Mol Cell Biochem (2014) 396:307–312 DOI 10.1007/s11010-014-2167-3
Clinical significance of serum epithelial cell adhesion molecule (EPCAM) levels in patients with lung cancer Faruk Tas • Senem Karabulut • Derya Duranyildiz
Received: 26 May 2014 / Accepted: 14 July 2014 / Published online: 25 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Cellular adhesion molecules might be good markers in some types of malignant tumors, thus providing useful information in diagnosis and prognosis. The objective of this study was to determine the clinical significance of the serum levels of epithelial cell adhesion molecule (EPCAM) in lung cancer patients. One hundred and thirty lung cancer patients were enrolled in this study. Serum EPCAM levels were determined by the solid-phase sandwich ELISA method. Age- and sex-matched 34 healthy controls were included in the analysis. The median age was 58 years, ranging 35–80 years. The majority of the patients had NSCLC (83.8 %) and stage IV disease (60.8 %). There was no significant difference in the serum EPCAM levels between lung cancer patients and healthy controls (p = 0.16). Moreover, known clinical variables including age of patient, gender, histology, stage of disease, and response to chemotherapy were not found to be correlated with serum EPCAM concentrations (p [ 0.05). Similarly, no prognostic role was found for outcome (1-year survival rate 62 vs. 65.1 %, p = 0.89). In conclusion, serum EPCAM concentrations have no diagnostic, predictive, and prognostic roles in lung cancer patients. Keywords
EPCAM Serum Lung cancer
Introduction Cellular adhesion molecules (CAMs) mediate a great variety of cellular interactions in normal and process of F. Tas (&) S. Karabulut D. Duranyildiz Institute of Oncology, University of Istanbul, Capa, 34390 Istanbul, Turkey e-mail: [email protected]
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 [2–9]. Although its function is still largely unknown, it is involved in also cellular signaling, cell migration, proliferation, and differentiation in addition to cell adhesion. In human, EPCAM is expressed exclusively in epithelia and epithelial-derived neoplasms [2–9]. Therefore, it can be used as a histologically diagnostic and differentiatial marker for epithelial tumors from non-epithelial tumors. EPCAM expression is not found in mesenchymal, muscular, neuroendocrine, and lymphoid tissues, and melanoma-originated tumors [2–9]. EPCAM is detected by immunohistochemistry (IHC) in histological tumor samples [2]. More specifically, EPCAM is detected at the basolateral cell membrane of normal human tissue [2–6]. 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, ovarian cancer, and lung cancer [2]. Although a high expression level of EPCAM has been reported in lung cancer, the clinical implication has not been determined fully [2–9]. While there are increasing reports about the role of EPCAM in various cancers, only a few reports of EPCAM expression in lung cancer have been published [2–9]. All of the studies concerning the influences of this CAM in lung cancer have been performed at tissue scale in the literature
123
308
[2–9]. Therefore, the objective of this study was to determine the value of circulating EPCAM in lung cancer patients.
Materials and methods
Mol Cell Biochem (2014) 396:307–312
during chemotherapy or every 12 weeks for no anticancer treatment. For comparison of serum levels of EPCAM, age- and sex-matched 34 healthy controls were included in the analysis. This study was approved by the ethics committee of the Institute of Oncology, Istanbul University. Written informed consent was obtained from the patients.
Patients Measurement of serum EPCAM levels This study comprised 130 patients admitted to Istanbul University, Institute of Oncology with histologically or cytologically confirmed non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) treated and followed up in our clinic were recorded from medical charts. Patients with bidimensionally measurable disease without history of chemo/radiotherapy in the last six months were included in the study. The staging of metastatic patients was done using various imaging modalities such as computed tomography, magnetic resonance imaging, and PET/CT scan. The pathological diagnosis of lung cancer was established in accordance with the revised World Health Organization classification of lung tumors and staged relying on the revised TNM staging for lung cancer. The pretreatment evaluation included detailed clinical history and physical examination with a series of complete blood cell counts and routine biochemistry tests, including urea, creatinine, transaminases, and lactate dehydrogenase. Those with ECOG performance status B2 and appropriate blood chemistry tests received chemotherapy on outpatient basis comprising platinum compounds with/without radiotherapy depending on the stage of disease. Patients were treated with various chemotherapy regimens as a single agent or combination therapy. Regimens of single or combination chemotherapy were selected based on performance status of patients and extension of disease. Drug schemas consist of platinum agents in combination with newer cytotoxic drugs, including paclitaxel, docetaxel, gemcitabine, and vinorelbine with standard doses in NSCLC patients for adjuvant- and primary-based chemotherapy. Additionally, cisplatin and etoposide combination chemotherapy regimen with standard dose and schedule was applied for the patients with SCLC. The response to chemotherapy was evaluated by radiologically after 2–3 cycles of chemotherapy according to international criteria, revised RECIST criteria. Nonresponder patients to chemotherapy and relapsed patients were treated with second-line chemotherapy if they had a good performance status. Chemotherapy was continued until disease progression or unacceptable toxicity. The follow-up programs consisted of clinical, laboratory, and radiological assessments performed at 8-week intervals
123
Serum samples were obtained on first admission before any adjuvant and metastatic treatment were given for follow-up patients. Blood samples were obtained from patients with lung cancer and healthy controls (n = 34) by venipuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at -20 °C until analysis. Serum EPCAM (Eastbiopharm, China) levels were determined by the solid-phase sandwich ELISA method. The EPCAM ELISA (Eastbiopharm, China) was used a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of Human EPCAM in samples. Serum samples and standards were added to the wells which were pre-coated with Human EPCAM monoclonal antibody. Following incubation, EPCAM antibodies labeled with biotin and combined with Streptavidin-HRP were added to form immune complex and allowed to incubate for 1 h. Unbound material was washed away, and then chromogen solution was 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 the effect of the acidic stop solution, the color became yellow. The colored reaction product was measured using an automated ELISA reader (Rayto, RT1904C Chemistry Analyzer, Atlanta GA, USA). The results were expressed as ng/mL. Statistical analysis Continuous variables were categorized using median values as cut-off point. Assessment of relationships and comparisons between various clinical/laboratory parameters were accomplished using Mann–Whitney U test and Kruskal–Wallis test for two and three groups, respectively. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. Kaplan–Meier method was used for estimation of survival distribution, and differences in survival were assessed by the log-rank statistics. A p value \0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software.
Mol Cell Biochem (2014) 396:307–312
309
Table 1 Patient and disease characteristics Variables
n
No. of patients
130
Age of patients C60
58
\60
72
Gender Male
119
Female
11
Histology NSCLC
109
Adeno
49
Epidermoid Undifferentiated
45 15
SCLC
21
Stage II
9
III
37
IV
63
Limited
5
Extended
16
Response to chemotherapy
Fig. 1 The value of serum EPCAM assay in lung cancer patients and healthy controls (p = 0.16)
Table 3 Results (median and range) of comparisons between serum EPCAM levels and various clinical variables Variables Age of patients (p)
Yes (Complete ? partial)
68
No (Stable ? progressive)
52
C60 \60 Gender (p)
Table 2 The values of serum EPCAM levels in lung cancer patients and healthy controls Assay
Serum EPCAM level (ng/mL)
Patients (n = 130)
Controls (n = 34)
Median
Range
Median
Range
9.1
0.6–25.4
9.7
1.0–24.5
p
Male Female Histology (p)
9.1 (0.6–24.2) 10.0 (0.8–25.4) 0.83 9.1 (0.6–25.4) 9.3 (1.0–24.2)
Adeno Epidermoid
0.83 9.0 (0.6–23.7) 9.2 (0.8–25.4) 0.37
II
9.4 (1.5–21.7)
III
8.8 (3.0–16.8)
IV
From June 2010 to July 2011, 130 patients with a pathologically confirmed diagnosis of lung cancer were enrolled into this study. Baseline histopathological characteristics and demographic features of patients are listed in Table 1. The median age at diagnosis was 58 years, ranging 35–80 years, where males constituted the majority of the group (n = 119, 91.5 %). The majority of the patients had NSCLC (n = 109, 83.8 %) and metastatic disease (n = 79, 60.8 %). The levels of serum EPCAM in patients with lung cancer and healthy controls are shown in Table 2. No significant difference was found in the serum EPCAM levels between lung cancer patients and healthy controls (9.1 versus 9.7 ng/mL, p = 0.16) (Fig. 1).
9.3 (0.8–25.4) 0.14
SCLC
Stage in NSCLC (p)
Results
0.26 9.0 (0.6–23.1)
NSCLC Histology in NSCLC (p) 0.16
EPCAM (ng/mL)
Stage in SCLC (p) Limited Extended Response to chemotherapy (p) Yes No
9.4 (0.6–23.7) 0.28 9.0 (3.0–16.8) 9.8 (1.0–24.2) 0.89 9.1 (0.8–25.4) 9.2 (0.6–23.7)
Table 3 shows the correlation between the serum levels of EPCAM assays and clinicopathological factors. The known clinical variables including age of patient, gender, histology, stage of disease, and response to chemotherapy were not found to be correlated with in serum EPCAM concentrations (p [ 0.05).
123
310
Mol Cell Biochem (2014) 396:307–312
overall survival rate was 63.7 (95 % CI 54.5–72.9). However, serum EPCAM level was found to have no prognostic role for outcome (1-year survival rate 62 versus 65.1 %, p = 0.89) (Table 4; Fig. 2).
Table 4 Univariate analysis of survival Variables
Median survival time ± SE (month)
1-year survival rate ± SD (%)
Age of patients
0.55
C60
15.0 (7.2)
21.0 (2.0)
\60
62.1 (7.2)
65.3 (6.1)
Gender 20.4 (1.5)
62.4 (4.9)
Female
21.7 (3.8)
77.1 (14.4)
Histology NSCLC
23.0 (5.1)
67.2 (4.9)
SCLC
11.0 (0.9)
0.002 44.1(12.9)
Histology in NSCLC
0.40
Adeno
24.6 (2.3)
78.8 (6.4)
Epidermoid
23.7 (2.3)
66.8 (7.7)
II–III
29.8 (2.0)
85.9 (5.4)
IV
14.9 (1.7)
51.5 (7.2)
18.0 (4.6)
66.7 (2.7)
10.4 (2.4)
38.2 (14.2)
\0.001
Stage in NSCLC
Stage in SCLC Extended Response to chemotherapy
Discussion 0.36
Male
Limited
p
0.20
\0.001
Yes
25.4 (1.8)
75.4 (5.5)
No
12.8 (1.5)
53.5 (7.9)
Serum EPCAM level (median)
0.89
Normal
22.0 (4.1)
65.1 (6.2)
Elevated
19.0 (2.9)
62.0 (6.9)
Fig. 2 Survival curves in lung cancer patients according to serum EPCAM levels (p = 0.89)
The median follow-up time was 10 months (range 0–37 months). Median survival for all patients was 21 ± 3.1 months (95 % CI 14.9–27.1 months). 1-year
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The results of the present study suggest that serum EPCAM level had neither diagnostic nor prognostic value in lung cancer patients. Moreover, serum EPCAM concentration did not correlate with response to chemotherapy. Finally, no significant associations were found between results of serum EPCAM level and clinicopathological parameters such as histology and stage of disease. Available studies of EPCAM have been limited to paraffin-embedded materials and cell line trials, so all data about EPCAM in lung cancer have been provided by tissue cultures. However, our study was performed in serum, the first in the literature, instead of tissue. So, herein, we discuss our results with findings that have been provided in tissue cultures. In normal bronchial epithelium, EPCAM shows only infrequent basolateral cytoplasmic membrane positivity [1]. It was not stained in alveolar pneumocytes [5]. On the other hand, EPCAM was expressed in NSCLC, and its staining along the cytoplasmic membrane was complete [1–8]. EPCAM is believed to be an early marker for premalignancies [1, 3, 5, 6]. In normal, EPCAM-negative epithelia, de novo expression has been described in dysplasia [3, 5, 6]. Enhanced EPCAM expression was found in preneoplastic epithelia [1]. The expression of EPCAM increases in a stepwise manner from uninvolved bronchial mucosa, epithelial hyperplasia to lung carcinoma [3, 5, 6]. Expression of EPCAM increased during the progression of the lung cancer and, therefore, may play a role in the carcinogenesis of this disease [3, 6]. The most striking feature of this study was the demonstration that serum EPCAM levels were no higher in patients with lung cancer than in healthy controls. Therefore, it showed that serum EPCAM level had no diagnostic value in these patients. However, previous studies demonstrated that EPCAM was frequently overexpressed in lung cancer [1–8]. In the subset of lung cancer array of a large study, 823 of 1,287 cases (63.9 %) showed a high level of EPCAM expression [1, 2]. Moreover, a high expression level of EPCAM was observed in human NSCLC cells by flow cytometry and RT-PCR [1, 2]. EPCAM expression was detected in 120 out of 234 (51.3 %) surgically resected adenocarcinoma tissues [6]. Interestingly, positive focal EPCAM staining was also seen in the normal bronchial epithelium in this study. In another study, EPCAM expression was elevated significantly by 18.3-fold
Mol Cell Biochem (2014) 396:307–312
in NSCLC cell lines compared to normal lung controls [7]. These results indicate that the majority of NSCLC cell lines overexpress EPCAM. These data show that no correlation was found between serum soluble EPCAM levels and tissue concentration of EPCAM in lung cancer patients. High EPCAM expression has also been reported in adenocarcinoma of lung cancer with 94.7 % [1, 2]. Large cell and squamous cell lung carcinoma had only slightly less EPCAM expression (84.9 and 82.7 %, respectively) than adenocarcinoma of the lung [1, 2]. In this study, the expression of EPCAM was also found to be high (96.7 %) in bronchoalveolar histology. In another trial, EPCAM overexpression was significantly more frequent in adenocarcinoma specimen than in bronchoalveolar carcinoma [7]. Contrarily, Pak et al. found that EPCAM was more frequently overexpressed in squamous cell cancer (85.9 % from 64 samples) than adenocarcinoma (39 % from 100 samples) [6]. In a small study, although in almost 50 % of the NSCLC specimen (n = 15), all tumor cells expressed EPCAM, more than 90 % of the tumor cells were positive for EPCAM in SCLC tumor specimen (n = 10) [8]. Similar to our study, clinicopathological parameters such as sex, age, T stage, or regional lymph node metastasis were not related to EPCAM expression [6]. In other trial, EPCAM overexpression was significantly associated with sex, tumor differentiation, pathologic T stage, lymph node metastasis, and stage in adenocarcinoma unlike squamous cell carcinoma [5]. In contrast, EPCAM overexpression was not significantly related to age at diagnosis and smoking status [5]. Piyathilake et al. found that the expression of EPCAM was significantly higher in poorly to moderately differentiated squamous cell carcinoma compared to that in well-differentiated squamous cell cancer in 60 human subjects [4]. An increase in the expression of EPCAM with increasing size or local extent of the primary tumor approached statistical significance. The expression of EPCAM increased significantly with the increasing involvement of regional lymph nodes and TNM stages. Overexpression of EPCAM was associated with an advanced stage of disease and linked to worse outcome in certain tumor types [1, 2]. For example, EPCAM expression was found to be associated with poor prognosis in patients with breast cancer, triple-negative breast cancer, ovarian cancer, prostate cancer, and gallbladder cancer [2]. Thus, EPCAM may serve as an additional prognostic marker in these patients. In contrast, EPCAM expression was associated with improved survival in patients with cell renal carcinoma, moderately differentiated stage II colon cancer, and NSCLC at stage pT2 [2]. No significant difference was found in disease-free or overall survival between EPCAM overexpressing and EPCAM-negative groups. EPCAM expression has no prognostic significance [7]. EPCAM overexpression showed no significance with
311
both disease-free and overall survivals in adenocarcinoma and squamous cell carcinoma. The results are consistent with previous reports showing no definite correlations between EPCAM expression and overall survival in any of the histological entities [2, 3, 5, 6]. Moreover, in the study of 60 squamous cell carcinoma subjects, no statistically significant association between patient survival and staining intensity of carcinomas for EPCAM is found [3]. However, the expression of EPCAM was significantly related to a favorable outcome in 130 paraffin-embedded specimens of small-sized, less than 2 cm in diameter, pulmonary adenocarcinoma [4]. These contradicting results of these several studies might be attributable to several factors. So far, no consensus exists on which tumors and methods should be used for testing EPCAM expressions. During recent decades, IHC has become a useful adjunctive method in diagnostic histopathology. There are a number of drawbacks with IHC, the most important of which are lack of assay standardization and variance in the interpretation of the IHC staining. In many cases, the studies were performed on a relatively small sample size, which may have been insufficient to show significant differences. A standardized method remains to be established and validated in a larger series of patients in prospective studies. In conclusion, determination of EPCAM level in serum was found to be of no diagnostic value compared with tissue investigation. Additionally, we found that it was also of no predictive and prognostic values. The present study contributes to the literature, because we performed it firstly in the literature. Further much large-scale studies are needed to determine the exact role of serum EPCAM levels in terms of clinical significance in lung cancer.
Conflict of interest
None.
References 1. Charalabopoulos K, Papalimneou V, Charalabopoulos A (2003) Adhesion molecules in lung cancer. Exp Oncol 25:16–21 2. Patriarca C, Macchi RM, Marschner AK, Mellstedt H (2012) Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat Rev 38:68–75 3. Went P, Vasei M, Bubendorf L, Terracciano L, Tornillo L, Riede U, Kononen J, Simon R, Sauter G, Baeuerle PA (2006) Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br J Cancer 94:128–135 4. Piyathilake CJ, Frost AR, Weiss H, Manne U, Heimburger DC, Grizzle WE (2000) The expression of Ep-CAM (17–1A) in squamous cell cancers of the lung. Hum Pathol 31:482–487 5. Kobayashi H, Minami Y, Anami Y, Kondou Y, Lijima T, Kano J, Morishita Y, Tsuta K, Hayashi S, Noguchi M (2010) Expression of the GA733 gene family and its relationship to prognosis in pulmonary adenocarcinoma. Virchows Arch 457:69–76
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312 6. Pak MG, Shin DH, Lee CH, Lee MK (2012) Significance of EpCAM and TROP2 expression in nonsmall cell lung cancer. World J Surg Oncol 10:53 7. Kim Y, Kim HS, Cui ZY, Lee HS, Ahn JS, Park CK, Park K, Ahn MJ (2009) Clinicopathological implications of EpCAM expression in adenocarcinoma of the lung. Anticancer Res 29:1817–1822 8. Hase T, Sato M, Yoshida K, Girard L, Takeyama Y, Horio M, Elshazley M, Oguri T, Sekido Y, Shames DS, Gazdar AF, Minna
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Mol Cell Biochem (2014) 396:307–312 JD, Kondo M, Hasegawa Y (2011) Pivotal role of epithelial cell adhesion molecule in the survival of lung cancer cells. Cancer Sci 101:1493–1500 9. Brezicka T (2005) Expression of epithelial-cell adhesion molecule (Ep-CAM) in small cell lung cancer as defined by monoclonal antibodies 17–1A and BerEP4. Acta Oncol 44:723–727
Clinical significance of serum epithelial cell adhesion molecule (EPCAM) levels in patients with lung cancer Faruk Tas • Senem Karabulut • Derya Duranyildiz
Received: 26 May 2014 / Accepted: 14 July 2014 / Published online: 25 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Cellular adhesion molecules might be good markers in some types of malignant tumors, thus providing useful information in diagnosis and prognosis. The objective of this study was to determine the clinical significance of the serum levels of epithelial cell adhesion molecule (EPCAM) in lung cancer patients. One hundred and thirty lung cancer patients were enrolled in this study. Serum EPCAM levels were determined by the solid-phase sandwich ELISA method. Age- and sex-matched 34 healthy controls were included in the analysis. The median age was 58 years, ranging 35–80 years. The majority of the patients had NSCLC (83.8 %) and stage IV disease (60.8 %). There was no significant difference in the serum EPCAM levels between lung cancer patients and healthy controls (p = 0.16). Moreover, known clinical variables including age of patient, gender, histology, stage of disease, and response to chemotherapy were not found to be correlated with serum EPCAM concentrations (p [ 0.05). Similarly, no prognostic role was found for outcome (1-year survival rate 62 vs. 65.1 %, p = 0.89). In conclusion, serum EPCAM concentrations have no diagnostic, predictive, and prognostic roles in lung cancer patients. Keywords
EPCAM Serum Lung cancer
Introduction Cellular adhesion molecules (CAMs) mediate a great variety of cellular interactions in normal and process of F. Tas (&) S. Karabulut D. Duranyildiz Institute of Oncology, University of Istanbul, Capa, 34390 Istanbul, Turkey e-mail: [email protected]
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 [2–9]. Although its function is still largely unknown, it is involved in also cellular signaling, cell migration, proliferation, and differentiation in addition to cell adhesion. In human, EPCAM is expressed exclusively in epithelia and epithelial-derived neoplasms [2–9]. Therefore, it can be used as a histologically diagnostic and differentiatial marker for epithelial tumors from non-epithelial tumors. EPCAM expression is not found in mesenchymal, muscular, neuroendocrine, and lymphoid tissues, and melanoma-originated tumors [2–9]. EPCAM is detected by immunohistochemistry (IHC) in histological tumor samples [2]. More specifically, EPCAM is detected at the basolateral cell membrane of normal human tissue [2–6]. 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, ovarian cancer, and lung cancer [2]. Although a high expression level of EPCAM has been reported in lung cancer, the clinical implication has not been determined fully [2–9]. While there are increasing reports about the role of EPCAM in various cancers, only a few reports of EPCAM expression in lung cancer have been published [2–9]. All of the studies concerning the influences of this CAM in lung cancer have been performed at tissue scale in the literature
123
308
[2–9]. Therefore, the objective of this study was to determine the value of circulating EPCAM in lung cancer patients.
Materials and methods
Mol Cell Biochem (2014) 396:307–312
during chemotherapy or every 12 weeks for no anticancer treatment. For comparison of serum levels of EPCAM, age- and sex-matched 34 healthy controls were included in the analysis. This study was approved by the ethics committee of the Institute of Oncology, Istanbul University. Written informed consent was obtained from the patients.
Patients Measurement of serum EPCAM levels This study comprised 130 patients admitted to Istanbul University, Institute of Oncology with histologically or cytologically confirmed non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) treated and followed up in our clinic were recorded from medical charts. Patients with bidimensionally measurable disease without history of chemo/radiotherapy in the last six months were included in the study. The staging of metastatic patients was done using various imaging modalities such as computed tomography, magnetic resonance imaging, and PET/CT scan. The pathological diagnosis of lung cancer was established in accordance with the revised World Health Organization classification of lung tumors and staged relying on the revised TNM staging for lung cancer. The pretreatment evaluation included detailed clinical history and physical examination with a series of complete blood cell counts and routine biochemistry tests, including urea, creatinine, transaminases, and lactate dehydrogenase. Those with ECOG performance status B2 and appropriate blood chemistry tests received chemotherapy on outpatient basis comprising platinum compounds with/without radiotherapy depending on the stage of disease. Patients were treated with various chemotherapy regimens as a single agent or combination therapy. Regimens of single or combination chemotherapy were selected based on performance status of patients and extension of disease. Drug schemas consist of platinum agents in combination with newer cytotoxic drugs, including paclitaxel, docetaxel, gemcitabine, and vinorelbine with standard doses in NSCLC patients for adjuvant- and primary-based chemotherapy. Additionally, cisplatin and etoposide combination chemotherapy regimen with standard dose and schedule was applied for the patients with SCLC. The response to chemotherapy was evaluated by radiologically after 2–3 cycles of chemotherapy according to international criteria, revised RECIST criteria. Nonresponder patients to chemotherapy and relapsed patients were treated with second-line chemotherapy if they had a good performance status. Chemotherapy was continued until disease progression or unacceptable toxicity. The follow-up programs consisted of clinical, laboratory, and radiological assessments performed at 8-week intervals
123
Serum samples were obtained on first admission before any adjuvant and metastatic treatment were given for follow-up patients. Blood samples were obtained from patients with lung cancer and healthy controls (n = 34) by venipuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at -20 °C until analysis. Serum EPCAM (Eastbiopharm, China) levels were determined by the solid-phase sandwich ELISA method. The EPCAM ELISA (Eastbiopharm, China) was used a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of Human EPCAM in samples. Serum samples and standards were added to the wells which were pre-coated with Human EPCAM monoclonal antibody. Following incubation, EPCAM antibodies labeled with biotin and combined with Streptavidin-HRP were added to form immune complex and allowed to incubate for 1 h. Unbound material was washed away, and then chromogen solution was 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 the effect of the acidic stop solution, the color became yellow. The colored reaction product was measured using an automated ELISA reader (Rayto, RT1904C Chemistry Analyzer, Atlanta GA, USA). The results were expressed as ng/mL. Statistical analysis Continuous variables were categorized using median values as cut-off point. Assessment of relationships and comparisons between various clinical/laboratory parameters were accomplished using Mann–Whitney U test and Kruskal–Wallis test for two and three groups, respectively. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. Kaplan–Meier method was used for estimation of survival distribution, and differences in survival were assessed by the log-rank statistics. A p value \0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software.
Mol Cell Biochem (2014) 396:307–312
309
Table 1 Patient and disease characteristics Variables
n
No. of patients
130
Age of patients C60
58
\60
72
Gender Male
119
Female
11
Histology NSCLC
109
Adeno
49
Epidermoid Undifferentiated
45 15
SCLC
21
Stage II
9
III
37
IV
63
Limited
5
Extended
16
Response to chemotherapy
Fig. 1 The value of serum EPCAM assay in lung cancer patients and healthy controls (p = 0.16)
Table 3 Results (median and range) of comparisons between serum EPCAM levels and various clinical variables Variables Age of patients (p)
Yes (Complete ? partial)
68
No (Stable ? progressive)
52
C60 \60 Gender (p)
Table 2 The values of serum EPCAM levels in lung cancer patients and healthy controls Assay
Serum EPCAM level (ng/mL)
Patients (n = 130)
Controls (n = 34)
Median
Range
Median
Range
9.1
0.6–25.4
9.7
1.0–24.5
p
Male Female Histology (p)
9.1 (0.6–24.2) 10.0 (0.8–25.4) 0.83 9.1 (0.6–25.4) 9.3 (1.0–24.2)
Adeno Epidermoid
0.83 9.0 (0.6–23.7) 9.2 (0.8–25.4) 0.37
II
9.4 (1.5–21.7)
III
8.8 (3.0–16.8)
IV
From June 2010 to July 2011, 130 patients with a pathologically confirmed diagnosis of lung cancer were enrolled into this study. Baseline histopathological characteristics and demographic features of patients are listed in Table 1. The median age at diagnosis was 58 years, ranging 35–80 years, where males constituted the majority of the group (n = 119, 91.5 %). The majority of the patients had NSCLC (n = 109, 83.8 %) and metastatic disease (n = 79, 60.8 %). The levels of serum EPCAM in patients with lung cancer and healthy controls are shown in Table 2. No significant difference was found in the serum EPCAM levels between lung cancer patients and healthy controls (9.1 versus 9.7 ng/mL, p = 0.16) (Fig. 1).
9.3 (0.8–25.4) 0.14
SCLC
Stage in NSCLC (p)
Results
0.26 9.0 (0.6–23.1)
NSCLC Histology in NSCLC (p) 0.16
EPCAM (ng/mL)
Stage in SCLC (p) Limited Extended Response to chemotherapy (p) Yes No
9.4 (0.6–23.7) 0.28 9.0 (3.0–16.8) 9.8 (1.0–24.2) 0.89 9.1 (0.8–25.4) 9.2 (0.6–23.7)
Table 3 shows the correlation between the serum levels of EPCAM assays and clinicopathological factors. The known clinical variables including age of patient, gender, histology, stage of disease, and response to chemotherapy were not found to be correlated with in serum EPCAM concentrations (p [ 0.05).
123
310
Mol Cell Biochem (2014) 396:307–312
overall survival rate was 63.7 (95 % CI 54.5–72.9). However, serum EPCAM level was found to have no prognostic role for outcome (1-year survival rate 62 versus 65.1 %, p = 0.89) (Table 4; Fig. 2).
Table 4 Univariate analysis of survival Variables
Median survival time ± SE (month)
1-year survival rate ± SD (%)
Age of patients
0.55
C60
15.0 (7.2)
21.0 (2.0)
\60
62.1 (7.2)
65.3 (6.1)
Gender 20.4 (1.5)
62.4 (4.9)
Female
21.7 (3.8)
77.1 (14.4)
Histology NSCLC
23.0 (5.1)
67.2 (4.9)
SCLC
11.0 (0.9)
0.002 44.1(12.9)
Histology in NSCLC
0.40
Adeno
24.6 (2.3)
78.8 (6.4)
Epidermoid
23.7 (2.3)
66.8 (7.7)
II–III
29.8 (2.0)
85.9 (5.4)
IV
14.9 (1.7)
51.5 (7.2)
18.0 (4.6)
66.7 (2.7)
10.4 (2.4)
38.2 (14.2)
\0.001
Stage in NSCLC
Stage in SCLC Extended Response to chemotherapy
Discussion 0.36
Male
Limited
p
0.20
\0.001
Yes
25.4 (1.8)
75.4 (5.5)
No
12.8 (1.5)
53.5 (7.9)
Serum EPCAM level (median)
0.89
Normal
22.0 (4.1)
65.1 (6.2)
Elevated
19.0 (2.9)
62.0 (6.9)
Fig. 2 Survival curves in lung cancer patients according to serum EPCAM levels (p = 0.89)
The median follow-up time was 10 months (range 0–37 months). Median survival for all patients was 21 ± 3.1 months (95 % CI 14.9–27.1 months). 1-year
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The results of the present study suggest that serum EPCAM level had neither diagnostic nor prognostic value in lung cancer patients. Moreover, serum EPCAM concentration did not correlate with response to chemotherapy. Finally, no significant associations were found between results of serum EPCAM level and clinicopathological parameters such as histology and stage of disease. Available studies of EPCAM have been limited to paraffin-embedded materials and cell line trials, so all data about EPCAM in lung cancer have been provided by tissue cultures. However, our study was performed in serum, the first in the literature, instead of tissue. So, herein, we discuss our results with findings that have been provided in tissue cultures. In normal bronchial epithelium, EPCAM shows only infrequent basolateral cytoplasmic membrane positivity [1]. It was not stained in alveolar pneumocytes [5]. On the other hand, EPCAM was expressed in NSCLC, and its staining along the cytoplasmic membrane was complete [1–8]. EPCAM is believed to be an early marker for premalignancies [1, 3, 5, 6]. In normal, EPCAM-negative epithelia, de novo expression has been described in dysplasia [3, 5, 6]. Enhanced EPCAM expression was found in preneoplastic epithelia [1]. The expression of EPCAM increases in a stepwise manner from uninvolved bronchial mucosa, epithelial hyperplasia to lung carcinoma [3, 5, 6]. Expression of EPCAM increased during the progression of the lung cancer and, therefore, may play a role in the carcinogenesis of this disease [3, 6]. The most striking feature of this study was the demonstration that serum EPCAM levels were no higher in patients with lung cancer than in healthy controls. Therefore, it showed that serum EPCAM level had no diagnostic value in these patients. However, previous studies demonstrated that EPCAM was frequently overexpressed in lung cancer [1–8]. In the subset of lung cancer array of a large study, 823 of 1,287 cases (63.9 %) showed a high level of EPCAM expression [1, 2]. Moreover, a high expression level of EPCAM was observed in human NSCLC cells by flow cytometry and RT-PCR [1, 2]. EPCAM expression was detected in 120 out of 234 (51.3 %) surgically resected adenocarcinoma tissues [6]. Interestingly, positive focal EPCAM staining was also seen in the normal bronchial epithelium in this study. In another study, EPCAM expression was elevated significantly by 18.3-fold
Mol Cell Biochem (2014) 396:307–312
in NSCLC cell lines compared to normal lung controls [7]. These results indicate that the majority of NSCLC cell lines overexpress EPCAM. These data show that no correlation was found between serum soluble EPCAM levels and tissue concentration of EPCAM in lung cancer patients. High EPCAM expression has also been reported in adenocarcinoma of lung cancer with 94.7 % [1, 2]. Large cell and squamous cell lung carcinoma had only slightly less EPCAM expression (84.9 and 82.7 %, respectively) than adenocarcinoma of the lung [1, 2]. In this study, the expression of EPCAM was also found to be high (96.7 %) in bronchoalveolar histology. In another trial, EPCAM overexpression was significantly more frequent in adenocarcinoma specimen than in bronchoalveolar carcinoma [7]. Contrarily, Pak et al. found that EPCAM was more frequently overexpressed in squamous cell cancer (85.9 % from 64 samples) than adenocarcinoma (39 % from 100 samples) [6]. In a small study, although in almost 50 % of the NSCLC specimen (n = 15), all tumor cells expressed EPCAM, more than 90 % of the tumor cells were positive for EPCAM in SCLC tumor specimen (n = 10) [8]. Similar to our study, clinicopathological parameters such as sex, age, T stage, or regional lymph node metastasis were not related to EPCAM expression [6]. In other trial, EPCAM overexpression was significantly associated with sex, tumor differentiation, pathologic T stage, lymph node metastasis, and stage in adenocarcinoma unlike squamous cell carcinoma [5]. In contrast, EPCAM overexpression was not significantly related to age at diagnosis and smoking status [5]. Piyathilake et al. found that the expression of EPCAM was significantly higher in poorly to moderately differentiated squamous cell carcinoma compared to that in well-differentiated squamous cell cancer in 60 human subjects [4]. An increase in the expression of EPCAM with increasing size or local extent of the primary tumor approached statistical significance. The expression of EPCAM increased significantly with the increasing involvement of regional lymph nodes and TNM stages. Overexpression of EPCAM was associated with an advanced stage of disease and linked to worse outcome in certain tumor types [1, 2]. For example, EPCAM expression was found to be associated with poor prognosis in patients with breast cancer, triple-negative breast cancer, ovarian cancer, prostate cancer, and gallbladder cancer [2]. Thus, EPCAM may serve as an additional prognostic marker in these patients. In contrast, EPCAM expression was associated with improved survival in patients with cell renal carcinoma, moderately differentiated stage II colon cancer, and NSCLC at stage pT2 [2]. No significant difference was found in disease-free or overall survival between EPCAM overexpressing and EPCAM-negative groups. EPCAM expression has no prognostic significance [7]. EPCAM overexpression showed no significance with
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both disease-free and overall survivals in adenocarcinoma and squamous cell carcinoma. The results are consistent with previous reports showing no definite correlations between EPCAM expression and overall survival in any of the histological entities [2, 3, 5, 6]. Moreover, in the study of 60 squamous cell carcinoma subjects, no statistically significant association between patient survival and staining intensity of carcinomas for EPCAM is found [3]. However, the expression of EPCAM was significantly related to a favorable outcome in 130 paraffin-embedded specimens of small-sized, less than 2 cm in diameter, pulmonary adenocarcinoma [4]. These contradicting results of these several studies might be attributable to several factors. So far, no consensus exists on which tumors and methods should be used for testing EPCAM expressions. During recent decades, IHC has become a useful adjunctive method in diagnostic histopathology. There are a number of drawbacks with IHC, the most important of which are lack of assay standardization and variance in the interpretation of the IHC staining. In many cases, the studies were performed on a relatively small sample size, which may have been insufficient to show significant differences. A standardized method remains to be established and validated in a larger series of patients in prospective studies. In conclusion, determination of EPCAM level in serum was found to be of no diagnostic value compared with tissue investigation. Additionally, we found that it was also of no predictive and prognostic values. The present study contributes to the literature, because we performed it firstly in the literature. Further much large-scale studies are needed to determine the exact role of serum EPCAM levels in terms of clinical significance in lung cancer.
Conflict of interest
None.
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