is a cell surface molecule sharing a segmental structural region consisting of 70–110 amino acids in the immunoglobulin chain [1]. VCAM-1 is constitutively expressed on many different types of endothelial and stromal cells, and mediates cellular adhesion. VCAM-1 appears to be of special interest because it may be involved in the process of invasion and metastatic spread. Although high expression of VCAM-1 has been shown to be associated with several cancers, and increased expression has been found to be related to progression of several cancers, its role in ovarian cancer is largely undefined [12–14]. Similar to other tumors, the expression of VCAM-1 has been also demonstrated histologically in tumor cell lines of EOC patients [15, 16]. Elevated serum levels of VCAM-1 have been identified in various malignant diseases, including breast cancer [17, 18] and colorectal cancer [19–21]. In these studies, the soluble VCAM-1 levels in the patients were significantly higher than those in the control subjects. The increased soluble levels of VCAM-1 were associated with advanced stage and prognostic significance. However, as yet, the serum levels of VCAM-1 have not been compared with tumor stage and tumor cell expression of VCAM-1in EOC patients. While there are increasing reports about the role of both EPCAM and VCAM-1 in various cancers, only a few reports of EPCAM and VCAM-1 expression in EOC have been published. The majority of the studies concerning the influences of these CAMs in EOC have been performed in tissue scale in the literature. Therefore, the objective of this study was to determine the value of circulating EPCAM and VCAM-1 in EOC patients.
Materials and methods Patients A total of 50 EOC patients with histologically proven diagnosis treated at Istanbul University, Institute of Oncology were enrolled into the study. The staging was established in accordance with the International Federation of Gynecologists and
Obstetricians (FIGO) classification. Patients with stage III with bulky disease or stage IV disease were initially treated with neoadjuvant chemotherapy and operated afterwards. The patients with operable stage III disease who had undergone primary surgery including total abdominal hysterectomy, bilateral salpingoopherectomy, appendectomy, omentectomy, and pelvic and/or para-aortic lymphadenectomy were treated with adjuvant chemotherapy. All patients received standard paclitaxel–carboplatin-containing chemotherapy regimen. For comparison of serum levels of EPCAM and VCAM-1, age- and sex-matched 30 healthy controls were included in the analysis. Informed consent was obtained from all patients, and the study was reviewed and approved by a local ethical committee. Measurement of serum EPCAM and VCAM-1 levels Serum samples were obtained on first admission before any adjuvant and metastatic treatment was given or before the patients were followed up. The blood samples were obtained from patients and healthy controls by venipuncture and clotted at room temperature. The sera were collected following centrifugation (10 min, 4,000 rpm, at room temperature) and frozen immediately at −20 °C until analysis. Serum EPCAM (Hangzhou Eastbiopharm Co., Ltd, China) and VCAM-1 (Bender MedSystems GmbH eBioscience, Austria) levels were determined by the solid-phase sandwich ELISA method. The EPCAM ELISA (Eastbiopharm, China) uses a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of human EPCAM in the samples. The serum samples and standards are added to the wells which are pre-coated with human EPCAM monoclonal antibody. Following incubation, the EPCAM antibodies labeled with biotin and combined with streptavidin–HRP are added to form immune complex and were allowed to incubate for 1 h. Unbound material is washed away, and then chromogen solution is added for the conversion of the colorless solution to a blue solution, the intensity of which is proportional to the amount of EPCAM in the sample. As an effect of the acidic stop solution, the color has become yellow. The colored reaction product is measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer, Atlanta, GA, USA). The results were expressed in ng/mL. The VCAM-1 ELISA (eBioscience) uses a doubleantibody sandwich enzyme-linked immunosorbent assay to determine the level of human VCAM-1 in the samples. The serum samples and standards are added to the wells which are pre-coated with human VCAM monoclonal antibody. Biotinconjugated anti-human VCAM-1 antibody and streptavidin– HRP are added. Biotin-conjugated anti-human VCAM-1 antibody binds to human VCAM-1 captured by the first antibody. Streptavidin–HRP binds to the biotin-conjugated antihuman VCAM-1 antibody. Following incubation, unbound
Tumor Biol.
streptavidin–HRP is removed during a wash step, and the substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of human VCAM-1 present in the sample or standard. The reaction is terminated by an addition of acid (stop solution), and absorbance is measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer). The results were expressed in ng/mL. Statistical analysis Continuous variables were categorized using median values as cutoff point. In addition to comparisons of the serum VCAM-1 and EPCAM levels in EOC patients and healthy controls, the comparison of the patient subgroups according to various clinical/laboratory parameters were carried out using Mann–Whitney U test. Overall survival (OS) was calculated from the date of first admission to the clinics to disease-related death or date of last contact with the patient or any family member. Progression-free survival (PFS) was calculated from the date of admission to the date of first radiologic progression with/without elevated serum tumor marker. Kaplan–Meier method was used for estimation of survival distribution, and differences in survival were assessed using log-rank statistics. A p value0.05). No correlation was found between the serum EPCAM and VCAM-1 levels in EOC patients (r s =0.105, n =50, p =0.362) (Fig. 2).
Table 1 Characteristics of the patients and disease Variables
Number
Patients Age of patients (year)
RESEARCH ARTICLE
Clinical significance of serum epithelial cell adhesion molecule (EPCAM) and vascular cell adhesion molecule-1 (VCAM-1) levels in patients with epithelial ovarian cancer Faruk Tas & Senem Karabulut & Murat Serilmez & Rumeysa Ciftci & Derya Duranyildiz
Received: 19 August 2013 / Accepted: 6 November 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract Cellular adhesion molecules might be good markers in some types of malignant tumors, useful information in diagnosis and prognosis. The objective of this study was to evaluate the serum levels of epithelial cell adhesion molecule (EPCAM) and vascular cell adhesion molecule-1 (VCAM-1) in epithelial ovarian cancer (EOC) patients. Fifty patients were enrolled into the study. Serum EPCAM and VCAM-1 levels were determined by the solid-phase sandwich ELISA method. Age- and sex-matched 30 healthy controls were included in the analysis. The median age of the patients was 56.5 years, range 22 to 83 years. Majority of the patients had advanced disease (stages III–IV) (90 %). The baseline serum EPCAM levels of the EOC patients were significantly higher than in those in the control group (p =0.03). However, there was no significant difference in the serum VCAM-1 level between EOC patients and controls (p = 0.24). Metastatic patients had higher serum VCAM-1 levels compared with the non-metastatic patients (p =0.03). Moreover, no other clinical variables including response to chemotherapy were found to be correlated with both serum assays (p >0.05). No correlation was found between serum EPCAM and VCAM-1 levels in EOC patients (r s =0.105, p =0.362). Neither serum EPCAM level nor serum VCAM-1 level had significant adverse effect on survival. In conclusion, the higher baseline serum levels of VCAM-1 were associated with metastatic disease, and serum EPCAM level was found to be a diagnostic marker in EOC patients. However, both serum assays had no prognostic roles on outcome. Keywords EPCAM . VCAM-1 . Serum . Ovarian cancer F. Tas (*) : S. Karabulut : M. Serilmez : R. Ciftci : D. Duranyildiz Institute of Oncology, University of Istanbul, Capa, 34390 Istanbul, Turkey e-mail: [email protected]
Introduction Epithelial ovarian cancer (EOC) is the fourth most frequent cause of death from cancer and the most fatal gynecologic malignancy in women. Because EOC typically cause few specific symptoms, more than two thirds of patients are diagnosed with advanced disease, 5-year survival rates are less than 30 %. Therefore, early detection of EOC has great promise to improve clinical outcome. Similar to serum CA 125, several EOC-related serum protein biomarkers lack sufficient sensitivity and specificity for detection of early stage disease. Thus, the need to develop a diagnostic assay in a large EOC diagnosed population still exists. Cellular adhesion molecules (CAMs) mediate a great variety of cellular interactions in normal tissue physiology and process of carcinogenesis [1]. In normal, CAMs participate in tissue growth and maintenance, blood coagulation, wound healing, and inflammation. Moreover, they are involved in invasion and metastasis in malign neoplastic transformation. In oncology practice, CAMs might be good markers in some types of malignant tumors, useful information in diagnosis and prognosis [1]. Epithelial cell adhesion molecule (EPCAM) is a transmembrane, epithelial-specific intercellular cell adhesion molecule [2–11]. Although its function is still largely unknown, it is involved also in cellular signaling, cell migration, proliferation, and differentiation in addition to cell adhesion. In human, EPCAM is expressed exclusively in epithelia and epithelialderived neoplasms. Therefore, it can be used as a histologically diagnostic and differentiative marker for epithelial tumors from non-epithelial tumors. EPCAM expression is not found in mesenchymal, muscular, neuroendocrine, lymphoid, and melanoma-originated tumor. EPCAM is detected by immunohistochemistry (IHC) in histological tumor samples [2]. More specifically, EPCAM is detected at the basolateral cell
Tumor Biol.
membrane of normal human tissues [2]. EPCAM is more intensely positive in tumor cells than in the normal tissue. High EPCAM expression has been reported in various epithelial tumors such as, colorectal, pancreas, stomach, breast, prostate, lung cancer, and ovarian cancer [2]. In other types of cancer including ovarian cancer, the role of EPCAM as diagnostic is not clear, and contradictory results have been reported (2–11). Although a high expression level of EPCAM has been reported in ovarian cancer, the clinical implication has not been determined fully [2–11]. So far, the diagnostic, prognostic, and predictive marker roles of EPCAM are unclear in ovarian cancer. Vascular cell adhesion molecule (VCAM) is a member of the immunoglobulin gene superfamily, which is a cell surface molecule sharing a segmental structural region consisting of 70–110 amino acids in the immunoglobulin chain [1]. VCAM-1 is constitutively expressed on many different types of endothelial and stromal cells, and mediates cellular adhesion. VCAM-1 appears to be of special interest because it may be involved in the process of invasion and metastatic spread. Although high expression of VCAM-1 has been shown to be associated with several cancers, and increased expression has been found to be related to progression of several cancers, its role in ovarian cancer is largely undefined [12–14]. Similar to other tumors, the expression of VCAM-1 has been also demonstrated histologically in tumor cell lines of EOC patients [15, 16]. Elevated serum levels of VCAM-1 have been identified in various malignant diseases, including breast cancer [17, 18] and colorectal cancer [19–21]. In these studies, the soluble VCAM-1 levels in the patients were significantly higher than those in the control subjects. The increased soluble levels of VCAM-1 were associated with advanced stage and prognostic significance. However, as yet, the serum levels of VCAM-1 have not been compared with tumor stage and tumor cell expression of VCAM-1in EOC patients. While there are increasing reports about the role of both EPCAM and VCAM-1 in various cancers, only a few reports of EPCAM and VCAM-1 expression in EOC have been published. The majority of the studies concerning the influences of these CAMs in EOC have been performed in tissue scale in the literature. Therefore, the objective of this study was to determine the value of circulating EPCAM and VCAM-1 in EOC patients.
Materials and methods Patients A total of 50 EOC patients with histologically proven diagnosis treated at Istanbul University, Institute of Oncology were enrolled into the study. The staging was established in accordance with the International Federation of Gynecologists and
Obstetricians (FIGO) classification. Patients with stage III with bulky disease or stage IV disease were initially treated with neoadjuvant chemotherapy and operated afterwards. The patients with operable stage III disease who had undergone primary surgery including total abdominal hysterectomy, bilateral salpingoopherectomy, appendectomy, omentectomy, and pelvic and/or para-aortic lymphadenectomy were treated with adjuvant chemotherapy. All patients received standard paclitaxel–carboplatin-containing chemotherapy regimen. For comparison of serum levels of EPCAM and VCAM-1, age- and sex-matched 30 healthy controls were included in the analysis. Informed consent was obtained from all patients, and the study was reviewed and approved by a local ethical committee. Measurement of serum EPCAM and VCAM-1 levels Serum samples were obtained on first admission before any adjuvant and metastatic treatment was given or before the patients were followed up. The blood samples were obtained from patients and healthy controls by venipuncture and clotted at room temperature. The sera were collected following centrifugation (10 min, 4,000 rpm, at room temperature) and frozen immediately at −20 °C until analysis. Serum EPCAM (Hangzhou Eastbiopharm Co., Ltd, China) and VCAM-1 (Bender MedSystems GmbH eBioscience, Austria) levels were determined by the solid-phase sandwich ELISA method. The EPCAM ELISA (Eastbiopharm, China) uses a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of human EPCAM in the samples. The serum samples and standards are added to the wells which are pre-coated with human EPCAM monoclonal antibody. Following incubation, the EPCAM antibodies labeled with biotin and combined with streptavidin–HRP are added to form immune complex and were allowed to incubate for 1 h. Unbound material is washed away, and then chromogen solution is added for the conversion of the colorless solution to a blue solution, the intensity of which is proportional to the amount of EPCAM in the sample. As an effect of the acidic stop solution, the color has become yellow. The colored reaction product is measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer, Atlanta, GA, USA). The results were expressed in ng/mL. The VCAM-1 ELISA (eBioscience) uses a doubleantibody sandwich enzyme-linked immunosorbent assay to determine the level of human VCAM-1 in the samples. The serum samples and standards are added to the wells which are pre-coated with human VCAM monoclonal antibody. Biotinconjugated anti-human VCAM-1 antibody and streptavidin– HRP are added. Biotin-conjugated anti-human VCAM-1 antibody binds to human VCAM-1 captured by the first antibody. Streptavidin–HRP binds to the biotin-conjugated antihuman VCAM-1 antibody. Following incubation, unbound
Tumor Biol.
streptavidin–HRP is removed during a wash step, and the substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of human VCAM-1 present in the sample or standard. The reaction is terminated by an addition of acid (stop solution), and absorbance is measured using an automated ELISA reader (Rayto, RT-1904C Chemistry Analyzer). The results were expressed in ng/mL. Statistical analysis Continuous variables were categorized using median values as cutoff point. In addition to comparisons of the serum VCAM-1 and EPCAM levels in EOC patients and healthy controls, the comparison of the patient subgroups according to various clinical/laboratory parameters were carried out using Mann–Whitney U test. Overall survival (OS) was calculated from the date of first admission to the clinics to disease-related death or date of last contact with the patient or any family member. Progression-free survival (PFS) was calculated from the date of admission to the date of first radiologic progression with/without elevated serum tumor marker. Kaplan–Meier method was used for estimation of survival distribution, and differences in survival were assessed using log-rank statistics. A p value0.05). No correlation was found between the serum EPCAM and VCAM-1 levels in EOC patients (r s =0.105, n =50, p =0.362) (Fig. 2).
Table 1 Characteristics of the patients and disease Variables
Number
Patients Age of patients (year)
