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Original Article
Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer Jun-Feng Shi ∗ , Shi-Xin Xu, Ping He, Zhi-Hui Xi Department of General Surgery, Jiangnan University Hospital (Wuxi Fourth People’s Hospital), No. 200, Huihe Road, Wuxi, Jiangsu 214000, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 26 December 2013 Accepted 10 March 2014 Keywords: Carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) Gastric adenocarcinoma Micro-vessel density Immunohistochemistry (IHC)
a b s t r a c t Objective: To examine the expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and CD34 in gastric adenocarcinoma, and to investigate their relations with clinical pathology-related factors. Methods: Immunohistochemistry (IHC) was used to analyze the expression of CEACAM1 and CD34, and micro-vessel density (MVD) marked by CD34 was calculated in 208 cases of human gastric adenocarcinoma and in 56 cases of normal human gastric tissues. Results: There was no expression of CEACAM1 in normal gastric mucosa. However, all of the gastric adenocarcinoma tissues expressed CEACAM1 with cytoplasmic and/or membranous staining. CEACAM1 expression was classified as high and low (137/208 vs. 71/208, 65.9% vs. 34.1%), the CD34-MVD value was significantly different between the two groups (P < 0.05). In addition, expressions of CEACAM1 and CD34 were significantly different from gastric adenocarcinoma to normal gastric tissues, respectively (P < 0.05). High CEACAM1 expression and high MVD value were positively associated with lymph nodes metastasis and TNM stage, but negatively related to pathological grade (P < 0.05). But they were irrelevant with tumor size, patients’ age and gender (P > 0.05). Conclusions: The up-regulation of CEACAM1 expression may participate in tumorous angiogenesis, especially high expression of CEACAM1 promoted its capability of invasion and metastasis. © 2014 Elsevier GmbH. All rights reserved.
Introduction Gastric cancer is the second leading cause of death from malignant diseases worldwide, with especially high mortality rates in East, South, and Central Asia [1]. The occurrence of cancer is a multifactor and multi-stage process [2]. A variety of environmental and genetic factors may synergisticlly or sequentially cause non-lethal DNA damage, leading to activation of oncogenes and/or inactivation of tumor suppressor gene, thus causing tumorigenesis [3]. Carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) is a member of the immunoglobulin superfamily, also known as C-CAM, BGP and CD66a, which has been found on quite different epithelial cells and various leukocytes [4,5]. It has been reported that the expression of CEACAM1 was found reduced in bladder, breast and prostate cancer [6–8]. Meanwhile, in some
∗ Corresponding author. Tel.: +86 0510 88682220. E-mail address: [email protected] (J.-F. Shi).
tumor cell lines, re-expression of CEACAM1 abolishes their oncogenicity in vivo, thus it has been suggested as a putative tumor suppressor [9]. However, Thies et al. [10] and Dango et al. [11] found an opposite role of CEACAM1 in melanoma and non-small cell lung carcinoma (NSCLC). Interestingly, up-regulation of CEACAM1 was considered as a risk factor of metastasis and an independent predictor of survival in melanoma and NSCLC patients. The growth and progression of all solid tumors rely on angiogenesis, which is regulated by angiogenic activators and inhibitors. Among them, cell adhesion molecules play a special and important role in vascular morphogenesis and endothelial signaling [12]. It has been reported that CEACAM1 can mediate VEGF-induced angiogenesis, and tube formation will be completely blocked by a monoclonal anti-CEACAM1 antibody (anti-CEACAM1 mAb) [13,14]. CD34, which is known as human hematopoietic progenitor cell antigen, can be detected in vascular endothelium. The anti-CD34 monoclonal antibody can recognize small caliber vessels in tumors. Meanwhile, micro-vessel density (MVD) is an important parameter of tumor angiogenesis and clinicopathological variables in gastric cancer. Detecting the proliferating tumor blood vessels by CD34
http://dx.doi.org/10.1016/j.prp.2014.03.014 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: J.-F. Shi, et al., Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.014
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antibodies is an efficient way to estimate the MVD value in gastric cancer tissue. At present, few papers have been published about the expression of CEACAM1 in gastric cancer. Thus, we analyzed the relationships among CEACAM1 expression, CD34-MVD value and clinicopathologic features of tumors to evaluate the role of CEACAM1 and CD34 in the occurrence and development of gastric cancer. Materials and methods Patients Gastric adenocarcinoma tissues were obtained from 208 patients who underwent primary surgical resection at Jiangnan University Hospital between 2004 and 2009. There were 138 men and 70 women. The average age of patients was (61.5 ± 6.7) (range from 37 to 85) years. Normal gastric tissues were obtained from 56 patients who received gastric endoscopic check and biopsy at Jiangnan University Hospital between 2004 and 2008. TNM staging was estimated by the 7th AJCC edition of gastric cancer. All patients had not received preoperative radiotherapy or chemotherapy. This study was admitted by the Ethical Committee of Jiangnan University Hospital.
Table 1 CEACAM1 expression and MVD by CD34 related gastric cancer with different clinicopathological parameters. Parameters
n
MVD (mean ± SD)
CEACAM1 Low
High
Gender Male Female
138 70
50 21
88 49
48.48 ± 10.71 51.95 ± 11.66
Age ≤60years >60years
117 91
36 35
81 56
50.57 ± 11.74 48.96 ± 10.87
Tumor size ≤5 cm >5 cm
159 49
57 14
102 35
49.27 ± 10.18 51.90 ± 11.31
36 85 87
31 31 9
5 54 78
35.04 ± 6.17 43.61 ± 6.29 60.38 ± 7.02
86 122
14 57
72 65
59.17 ± 8.24 55.84 ± 8.78
49 96 63
43 23 5
6 73 58
37.97 ± 6.85 49.01 ± 6.12 59.97 ± 8.45
Differentiation Well Moderate Poorly LN metastasis + − Stage I II III
Immunohistochemistry (IHC) All specimens were fixed in formalin, performed on 4-mm-thick routinely processed paraffin sections, attached to slides pretreated with 1% of polylysine in series. After the sections were rinsed in phosphate-buffered saline (PBS), endogenous peroxidase was blocked with 0.3% H2 O2 in methanol for 30 min. Antigens were retrieved by autoclaving sections on slides in 0.01 mmol/l citrate buffer (pH 6.0) for 25 min for CEACAM1, or by incubation with Proteinase K for 15 min for CD34. After rinsing in PBS, the sections were incubated with each primary antibody overnight at 4 ◦ C. Negative controls were performed in which the primary antibody was omitted. The primary antibodies were mouse monoclonal anti-CEACAM1 antibody (abcam, dilution 1:75) and rabbit monoclonal anti-CD34 antibody (Novocastra Laboratories, dilution 1:100). Evaluation of CEACAM1 staining All specimens were examined by two pathologists independently. They examined the sections at 100× magnification. For each case, at least 1000 carcinoma cells were evaluated for CEACAM1 expression, and carcinoma cells with membranous and/or cytoplasmic staining were regarded as positive expression. In addition, according to the percentage of stained tumor cells, CEACAM1 expression was classified as negative expression (50% positive cells). Evaluation of MVD For MVD assessment, micro-vessels were counted using light microscopy, which was described by Weidner et al. [15]. After scanning the whole section at low magnifications (40×), three tumor areas with the greatest number of distinctly highlighted micro-vessels (hot spot) were selected. The number of vessels was expressed as the average value of counted micro-vessel in three hot spots at high magnifications (100×). Data were shown as mean ± SD.
Statistical analysis All statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The student’s t-test, Chi-square test and one-way (ANOVA) were used to analyze the data. The relationships of data were analyzed by Spearman rank correlation analysis. Differences at p < 0.05 were considered significant.
Results CEACAM1 staining was hardly observed in any gastric mucosa (Fig. 1A). CEACAM1 was expressed in all gastric adenocarcinomas with membranous and/or cytoplasmic staining. In addition, all 208 specimens were categorized into high expression group (n = 137) and low expression group (n = 71) according to the percentage of positive tumor cells. CEACAM1 expression in well, moderately and poorly differentiated tumors showed significant differences (P < 0.05), and high CEACAM1 expression showed negative relationship with pathological grade (Fig. 1B–E). Moreover, lymph node metastasis and higher TNM stage also strongly expressed CEACAM1 (Table 1). CD34-MVD value in normal gastric mucosa and gastric adenocarcinomas were significantly different (18.65 ± 5.41 vs. 47.01 ± 5.92, P < 0.05). Compared with well differentiated tumors (35.04 ± 6.17), moderately (43.61 ± 6.29) and poorly (60.38 ± 7.02) differentiated tumors showed more CD34-positive vessels (Fig. 1F–H). CD34-MVD value was higher in the lymph nodes metastasis group in comparison with no LN metastasis group (59.17 ± 8.24 vs. 55.84 ± 8.78, P < 0.05). Moreover, value of CD34-MVD showed differences among TNM stage I, II, III (37.97 ± 6.85 vs.49.01 ± 6.12 vs. 59.97 ± 8.45, P < 0.05) (Table 1). However, CEACAM1 expression and CD34-MVD showed irrelevance with tumor size, patients’ age and gender (Table 1). Furthermore, CD34-MVD showed a significant difference in the low and high CEACAM1 group (48.26 ± 7.25 vs. 59.32 ± 6.96, P < 0.05) (Table 2).
Please cite this article in press as: J.-F. Shi, et al., Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.014
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Fig. 1. Normal gastric mucosa was rarely detected in CEACAM1 staining (A). CEACAM1 was expressed in well (B), moderately (C) and poorly (D) differentiated gastric adenocarcinoma. Gastric adenocarcinoma cell in LN with metastasis showed CEACAM1 staining (E). For well (F), moderately (G) and poorly (H) differentiated gastric adenocarcinoma tissues, endothelial cells of small capillary-like vessels were stained with CD34 (Original magnification 100×).
Discussion The role of CEACAM1 in different tumor cells was apparently contradictory. Some studies have revealed that CEACAM1 was down-regulated in bladder, breast, prostate [6–8], and the reduction or loss of CEACAM1 expression was associated with malignant
tumor grading and loss of cell polarity, which may promote tumor growth and metastasis [16,17]. However, several studies have challenged the previously postulated concept as a tumor-suppressive effect of CEACAM1. A large body of evidence demonstrated that CEACAM1 was a marker for progression and metastatization of NSCLC and melanoma [10,11].
Please cite this article in press as: J.-F. Shi, et al., Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.014
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Table 2 Correlation analysis of CEACAM1 expression and MVD by CD34 in gastric cancer. Parameters
Gender Age Tumor size Differentiation LN metastasis Stage CEACAM1 CD34-MVD
CEACAM1
of survival in gastric cancer, and thereby may help to refine therapeutic decisions in gastric cancer.
CD34-MVD
r value
P value
r value
P value
0.062 −0.080 0.065 0.523 0.154 0.573 – 0.238
0.370 0.246 0.348 0.000 0.000 0.000 – 0.002
−0.074 0.063 0.090 0.442 0.295 0.320 0.238 –
0.241 0.450 0.376 0.010 0.000 0.027 0.002 –
Oliveira-Ferrer et al. [18] observed that CEACAM1 was expressed in umbrella cells of bladder urothelium ubiquitously. But CEACAM1 was down-regulated in superficial bladder cancer; interestingly it was up-regulated in the endothelia of adjacent angiogenic blood vessels concurrently. The differential switch and the epithelial down-regulation of CEACAM1 induced angiogenesis via increased expression of VEGF-C and VEGF-D. As a result, CEACAM1 appeared to be a promising endothelial target for bladder cancer therapy. Dango et al. [11] showed that increased CEACAM1 expression in the tumor cells stimulates angiogenesis and it had an important role in the development and progression of cancer. Our results showed that CEACAM1 was a tumor promoter in gastric adenocarcinoma. Compared with well differentiated carcinoma, higher CEACAM1 expression and CD34-MVD value were observed more frequently in poor differentiated carcinoma. Meanwhile, the patients with lymph node metastasis showed high expression of CEACAM1 and CD34-MVD value. In addition, high expression of CEACAM1 often implied a higher TNM staging, as well as a high CD34-MVD value. On the one hand, high expression of CEACAM1 decreased the adhesion of carcinoma cells to extracellular matrix, and then facilitated the infiltration and metastasis of carcinoma cells. On the other hand, high expression of CEACAM1 stimulated the secretion of vascular growth factors which would promote tumor angiogenesis, and new born blood vessels may facilitate the invasion and metastasis of tumor. In conclusion, the up-regulation of CEACAM1 is an important biological event in the initiation and progression of gastric adenocarcinoma. Also, increased CEACAM1 expression in the tumor cells stimulates angiogenesis. Proliferation of blood vessels may provide nutrients and pathways for tumor cells and improve its ability of invasion and metastasis, thus affecting the biological behavior of gastric adenocarcinoma. CEACAM1 may be a useful predictor
References [1] J. Ferlay, H.R. Shin, F. Bray, et al., Estimates of worldwide burden of cancer in 2008: GLOBOCAN, Int. J. Cancer 127 (2893–2917) (2010) 2008. [2] R.A. Weinberg, Oncogenes, antioncogenes, and the molecular bases of multistep carcinogenesis, Cancer Res. 49 (1989) 3713–3721. [3] T.G. Strohmeyer, D.J. Slamon, Proto-oncogenes and tumor suppressor genes in human urological malignancies, J. Urol. 151 (1994) 1479–1497. [4] D. Chen, H. Iijima, T. Nagaishi, et al., Carcinoembryonic antigen-related cellular adhesion molecule 1 isoforms alternatively inhibit and costimulate human T cell function, J. Immunol. 172 (2004) 3535–3543. [5] B.B. Singer, I. Scheffrahn, B. Obrink, The tumor growth inhibiting cell adhesion molecule CEACAM1 (C-CAM) is differently expressed in proliferating and quiescent epithelial cells and regulates cell proliferation, Cancer Res. 60 (2000) 1236–1244. [6] D. Tilki, B.B. Singer, S.F. Shariat, et al., CEACAM1: a novel urinary marker for bladder cancer detection, Eur. Urol. 57 (4) (2010) 648–654. [7] L. Riethdorf, B.W. Lisboa, U. Henkel, et al., Differential expression of CD66a(BGP), a cell adhesion molecule of the carcinoembryonic antigen family, in benign, premalignant, and malignant lesions of the human mammary gland, Histochem. Cytochem. 45 (7) (1997) 957–963. [8] D.I. Kleinerman, P. Troncoso, Sll Lin, et al., Consistent expression of an epithelial cell adhesion molecule (C-CAM) during human prostate development and loss of expression in prostate cancer: implication as a tumor suppressor, Cancer Res. 55 (6) (1995) 1215–1220. [9] R. Kammerer, R. Riesenberg, C. Weiler, et al., The tumor suppressor gene CEACAM1 is completely but reversibly down regulated in renal cell carcinoma, J. Pathol. 204 (2004) 258–267. [10] A. Thies, I. Moll, J. Berger, C. Wagener, et al., CEACAM-1 expression in cutaneous malignant melanoma predicts the development of metastatic disease, J. Clin. Oncol. 20 (2002) 2530–2536. [11] S. Dango, W. Sienel, M. Schreiber, et al., Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) is associated with increased angiogenic potential in non-small-cell lung cancer, Lung Cancer 60 (2008) 426–433. [12] E. Dejana, R. Spagnuolo, G. Bazzoni, Interendothelial junctions and their role in the control of angiogenesis, vascular permeability and leukocyte transmigration, Thromb. Haemost. 86 (2001) 308–315. [13] S. Ergun, N. Kilik, G. Ziegeler, et al., CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor, Mol. Cell 5 (2000) 311–320. [14] N. Kilic, L. Oliveira-Ferrer, J.H. Wurmbach, et al., Pro-angiogenic signaling by the endothelial presence of CEACAM1, J. Biol. Chem. 280 (2005) 2361–2369. [15] N. Weidner, J.P. Semple, W.R. Weich, et al., Tumor angiogenesis and metastasis – correlation in invasive breast carcinoma, N. Engl. J. Med. 324 (1991) 1–8. [16] T. Okegawa, R.C. Pong, Y. Li, et al., The role of cell adhesion molecule in cancer progression and its application in cancer therapy, Acta Biochim. Pol. 51 (2004) 445–457. [17] C. Bush, T.A. Hanssen, C. Wagener, et al., Down-regulation of CEACAM in human prostate cancer: correlation with loss of cell polarity, increased proliferation rate, and Gleason gade 3 to 4 transition, Hum. Pathol. 33 (2002) 290–298. [18] L. Oliveira-Ferrer, D. Tilki, G. Ziegeler, et al., Dual role of carcinoembryonic antigen-related cell adhesion molecule 1 in angiogenesis and invasion of human urinary bladder cancer, Cancer Res. 64 (24) (2004) 8932–8938.
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Original Article
Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer Jun-Feng Shi ∗ , Shi-Xin Xu, Ping He, Zhi-Hui Xi Department of General Surgery, Jiangnan University Hospital (Wuxi Fourth People’s Hospital), No. 200, Huihe Road, Wuxi, Jiangsu 214000, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 26 December 2013 Accepted 10 March 2014 Keywords: Carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) Gastric adenocarcinoma Micro-vessel density Immunohistochemistry (IHC)
a b s t r a c t Objective: To examine the expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and CD34 in gastric adenocarcinoma, and to investigate their relations with clinical pathology-related factors. Methods: Immunohistochemistry (IHC) was used to analyze the expression of CEACAM1 and CD34, and micro-vessel density (MVD) marked by CD34 was calculated in 208 cases of human gastric adenocarcinoma and in 56 cases of normal human gastric tissues. Results: There was no expression of CEACAM1 in normal gastric mucosa. However, all of the gastric adenocarcinoma tissues expressed CEACAM1 with cytoplasmic and/or membranous staining. CEACAM1 expression was classified as high and low (137/208 vs. 71/208, 65.9% vs. 34.1%), the CD34-MVD value was significantly different between the two groups (P < 0.05). In addition, expressions of CEACAM1 and CD34 were significantly different from gastric adenocarcinoma to normal gastric tissues, respectively (P < 0.05). High CEACAM1 expression and high MVD value were positively associated with lymph nodes metastasis and TNM stage, but negatively related to pathological grade (P < 0.05). But they were irrelevant with tumor size, patients’ age and gender (P > 0.05). Conclusions: The up-regulation of CEACAM1 expression may participate in tumorous angiogenesis, especially high expression of CEACAM1 promoted its capability of invasion and metastasis. © 2014 Elsevier GmbH. All rights reserved.
Introduction Gastric cancer is the second leading cause of death from malignant diseases worldwide, with especially high mortality rates in East, South, and Central Asia [1]. The occurrence of cancer is a multifactor and multi-stage process [2]. A variety of environmental and genetic factors may synergisticlly or sequentially cause non-lethal DNA damage, leading to activation of oncogenes and/or inactivation of tumor suppressor gene, thus causing tumorigenesis [3]. Carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) is a member of the immunoglobulin superfamily, also known as C-CAM, BGP and CD66a, which has been found on quite different epithelial cells and various leukocytes [4,5]. It has been reported that the expression of CEACAM1 was found reduced in bladder, breast and prostate cancer [6–8]. Meanwhile, in some
∗ Corresponding author. Tel.: +86 0510 88682220. E-mail address: [email protected] (J.-F. Shi).
tumor cell lines, re-expression of CEACAM1 abolishes their oncogenicity in vivo, thus it has been suggested as a putative tumor suppressor [9]. However, Thies et al. [10] and Dango et al. [11] found an opposite role of CEACAM1 in melanoma and non-small cell lung carcinoma (NSCLC). Interestingly, up-regulation of CEACAM1 was considered as a risk factor of metastasis and an independent predictor of survival in melanoma and NSCLC patients. The growth and progression of all solid tumors rely on angiogenesis, which is regulated by angiogenic activators and inhibitors. Among them, cell adhesion molecules play a special and important role in vascular morphogenesis and endothelial signaling [12]. It has been reported that CEACAM1 can mediate VEGF-induced angiogenesis, and tube formation will be completely blocked by a monoclonal anti-CEACAM1 antibody (anti-CEACAM1 mAb) [13,14]. CD34, which is known as human hematopoietic progenitor cell antigen, can be detected in vascular endothelium. The anti-CD34 monoclonal antibody can recognize small caliber vessels in tumors. Meanwhile, micro-vessel density (MVD) is an important parameter of tumor angiogenesis and clinicopathological variables in gastric cancer. Detecting the proliferating tumor blood vessels by CD34
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antibodies is an efficient way to estimate the MVD value in gastric cancer tissue. At present, few papers have been published about the expression of CEACAM1 in gastric cancer. Thus, we analyzed the relationships among CEACAM1 expression, CD34-MVD value and clinicopathologic features of tumors to evaluate the role of CEACAM1 and CD34 in the occurrence and development of gastric cancer. Materials and methods Patients Gastric adenocarcinoma tissues were obtained from 208 patients who underwent primary surgical resection at Jiangnan University Hospital between 2004 and 2009. There were 138 men and 70 women. The average age of patients was (61.5 ± 6.7) (range from 37 to 85) years. Normal gastric tissues were obtained from 56 patients who received gastric endoscopic check and biopsy at Jiangnan University Hospital between 2004 and 2008. TNM staging was estimated by the 7th AJCC edition of gastric cancer. All patients had not received preoperative radiotherapy or chemotherapy. This study was admitted by the Ethical Committee of Jiangnan University Hospital.
Table 1 CEACAM1 expression and MVD by CD34 related gastric cancer with different clinicopathological parameters. Parameters
n
MVD (mean ± SD)
CEACAM1 Low
High
Gender Male Female
138 70
50 21
88 49
48.48 ± 10.71 51.95 ± 11.66
Age ≤60years >60years
117 91
36 35
81 56
50.57 ± 11.74 48.96 ± 10.87
Tumor size ≤5 cm >5 cm
159 49
57 14
102 35
49.27 ± 10.18 51.90 ± 11.31
36 85 87
31 31 9
5 54 78
35.04 ± 6.17 43.61 ± 6.29 60.38 ± 7.02
86 122
14 57
72 65
59.17 ± 8.24 55.84 ± 8.78
49 96 63
43 23 5
6 73 58
37.97 ± 6.85 49.01 ± 6.12 59.97 ± 8.45
Differentiation Well Moderate Poorly LN metastasis + − Stage I II III
Immunohistochemistry (IHC) All specimens were fixed in formalin, performed on 4-mm-thick routinely processed paraffin sections, attached to slides pretreated with 1% of polylysine in series. After the sections were rinsed in phosphate-buffered saline (PBS), endogenous peroxidase was blocked with 0.3% H2 O2 in methanol for 30 min. Antigens were retrieved by autoclaving sections on slides in 0.01 mmol/l citrate buffer (pH 6.0) for 25 min for CEACAM1, or by incubation with Proteinase K for 15 min for CD34. After rinsing in PBS, the sections were incubated with each primary antibody overnight at 4 ◦ C. Negative controls were performed in which the primary antibody was omitted. The primary antibodies were mouse monoclonal anti-CEACAM1 antibody (abcam, dilution 1:75) and rabbit monoclonal anti-CD34 antibody (Novocastra Laboratories, dilution 1:100). Evaluation of CEACAM1 staining All specimens were examined by two pathologists independently. They examined the sections at 100× magnification. For each case, at least 1000 carcinoma cells were evaluated for CEACAM1 expression, and carcinoma cells with membranous and/or cytoplasmic staining were regarded as positive expression. In addition, according to the percentage of stained tumor cells, CEACAM1 expression was classified as negative expression (50% positive cells). Evaluation of MVD For MVD assessment, micro-vessels were counted using light microscopy, which was described by Weidner et al. [15]. After scanning the whole section at low magnifications (40×), three tumor areas with the greatest number of distinctly highlighted micro-vessels (hot spot) were selected. The number of vessels was expressed as the average value of counted micro-vessel in three hot spots at high magnifications (100×). Data were shown as mean ± SD.
Statistical analysis All statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The student’s t-test, Chi-square test and one-way (ANOVA) were used to analyze the data. The relationships of data were analyzed by Spearman rank correlation analysis. Differences at p < 0.05 were considered significant.
Results CEACAM1 staining was hardly observed in any gastric mucosa (Fig. 1A). CEACAM1 was expressed in all gastric adenocarcinomas with membranous and/or cytoplasmic staining. In addition, all 208 specimens were categorized into high expression group (n = 137) and low expression group (n = 71) according to the percentage of positive tumor cells. CEACAM1 expression in well, moderately and poorly differentiated tumors showed significant differences (P < 0.05), and high CEACAM1 expression showed negative relationship with pathological grade (Fig. 1B–E). Moreover, lymph node metastasis and higher TNM stage also strongly expressed CEACAM1 (Table 1). CD34-MVD value in normal gastric mucosa and gastric adenocarcinomas were significantly different (18.65 ± 5.41 vs. 47.01 ± 5.92, P < 0.05). Compared with well differentiated tumors (35.04 ± 6.17), moderately (43.61 ± 6.29) and poorly (60.38 ± 7.02) differentiated tumors showed more CD34-positive vessels (Fig. 1F–H). CD34-MVD value was higher in the lymph nodes metastasis group in comparison with no LN metastasis group (59.17 ± 8.24 vs. 55.84 ± 8.78, P < 0.05). Moreover, value of CD34-MVD showed differences among TNM stage I, II, III (37.97 ± 6.85 vs.49.01 ± 6.12 vs. 59.97 ± 8.45, P < 0.05) (Table 1). However, CEACAM1 expression and CD34-MVD showed irrelevance with tumor size, patients’ age and gender (Table 1). Furthermore, CD34-MVD showed a significant difference in the low and high CEACAM1 group (48.26 ± 7.25 vs. 59.32 ± 6.96, P < 0.05) (Table 2).
Please cite this article in press as: J.-F. Shi, et al., Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.014
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Fig. 1. Normal gastric mucosa was rarely detected in CEACAM1 staining (A). CEACAM1 was expressed in well (B), moderately (C) and poorly (D) differentiated gastric adenocarcinoma. Gastric adenocarcinoma cell in LN with metastasis showed CEACAM1 staining (E). For well (F), moderately (G) and poorly (H) differentiated gastric adenocarcinoma tissues, endothelial cells of small capillary-like vessels were stained with CD34 (Original magnification 100×).
Discussion The role of CEACAM1 in different tumor cells was apparently contradictory. Some studies have revealed that CEACAM1 was down-regulated in bladder, breast, prostate [6–8], and the reduction or loss of CEACAM1 expression was associated with malignant
tumor grading and loss of cell polarity, which may promote tumor growth and metastasis [16,17]. However, several studies have challenged the previously postulated concept as a tumor-suppressive effect of CEACAM1. A large body of evidence demonstrated that CEACAM1 was a marker for progression and metastatization of NSCLC and melanoma [10,11].
Please cite this article in press as: J.-F. Shi, et al., Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.014
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Table 2 Correlation analysis of CEACAM1 expression and MVD by CD34 in gastric cancer. Parameters
Gender Age Tumor size Differentiation LN metastasis Stage CEACAM1 CD34-MVD
CEACAM1
of survival in gastric cancer, and thereby may help to refine therapeutic decisions in gastric cancer.
CD34-MVD
r value
P value
r value
P value
0.062 −0.080 0.065 0.523 0.154 0.573 – 0.238
0.370 0.246 0.348 0.000 0.000 0.000 – 0.002
−0.074 0.063 0.090 0.442 0.295 0.320 0.238 –
0.241 0.450 0.376 0.010 0.000 0.027 0.002 –
Oliveira-Ferrer et al. [18] observed that CEACAM1 was expressed in umbrella cells of bladder urothelium ubiquitously. But CEACAM1 was down-regulated in superficial bladder cancer; interestingly it was up-regulated in the endothelia of adjacent angiogenic blood vessels concurrently. The differential switch and the epithelial down-regulation of CEACAM1 induced angiogenesis via increased expression of VEGF-C and VEGF-D. As a result, CEACAM1 appeared to be a promising endothelial target for bladder cancer therapy. Dango et al. [11] showed that increased CEACAM1 expression in the tumor cells stimulates angiogenesis and it had an important role in the development and progression of cancer. Our results showed that CEACAM1 was a tumor promoter in gastric adenocarcinoma. Compared with well differentiated carcinoma, higher CEACAM1 expression and CD34-MVD value were observed more frequently in poor differentiated carcinoma. Meanwhile, the patients with lymph node metastasis showed high expression of CEACAM1 and CD34-MVD value. In addition, high expression of CEACAM1 often implied a higher TNM staging, as well as a high CD34-MVD value. On the one hand, high expression of CEACAM1 decreased the adhesion of carcinoma cells to extracellular matrix, and then facilitated the infiltration and metastasis of carcinoma cells. On the other hand, high expression of CEACAM1 stimulated the secretion of vascular growth factors which would promote tumor angiogenesis, and new born blood vessels may facilitate the invasion and metastasis of tumor. In conclusion, the up-regulation of CEACAM1 is an important biological event in the initiation and progression of gastric adenocarcinoma. Also, increased CEACAM1 expression in the tumor cells stimulates angiogenesis. Proliferation of blood vessels may provide nutrients and pathways for tumor cells and improve its ability of invasion and metastasis, thus affecting the biological behavior of gastric adenocarcinoma. CEACAM1 may be a useful predictor
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Please cite this article in press as: J.-F. Shi, et al., Expression of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) and its correlation with angiogenesis in gastric cancer, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.014
