- Email: [email protected]
CCL2 expression correlates with Snail expression and affects the prognosis of patients with gastric cancer
Accepted Manuscript Title: CCL2 expression correlates with Snail expression and affects the prognosis of patients with gastric cancer Author: Jia Zhang Yan Yan Xiaohai Cui Jing Zhang Ya Yang Huajing Li Hongpei Wu Junhai Li Li Wang Min Li Xu Liu Jiansheng Wang Xiaoyi Duan PII: DOI: Reference:
S0344-0338(16)30363-6 http://dx.doi.org/doi:10.1016/j.prp.2016.12.013 PRP 51700
To appear in: Received date:
22-8-2016
Please cite this article as: Jia Zhang, Yan Yan, Xiaohai Cui, Jing Zhang, Ya Yang, Huajing Li, Hongpei Wu, Junhai Li, Li Wang, Min Li, Xu Liu, Jiansheng Wang, Xiaoyi Duan, CCL2 expression correlates with Snail expression and affects the prognosis of patients with gastric cancer, Pathology - Research and Practice http://dx.doi.org/10.1016/j.prp.2016.12.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
CCL2 expression correlates with Snail expression and affects the prognosis of patients with gastric cancer
Jia Zhang
a, 1
, Yan Yan
a, 1
, Xiaohai Cui
a, 1
, Jing Zhang a, Ya Yang a, Huajing Li b,
Hongpei Wu c, Junhai Li c, Li Wang a, Min Li a, Xu Liu a, Jiansheng Wang a, Xiaoyi Duand, *
a
The Second Department of Thoracic Surgery, The First Affiliated Hospital of Xi’an
Jiaotong University, Xi’an, Shaanxi, China b
Department of Otorhinolaryngology, The First Affiliated Hospital of Xi’an Jiaotong
University, Xi’an, Shaanxi, China c
Department of Thoracic Surgery, The Nucleus Industry 215 Hospital, Xianyang,
Shaanxi, China. d
Department of Nuclear Medicine, The First Affiliated Hospital of Xi’an Jiaotong
University, Xi’an, Shaanxi, China 1
These authors have contributed equally to this work
Correspondence to: Professor Xiaoyi Duan, Department of Nuclear Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, 277 West Yanta Road, Xi’an 710061, Shaanxi, China E-mail: [email protected]
Abstract We aim to explore the associations of CCL2 and Snail in gastric cancer to the clinicopathologic features and prognosis of gastric cancer (GC). In our study, the expression of CCL2 and Snail in clinical specimens of 178 GC patients was detected by immunohistochemistry. High expression of CCL2 and Snail were closely related to the clinicopathologic features. The results showed there is a link between CCL2 and Snail expression at protein levels (Pearson Χ2 =40.751, P<0.001). The Kaplan-Meier survival analysis showed that CCL2 or Snail expression was correlated with 5-year survival rate (P<0.001, P<0.001, respectively). Univariate analysis showed that CCL2, Snail, pTNM stage, depth of invasion, nodal involvement, metastasis and tumor diameter were significantly associated with 5-year survival rate respectively. Multivariate Cox analysis showed that the CCL2, Snail and nodal involvement were independent prognostic factor for patients with GC. In conclusion, the expression of CCL2 is significantly correlated with Snail expression and may be used as a predictive co-biomarker for patient prognosis and tumor aggressiveness in GC. The exactly mechanism between CCL2 and Snail in the process of EMT in GC need further investigation.
Keywords Gastric cancer﹒Prognosis﹒CCL2﹒Snail﹒EMT
Introduction Gastric cancer (GC) is one of the most common malignancies worldwide, and is the second leading cause of cancer related death worldwide, second only to lung cancer[24]. Despite of our treatment of GC has improved in the last decade, the prognosis for patients with advanced GC remains poor[25]. Owing to the majority of patients with GC present with metastatic dissemination at the time of diagnosis[28]. Thus, the better understanding the molecular basis of GC, in particular identification of reliable metastasis-associated prognostic markers, may improve the prevention and treatment of GC. Chemokines are a family of small, secreted proteins that have pleiotropic roles in
inflammation-related pathological diseases, including cancer[19]. Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP1) , is a member of the CC chemokine family[18]. Aberrant expression of CCL2 has been reported in a variety of malignancies and is associated with adverse prognosis such as breast cancer, colorectal cancer, prostate cancer, cervix cancer, thyroid cancer, renal cell carcinoma, pancreatic cancer, lung cancer and gastric cancer[4, 10, 11, 14, 20, 21, 26, 29, 31]. CCL2 has been shown to play a critical role in recruiting monocytes/macrophages and promoting their survival[17]. Our previous study has shown high infiltration of tumorassociated macrophages influence poor prognosis in human GC patients and associate with the phenomenon of epithelial-mesenchymal transition (EMT)[30]. EMT is the crucial mechanism by which cancer cells are reprogrammed to possess migratory and tumor-initiating capacity[2, 12]. It is characterized by disruption of apical-basal polarity and loss of E-cadherin expression[22, 23]. The Snail family of transcription factors are core inducers of EMT[16]. Snail is one of the important Snail family members and a zinc-finger transcription regulator[7]. However, the correlation between the CCL2 and EMT in GC is still poorly understood. In the present study, we investigated the correlation of CCL2 and Snail expression and its relevance to clinical parameters in GC patients using immunohistochemistry (IHC). Furthermore, we evaluated the prognostic value of CCL2 and Snail expression in GC patients. Our findings may uncover the clinical significance of the CCL2 and EMT in GC. Materials and Methods Ethics statement This study was approved by the Protection of Human Subjects Committee of First Affiliated Hospital, Xi’an Jiaotong University and complies with the Helsinki declaration. Tissue samples We collected tissues specimens of 178 gastric cancer patients from consecutive
surgical cases in Department of Surgical Oncology, The First Affiliated Hospital, Xi’an Jiaotong University and Department of Surgical Oncology, the 215th Hospital of Shaanxi province between 2004 and 2009. The patients included 125 male and 53 female patients (ranging from 25 to 81 years of age). The p-TNM staging system of the International Union Against Cancer (7th Edition) was used to classify specimens as stages. None of these 178 patients received neoadjuvant or adjuvant chemotherapy before the operation. Immunohistochemical staining The tissues specimens were fixed in neutral buffered formalin and embedded in paraffin wax. The sections of 4-mm thickness were cut and mounted on charged glass slides. Antigen retrieval was performed using citrate buffer at pH 6.0. Immunohistochemical staining was performed with rabbit anti-CCL2 antibody (1:100 dilution) (bs-20201R, Boaosen Int, Beijing, China) and rabbit anti-Snail antibody (1:100 dilution) (bs-1371R, Boaosen Int, Beijing, China). The streptavidin-peroxidase technique (SP-9001 Golden Bridge Int, Beijing, China) was used. An irrelevant rabbit antiserum served as a negative control. The sections were stained with 0.02% diaminobenzidine (DAB) solution followed by counterstaining with hematoxylin. Evaluation of immunohistochemical analysis The staining results of CCL2 and Snail were scored semi-quantitatively by calculating the immunostaining intensity and the percentage of positive malignant cells. The percentage of positive malignant cells was determined in at least 5 areas under 400 × magnifications and averaged. The mean percentage was scored as follows: 0 (0-5%); 1(6-25%); 2(26-50%); 3(51-75%), and 4 (76-100%). Intensity score 0: no staining; 1-weak staining, 2-moderate staining and 3-strong staining. Finally, the staining score was obtained by calculating the product of the staining intensity and the positive cell percentage, where ≤ 5 was defined as low expression and ≥ 6 as high expression. Statistical analysis Statistical analysis was performed using the SPSS software package (Version16.0,
Chicago, IL, USA). Overall survival was defined as the time from the date of surgery to the date of the last follow-up or death from any cause. The Χ2 test was used to analyze the correlation between CCL2 and Snail expression and its relevance to clinical parameters. Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test. For multivariate analysis, the prognostic factors were analyzed using Cox’s proportional hazard model. The results were considered statistically significant if P < 0.05(P< 0.05*, P < 0.01 **). Results Expression of CCL2 and Snail in GC tissues Respective photomicrographs of immunohistochemically staining of CCL2 and Snail are shown in Figure 1. Of the 178 tissues analyzed, 100(56.2 %) were high expression of CCL2, 87 (48.9%) were high expression of Snail, 70 (39.3 %) were high expression of both markers, and 61 (34.3%) were low expression of both markers. Association of CCL2 and Snail expression with clinicopathologic features of tumors As shown in Table 1, High-expression of CCL2 was closely related to the pTNM stage(P<0.001), depth of invasion(P<0.001), nodal involvement(P<0.001), metastasis(P=0.002), and tumor diameter(P=0.001), but not to the patients’ gender(P=0.115), age(P=0.584), the Borrmann types(P=0.197) and differentiation(P=0.109). High-expression of Snail was closely related to the pTNM stage(P<0.001), depth of invasion(P<0.001), nodal involvement(P<0.001), metastasis(P=0.001), the Borrmann types(P=0.016), differentiation(P=0.037) and tumor diameter(P<0.001), but not to the patients’ gender(P=0.053) and age(P=0.074). Association of CCL2 with expression of Snail The relationships between expression of CCL2 and Snail were calculated and have been outlined in Table 2. The result showed that high expression of CCL2 correlated with anomalous positivity of Snail expression (Χ2 = 40.751, P<0.001) in clinical GC samples. Expression of CCL2 and Snail with clinical outcomes
The patients with high CCL2 or Snail expression showed a more unfavorable prognosis than those with low expression (P<0.001) (Fig. 2 and Table 3). According to the univariate analysis (Table 2), CCL2(P<0.001), Snail(P<0.001), pTNM stage(P<0.001), depth of invasion(P=0.002), nodal involvement(P<0.001), metastasis(P<0.001) and tumor diameter(P=0.013) were significantly associated with 5-year OS respectively. Moreover, as seen in Table 4, multivariate Cox analysis showed that the CCL2 (HR 2.279, 95 % CI 1.522-3.411, P<0.001), Snail (HR 2.270, 95 % CI 1.529-3.369, P<0.001) and nodal involvement (HR 1.323, 95 % CI 1.118– 1.566, P=0.001) were independent prognostic factor for patients with GC. Discussion In this study, we demonstrated that patients with high expression of CCL2 or Snail in GC tissue had unfavorable outcomes. CCL2 expression was correlated with Snail expression in patients with GC. CCL2, and Snail were independent prognostic factor for patients with GC. This is the first report to clarify the relationships between CCL2, and Snail in GC. CCL2 was purified and cloned in 1989 from human gliomas and myelomonocytic cells in view of its ability to chemoattract monocytes[13, 27]. The roles of CCL2 have been clarified in the pathogenesis of various diseases that associate with monocyte infiltration, such as rheumatoid arthritis, atherosclerosis, and multiple types of cancer[3]. In GC patients, previous study indicated the increased expression of CCL2 correlate with the clinicopathological characteristics of the patients and associate with poor prognosis[11]. Our previous study has shown high infiltration of tumor-associated macrophages influence poor prognosis in human GC patients and associate with EMT. The essential of CCL2 as a chemokine of monocyte/macrophage chemotaxis made us to assume if there is a correlation between CCL2 and EMT. Snail could directly bind to the E-boxes of E-cadherin gene promoter to repress its transcription, and subsequently convert normal epithelial cells into mesenchymal cell phenotype[1]. Because of this, researches regard it as an ideal target for the study of EMT. In 2008, evidence indicated CCL2 may induce the EMT of renal proximal tubular
epithelial cells in vitro partly by upregulate Snail mRNA expression[9]. CCL2 is involved in peritoneal dialysis-related EMT of peritoneal mesothelial cells, increased expression of Snail has been seen in this process[8]. It has been demonstrated that CCL2 is a critical determinant for both tumor metastasis and immunosuppression induced by Snail (+) tumor cells[6]. Acetylation of snail activates cytokinome including CCL2, which promote the recruitment of tumor-associated macrophages[5]. Nuclear translocation of Snail and enhanced the secretion CCL2 has been shown in EBV infected human corneal epithelial cells[15]. Recently, in MCF-7 human breast carcinoma cells, the result indicates that MCP-1-induced ERK/GSK-3β/Snail signaling facilitate EMT and promotes the migration. However, the correlation between the CCL2, Snail and EMT in GC is still poorly understood. In the present study, we find that high expression of CCL2 and Snail are closely related to the clinicopathologic features. The results showed there is a link between CCL2 and Snail expression at protein levels (Pearson Χ2 =40.751, P<0.001). The Kaplan-Meier survival analysis showed that CCL2 or Snail expression was correlated with 5-year survival rate (P<0.001, P<0.001, respectively). Univariate analysis showed that CCL2, Snail, pTNM stage, depth of invasion, nodal involvement, metastasis and tumor diameter were significantly associated with 5-year survival rate respectively. Multivariate Cox analysis showed that the CCL2, Snail and nodal involvement were independent prognostic factor for patients with GC. In conclusion, CCL2 expression is correlated with Snail expression and affects the prognosis of patients with GC. To the best of our knowledge, this is the first report to clarify the correlation between CCL2 and Snail in GC. The exactly mechanism between CCL2 and Snail in the process of EMT in GC need further investigation. Conflicts of interest None Acknowledgment This work was supported by the Fundamental Research Funds for the Central Universities.
References [1] E. Batlle, E. Sancho, C. Franci, D. Dominguez, M. Monfar, J. Baulida, A. Garcia De Herreros, The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature cell biology 2 (2000) 84-89. [2] B. De Craene, G. Berx, Regulatory networks defining EMT during cancer initiation and progression. Nature reviews. Cancer 13 (2013) 97-110. [3] S.L. Deshmane, S. Kremlev, S. Amini, B.E. Sawaya, Monocyte chemoattractant protein-1 (MCP-1): an overview. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 29 (2009) 313-326. [4] B. Hemmerlein, U. Johanns, A. Kugler, M. Reffelmann, H.J. Radzun, Quantification and in situ localization of MCP-1 mRNA and its relation to the immune response of renal cell carcinoma. Cytokine 13 (2001) 227-233. [5] D.S. Hsu, H.J. Wang, S.K. Tai, C.H. Chou, C.H. Hsieh, P.H. Chiu, N.J. Chen, M.H. Yang, Acetylation of snail modulates the cytokinome of cancer cells to enhance the recruitment of macrophages. Cancer cell 26 (2014) 534-548. [6] C. Kudo-Saito, H. Shirako, M. Ohike, N. Tsukamoto, Y. Kawakami, CCL2 is critical for immunosuppression to promote cancer metastasis. Clinical & experimental metastasis 30 (2013) 393-405. [7] M.Y. Lee, M.R. Shen, Epithelial-mesenchymal transition in cervical carcinoma. American journal of translational research 4 (2012) 1-13. [8] S.H. Lee, H.Y. Kang, K.S. Kim, B.Y. Nam, J. Paeng, S. Kim, J.J. Li, J.T. Park, D.K. Kim, S.H. Han, T.H. Yoo, S.W. Kang, The monocyte chemoattractant protein-1 (MCP-1)/CCR2
system is involved in peritoneal dialysis-related epithelial-mesenchymal transition of peritoneal mesothelial cells. Laboratory investigation; a journal of technical methods and pathology 92 (2012) 1698-1711. [9] H.L. Li, F.L. Zheng, B. Zhao, [Effects of induction of tubular epithelial-myofibroblast transition by monocyte chemoattractant protein-1 and mechanism thereof: an in vitro experiment]. Zhonghua yi xue za zhi 88 (2008) 400-405. [10] X. Li, H.H. Tai, Activation of thromboxane A2 receptor (TP) increases the expression of monocyte chemoattractant protein -1 (MCP-1)/chemokine (C-C motif) ligand 2 (CCL2) and recruits macrophages to promote invasion of lung cancer cells. PloS one 8 (2013) e54073. [11] H. Liu, Z. Shen, X. Wang, H. Zhang, J. Qin, X. Qin, J. Xu, Y. Sun, Increased expression of C-C motif ligand 2 associates with poor prognosis in patients with gastric cancer after gastrectomy. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine (2015). [12] S.A. Mani, W. Guo, M.J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, L.L. Campbell, K. Polyak, C. Brisken, J. Yang, R.A. Weinberg, The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133 (2008) 704-715. [13] K. Matsushima, C.G. Larsen, G.C. DuBois, J.J. Oppenheim, Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line. The Journal of experimental medicine 169 (1989) 1485-1490. [14] P. Monti, B.E. Leone, F. Marchesi, G. Balzano, A. Zerbi, F. Scaltrini, C. Pasquali, G. Calori, F. Pessi, C. Sperti, V. Di Carlo, P. Allavena, L. Piemonti, The CC chemokine MCP-
1/CCL2 in pancreatic cancer progression: regulation of expression and potential mechanisms of antimalignant activity. Cancer research 63 (2003) 7451-7461. [15] G.B. Park, D. Kim, Y.S. Kim, S. Kim, H.K. Lee, J.W. Yang, D.Y. Hur, The Epstein-Barr virus causes epithelial-mesenchymal transition in human corneal epithelial cells via Syk/src and Akt/Erk signaling pathways. Investigative ophthalmology & visual science 55 (2014) 1770-1779. [16] H. Peinado, D. Olmeda, A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nature reviews. Cancer 7 (2007) 415-428. [17] H. Roca, Z.S. Varsos, S. Sud, M.J. Craig, C. Ying, K.J. Pienta, CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization. The Journal of biological chemistry 284 (2009) 34342-34354. [18] B.J. Rollins, A. Walz, M. Baggiolini, Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes. Blood 78 (1991) 1112-1116. [19] E.T. Roussos, J.S. Condeelis, A. Patsialou, Chemotaxis in cancer. Nature reviews. Cancer 11 (2011) 573-587. [20] G. Soria, M. Ofri-Shahak, I. Haas, N. Yaal-Hahoshen, L. Leider-Trejo, T. LeibovichRivkin, P. Weitzenfeld, T. Meshel, E. Shabtai, M. Gutman, A. Ben-Baruch, Inflammatory mediators in breast cancer: coordinated expression of TNFalpha & IL-1beta with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition. BMC cancer 11 (2011) 130. [21] K. Tanaka, J. Kurebayashi, M. Sohda, T. Nomura, U. Prabhakar, L. Yan, H. Sonoo, The expression of monocyte chemotactic protein-1 in papillary thyroid carcinoma is correlated with lymph node metastasis and tumor recurrence. Thyroid : official journal of the American
Thyroid Association 19 (2009) 21-25. [22] J.P. Thiery, H. Acloque, R.Y. Huang, M.A. Nieto, Epithelial-mesenchymal transitions in development and disease. Cell 139 (2009) 871-890. [23] J.P. Thiery, J.P. Sleeman, Complex networks orchestrate epithelial-mesenchymal transitions. Nature reviews. Molecular cell biology 7 (2006) 131-142. [24] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012. CA Cancer J Clin 65 (2015) 87-108. [25] A.D. Wagner, W. Grothe, J. Haerting, G. Kleber, A. Grothey, W.E. Fleig, Chemotherapy in advanced gastric cancer: a systematic review and meta-analysis based on aggregate data. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 24 (2006) 2903-2909. [26] H. Yoshidome, H. Kohno, T. Shida, F. Kimura, H. Shimizu, M. Ohtsuka, Y. Nakatani, M. Miyazaki, Significance of monocyte chemoattractant protein-1 in angiogenesis and survival in colorectal liver metastases. International journal of oncology 34 (2009) 923-930. [27] T. Yoshimura, E.A. Robinson, S. Tanaka, E. Appella, J. Kuratsu, E.J. Leonard, Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants. The Journal of experimental medicine 169 (1989) 1449-1459. [28] G. Yu, J. Wang, Y. Chen, X. Wang, J. Pan, G. Li, Z. Jia, Q. Li, J.C. Yao, K. Xie, Overexpression of phosphorylated mammalian target of rapamycin predicts lymph node metastasis and prognosis of chinese patients with gastric cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 15 (2009) 1821-1829. [29] J. Zhang, L. Patel, K.J. Pienta, CC chemokine ligand 2 (CCL2) promotes prostate cancer
tumorigenesis and metastasis. Cytokine & growth factor reviews 21 (2010) 41-48. [30] J. Zhang, Y. Yan, Y. Yang, L. Wang, M. Li, J. Wang, X. Liu, X. Duan, J. Wang, High Infiltration of Tumor-Associated Macrophages Influences Poor Prognosis in Human Gastric Cancer Patients, Associates With the Phenomenon of EMT. Medicine 95 (2016) e2636. [31] H.J. Zijlmans, G.J. Fleuren, H.J. Baelde, P.H. Eilers, G.G. Kenter, A. Gorter, The absence of CCL2 expression in cervical carcinoma is associated with increased survival and loss of heterozygosity at 17q11.2. The Journal of pathology 208 (2006) 507-517.
Fig.1 Immunohistochemical staining of CCL2 and Snail level in GC tissue. A High CCL2 staining in GC tissue. B High Snail staining in GC tissue. C Low CCL2 staining in GC tissue. D Low Snail staining in GC tissue. Original magnifications, ×400.
Fig.2 Expression of CCL2 and Snail associated predict poor prognosis of GC. A Patients that high expression CCL2 demonstrated shorter OS than those in the low expression group (P <0.001). B Patients that high expression Snail demonstrated shorter OS than those in the low expression group (P <0.001).
Table 1 Correlation of CCL2 and Snail expression with clinicopathologic characteristics of gastric cancer Variables
CCL2 N
high
Snail low
P
high
low
0.115
Gender
0.053
male
125
75
50
67
58
female
53
25
28
20
33
0.584
Age
0.074
≤60
94
51
43
40
54
＞60
84
49
35
47
37
<0.001
TNM
<0.001
Ⅰ
21
6
15
0
21
Ⅱ
34
10
24
5
29
Ⅲ
83
53
30
53
30
Ⅳ
40
31
9
29
11
<0.001
Depth of invasion
<0.001
T1
16
5
11
0
16
T2
18
7
11
1
17
T3
67
30
37
25
42
T4
77
58
19
61
16
<0.001
Nodal involvement
<0.001
N0
49
14
35
7
42
N1
36
25
11
22
14
N2
35
22
13
20
15
N3
58
39
19
38
20
0.002
Metastasis M0
138
69
69
P
0.001 58
80
M1
40
31
29
9
11
0.197
Borrmann type
0.016
Ⅰ
22
8
14
6
16
Ⅱ
55
30
25
22
33
Ⅲ
62
38
24
34
28
Ⅳ
39
24
15
25
14
0.109
Differentiation
0.037
well
12
4
8
3
9
moderate
57
29
28
23
34
poor
109
67
42
61
48
0.001
Tumor diameter
<0.001
＜3cm
44
14
30
7
37
3cm-5cm
54
36
18
32
22
＞5cm
80
50
30
48
32
Table 2 The association between CCL2 and Snail CCL2
Pearson Χ2
high
low
high
70
17
low
30
61
P
Snail 40.751
<0.001
Table 3 Univariate Analysis for Overall Survival UNIVARIATE Variables
HR
95%CI
P
CCL2
3.309
2.273-4.817
<0.001
Snail
3.586
2.497-5.150
<0.001
Gender
1.070
0.735-1.557
0.725
Age
1.279
0.906-1.806
0.161
TNM
1.751
1.415-2.168
<0.001
Depth of invasion
1.452
1.184-1.782
<0.001
Nodal involvement
1.469
1.263-1.708
<0.001
Metastasis
2.594
1.761-3.820
<0.001
Borrmann type
1.167
0.971-1.403
0.100
Differentiation
1.350
1.009-1.807
0.043
Tumor diameter
1.369
1.101-1.702
0.005
Table 4 Multivariate Cox Proportional Hazards Analysis for Overall Survival Overall survival Variables
HR
95%CI
P
CCL2
2.279
1.522-3.411
<0.001
Snail
2.270
1.529-3.369
<0.001
Nodal involvement
1.323
1.118-1.566
0.001
S0344-0338(16)30363-6 http://dx.doi.org/doi:10.1016/j.prp.2016.12.013 PRP 51700
To appear in: Received date:
22-8-2016
Please cite this article as: Jia Zhang, Yan Yan, Xiaohai Cui, Jing Zhang, Ya Yang, Huajing Li, Hongpei Wu, Junhai Li, Li Wang, Min Li, Xu Liu, Jiansheng Wang, Xiaoyi Duan, CCL2 expression correlates with Snail expression and affects the prognosis of patients with gastric cancer, Pathology - Research and Practice http://dx.doi.org/10.1016/j.prp.2016.12.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
CCL2 expression correlates with Snail expression and affects the prognosis of patients with gastric cancer
Jia Zhang
a, 1
, Yan Yan
a, 1
, Xiaohai Cui
a, 1
, Jing Zhang a, Ya Yang a, Huajing Li b,
Hongpei Wu c, Junhai Li c, Li Wang a, Min Li a, Xu Liu a, Jiansheng Wang a, Xiaoyi Duand, *
a
The Second Department of Thoracic Surgery, The First Affiliated Hospital of Xi’an
Jiaotong University, Xi’an, Shaanxi, China b
Department of Otorhinolaryngology, The First Affiliated Hospital of Xi’an Jiaotong
University, Xi’an, Shaanxi, China c
Department of Thoracic Surgery, The Nucleus Industry 215 Hospital, Xianyang,
Shaanxi, China. d
Department of Nuclear Medicine, The First Affiliated Hospital of Xi’an Jiaotong
University, Xi’an, Shaanxi, China 1
These authors have contributed equally to this work
Correspondence to: Professor Xiaoyi Duan, Department of Nuclear Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, 277 West Yanta Road, Xi’an 710061, Shaanxi, China E-mail: [email protected]
Abstract We aim to explore the associations of CCL2 and Snail in gastric cancer to the clinicopathologic features and prognosis of gastric cancer (GC). In our study, the expression of CCL2 and Snail in clinical specimens of 178 GC patients was detected by immunohistochemistry. High expression of CCL2 and Snail were closely related to the clinicopathologic features. The results showed there is a link between CCL2 and Snail expression at protein levels (Pearson Χ2 =40.751, P<0.001). The Kaplan-Meier survival analysis showed that CCL2 or Snail expression was correlated with 5-year survival rate (P<0.001, P<0.001, respectively). Univariate analysis showed that CCL2, Snail, pTNM stage, depth of invasion, nodal involvement, metastasis and tumor diameter were significantly associated with 5-year survival rate respectively. Multivariate Cox analysis showed that the CCL2, Snail and nodal involvement were independent prognostic factor for patients with GC. In conclusion, the expression of CCL2 is significantly correlated with Snail expression and may be used as a predictive co-biomarker for patient prognosis and tumor aggressiveness in GC. The exactly mechanism between CCL2 and Snail in the process of EMT in GC need further investigation.
Keywords Gastric cancer﹒Prognosis﹒CCL2﹒Snail﹒EMT
Introduction Gastric cancer (GC) is one of the most common malignancies worldwide, and is the second leading cause of cancer related death worldwide, second only to lung cancer[24]. Despite of our treatment of GC has improved in the last decade, the prognosis for patients with advanced GC remains poor[25]. Owing to the majority of patients with GC present with metastatic dissemination at the time of diagnosis[28]. Thus, the better understanding the molecular basis of GC, in particular identification of reliable metastasis-associated prognostic markers, may improve the prevention and treatment of GC. Chemokines are a family of small, secreted proteins that have pleiotropic roles in
inflammation-related pathological diseases, including cancer[19]. Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP1) , is a member of the CC chemokine family[18]. Aberrant expression of CCL2 has been reported in a variety of malignancies and is associated with adverse prognosis such as breast cancer, colorectal cancer, prostate cancer, cervix cancer, thyroid cancer, renal cell carcinoma, pancreatic cancer, lung cancer and gastric cancer[4, 10, 11, 14, 20, 21, 26, 29, 31]. CCL2 has been shown to play a critical role in recruiting monocytes/macrophages and promoting their survival[17]. Our previous study has shown high infiltration of tumorassociated macrophages influence poor prognosis in human GC patients and associate with the phenomenon of epithelial-mesenchymal transition (EMT)[30]. EMT is the crucial mechanism by which cancer cells are reprogrammed to possess migratory and tumor-initiating capacity[2, 12]. It is characterized by disruption of apical-basal polarity and loss of E-cadherin expression[22, 23]. The Snail family of transcription factors are core inducers of EMT[16]. Snail is one of the important Snail family members and a zinc-finger transcription regulator[7]. However, the correlation between the CCL2 and EMT in GC is still poorly understood. In the present study, we investigated the correlation of CCL2 and Snail expression and its relevance to clinical parameters in GC patients using immunohistochemistry (IHC). Furthermore, we evaluated the prognostic value of CCL2 and Snail expression in GC patients. Our findings may uncover the clinical significance of the CCL2 and EMT in GC. Materials and Methods Ethics statement This study was approved by the Protection of Human Subjects Committee of First Affiliated Hospital, Xi’an Jiaotong University and complies with the Helsinki declaration. Tissue samples We collected tissues specimens of 178 gastric cancer patients from consecutive
surgical cases in Department of Surgical Oncology, The First Affiliated Hospital, Xi’an Jiaotong University and Department of Surgical Oncology, the 215th Hospital of Shaanxi province between 2004 and 2009. The patients included 125 male and 53 female patients (ranging from 25 to 81 years of age). The p-TNM staging system of the International Union Against Cancer (7th Edition) was used to classify specimens as stages. None of these 178 patients received neoadjuvant or adjuvant chemotherapy before the operation. Immunohistochemical staining The tissues specimens were fixed in neutral buffered formalin and embedded in paraffin wax. The sections of 4-mm thickness were cut and mounted on charged glass slides. Antigen retrieval was performed using citrate buffer at pH 6.0. Immunohistochemical staining was performed with rabbit anti-CCL2 antibody (1:100 dilution) (bs-20201R, Boaosen Int, Beijing, China) and rabbit anti-Snail antibody (1:100 dilution) (bs-1371R, Boaosen Int, Beijing, China). The streptavidin-peroxidase technique (SP-9001 Golden Bridge Int, Beijing, China) was used. An irrelevant rabbit antiserum served as a negative control. The sections were stained with 0.02% diaminobenzidine (DAB) solution followed by counterstaining with hematoxylin. Evaluation of immunohistochemical analysis The staining results of CCL2 and Snail were scored semi-quantitatively by calculating the immunostaining intensity and the percentage of positive malignant cells. The percentage of positive malignant cells was determined in at least 5 areas under 400 × magnifications and averaged. The mean percentage was scored as follows: 0 (0-5%); 1(6-25%); 2(26-50%); 3(51-75%), and 4 (76-100%). Intensity score 0: no staining; 1-weak staining, 2-moderate staining and 3-strong staining. Finally, the staining score was obtained by calculating the product of the staining intensity and the positive cell percentage, where ≤ 5 was defined as low expression and ≥ 6 as high expression. Statistical analysis Statistical analysis was performed using the SPSS software package (Version16.0,
Chicago, IL, USA). Overall survival was defined as the time from the date of surgery to the date of the last follow-up or death from any cause. The Χ2 test was used to analyze the correlation between CCL2 and Snail expression and its relevance to clinical parameters. Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test. For multivariate analysis, the prognostic factors were analyzed using Cox’s proportional hazard model. The results were considered statistically significant if P < 0.05(P< 0.05*, P < 0.01 **). Results Expression of CCL2 and Snail in GC tissues Respective photomicrographs of immunohistochemically staining of CCL2 and Snail are shown in Figure 1. Of the 178 tissues analyzed, 100(56.2 %) were high expression of CCL2, 87 (48.9%) were high expression of Snail, 70 (39.3 %) were high expression of both markers, and 61 (34.3%) were low expression of both markers. Association of CCL2 and Snail expression with clinicopathologic features of tumors As shown in Table 1, High-expression of CCL2 was closely related to the pTNM stage(P<0.001), depth of invasion(P<0.001), nodal involvement(P<0.001), metastasis(P=0.002), and tumor diameter(P=0.001), but not to the patients’ gender(P=0.115), age(P=0.584), the Borrmann types(P=0.197) and differentiation(P=0.109). High-expression of Snail was closely related to the pTNM stage(P<0.001), depth of invasion(P<0.001), nodal involvement(P<0.001), metastasis(P=0.001), the Borrmann types(P=0.016), differentiation(P=0.037) and tumor diameter(P<0.001), but not to the patients’ gender(P=0.053) and age(P=0.074). Association of CCL2 with expression of Snail The relationships between expression of CCL2 and Snail were calculated and have been outlined in Table 2. The result showed that high expression of CCL2 correlated with anomalous positivity of Snail expression (Χ2 = 40.751, P<0.001) in clinical GC samples. Expression of CCL2 and Snail with clinical outcomes
The patients with high CCL2 or Snail expression showed a more unfavorable prognosis than those with low expression (P<0.001) (Fig. 2 and Table 3). According to the univariate analysis (Table 2), CCL2(P<0.001), Snail(P<0.001), pTNM stage(P<0.001), depth of invasion(P=0.002), nodal involvement(P<0.001), metastasis(P<0.001) and tumor diameter(P=0.013) were significantly associated with 5-year OS respectively. Moreover, as seen in Table 4, multivariate Cox analysis showed that the CCL2 (HR 2.279, 95 % CI 1.522-3.411, P<0.001), Snail (HR 2.270, 95 % CI 1.529-3.369, P<0.001) and nodal involvement (HR 1.323, 95 % CI 1.118– 1.566, P=0.001) were independent prognostic factor for patients with GC. Discussion In this study, we demonstrated that patients with high expression of CCL2 or Snail in GC tissue had unfavorable outcomes. CCL2 expression was correlated with Snail expression in patients with GC. CCL2, and Snail were independent prognostic factor for patients with GC. This is the first report to clarify the relationships between CCL2, and Snail in GC. CCL2 was purified and cloned in 1989 from human gliomas and myelomonocytic cells in view of its ability to chemoattract monocytes[13, 27]. The roles of CCL2 have been clarified in the pathogenesis of various diseases that associate with monocyte infiltration, such as rheumatoid arthritis, atherosclerosis, and multiple types of cancer[3]. In GC patients, previous study indicated the increased expression of CCL2 correlate with the clinicopathological characteristics of the patients and associate with poor prognosis[11]. Our previous study has shown high infiltration of tumor-associated macrophages influence poor prognosis in human GC patients and associate with EMT. The essential of CCL2 as a chemokine of monocyte/macrophage chemotaxis made us to assume if there is a correlation between CCL2 and EMT. Snail could directly bind to the E-boxes of E-cadherin gene promoter to repress its transcription, and subsequently convert normal epithelial cells into mesenchymal cell phenotype[1]. Because of this, researches regard it as an ideal target for the study of EMT. In 2008, evidence indicated CCL2 may induce the EMT of renal proximal tubular
epithelial cells in vitro partly by upregulate Snail mRNA expression[9]. CCL2 is involved in peritoneal dialysis-related EMT of peritoneal mesothelial cells, increased expression of Snail has been seen in this process[8]. It has been demonstrated that CCL2 is a critical determinant for both tumor metastasis and immunosuppression induced by Snail (+) tumor cells[6]. Acetylation of snail activates cytokinome including CCL2, which promote the recruitment of tumor-associated macrophages[5]. Nuclear translocation of Snail and enhanced the secretion CCL2 has been shown in EBV infected human corneal epithelial cells[15]. Recently, in MCF-7 human breast carcinoma cells, the result indicates that MCP-1-induced ERK/GSK-3β/Snail signaling facilitate EMT and promotes the migration. However, the correlation between the CCL2, Snail and EMT in GC is still poorly understood. In the present study, we find that high expression of CCL2 and Snail are closely related to the clinicopathologic features. The results showed there is a link between CCL2 and Snail expression at protein levels (Pearson Χ2 =40.751, P<0.001). The Kaplan-Meier survival analysis showed that CCL2 or Snail expression was correlated with 5-year survival rate (P<0.001, P<0.001, respectively). Univariate analysis showed that CCL2, Snail, pTNM stage, depth of invasion, nodal involvement, metastasis and tumor diameter were significantly associated with 5-year survival rate respectively. Multivariate Cox analysis showed that the CCL2, Snail and nodal involvement were independent prognostic factor for patients with GC. In conclusion, CCL2 expression is correlated with Snail expression and affects the prognosis of patients with GC. To the best of our knowledge, this is the first report to clarify the correlation between CCL2 and Snail in GC. The exactly mechanism between CCL2 and Snail in the process of EMT in GC need further investigation. Conflicts of interest None Acknowledgment This work was supported by the Fundamental Research Funds for the Central Universities.
References [1] E. Batlle, E. Sancho, C. Franci, D. Dominguez, M. Monfar, J. Baulida, A. Garcia De Herreros, The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature cell biology 2 (2000) 84-89. [2] B. De Craene, G. Berx, Regulatory networks defining EMT during cancer initiation and progression. Nature reviews. Cancer 13 (2013) 97-110. [3] S.L. Deshmane, S. Kremlev, S. Amini, B.E. Sawaya, Monocyte chemoattractant protein-1 (MCP-1): an overview. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 29 (2009) 313-326. [4] B. Hemmerlein, U. Johanns, A. Kugler, M. Reffelmann, H.J. Radzun, Quantification and in situ localization of MCP-1 mRNA and its relation to the immune response of renal cell carcinoma. Cytokine 13 (2001) 227-233. [5] D.S. Hsu, H.J. Wang, S.K. Tai, C.H. Chou, C.H. Hsieh, P.H. Chiu, N.J. Chen, M.H. Yang, Acetylation of snail modulates the cytokinome of cancer cells to enhance the recruitment of macrophages. Cancer cell 26 (2014) 534-548. [6] C. Kudo-Saito, H. Shirako, M. Ohike, N. Tsukamoto, Y. Kawakami, CCL2 is critical for immunosuppression to promote cancer metastasis. Clinical & experimental metastasis 30 (2013) 393-405. [7] M.Y. Lee, M.R. Shen, Epithelial-mesenchymal transition in cervical carcinoma. American journal of translational research 4 (2012) 1-13. [8] S.H. Lee, H.Y. Kang, K.S. Kim, B.Y. Nam, J. Paeng, S. Kim, J.J. Li, J.T. Park, D.K. Kim, S.H. Han, T.H. Yoo, S.W. Kang, The monocyte chemoattractant protein-1 (MCP-1)/CCR2
system is involved in peritoneal dialysis-related epithelial-mesenchymal transition of peritoneal mesothelial cells. Laboratory investigation; a journal of technical methods and pathology 92 (2012) 1698-1711. [9] H.L. Li, F.L. Zheng, B. Zhao, [Effects of induction of tubular epithelial-myofibroblast transition by monocyte chemoattractant protein-1 and mechanism thereof: an in vitro experiment]. Zhonghua yi xue za zhi 88 (2008) 400-405. [10] X. Li, H.H. Tai, Activation of thromboxane A2 receptor (TP) increases the expression of monocyte chemoattractant protein -1 (MCP-1)/chemokine (C-C motif) ligand 2 (CCL2) and recruits macrophages to promote invasion of lung cancer cells. PloS one 8 (2013) e54073. [11] H. Liu, Z. Shen, X. Wang, H. Zhang, J. Qin, X. Qin, J. Xu, Y. Sun, Increased expression of C-C motif ligand 2 associates with poor prognosis in patients with gastric cancer after gastrectomy. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine (2015). [12] S.A. Mani, W. Guo, M.J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, L.L. Campbell, K. Polyak, C. Brisken, J. Yang, R.A. Weinberg, The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133 (2008) 704-715. [13] K. Matsushima, C.G. Larsen, G.C. DuBois, J.J. Oppenheim, Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line. The Journal of experimental medicine 169 (1989) 1485-1490. [14] P. Monti, B.E. Leone, F. Marchesi, G. Balzano, A. Zerbi, F. Scaltrini, C. Pasquali, G. Calori, F. Pessi, C. Sperti, V. Di Carlo, P. Allavena, L. Piemonti, The CC chemokine MCP-
1/CCL2 in pancreatic cancer progression: regulation of expression and potential mechanisms of antimalignant activity. Cancer research 63 (2003) 7451-7461. [15] G.B. Park, D. Kim, Y.S. Kim, S. Kim, H.K. Lee, J.W. Yang, D.Y. Hur, The Epstein-Barr virus causes epithelial-mesenchymal transition in human corneal epithelial cells via Syk/src and Akt/Erk signaling pathways. Investigative ophthalmology & visual science 55 (2014) 1770-1779. [16] H. Peinado, D. Olmeda, A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nature reviews. Cancer 7 (2007) 415-428. [17] H. Roca, Z.S. Varsos, S. Sud, M.J. Craig, C. Ying, K.J. Pienta, CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization. The Journal of biological chemistry 284 (2009) 34342-34354. [18] B.J. Rollins, A. Walz, M. Baggiolini, Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes. Blood 78 (1991) 1112-1116. [19] E.T. Roussos, J.S. Condeelis, A. Patsialou, Chemotaxis in cancer. Nature reviews. Cancer 11 (2011) 573-587. [20] G. Soria, M. Ofri-Shahak, I. Haas, N. Yaal-Hahoshen, L. Leider-Trejo, T. LeibovichRivkin, P. Weitzenfeld, T. Meshel, E. Shabtai, M. Gutman, A. Ben-Baruch, Inflammatory mediators in breast cancer: coordinated expression of TNFalpha & IL-1beta with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition. BMC cancer 11 (2011) 130. [21] K. Tanaka, J. Kurebayashi, M. Sohda, T. Nomura, U. Prabhakar, L. Yan, H. Sonoo, The expression of monocyte chemotactic protein-1 in papillary thyroid carcinoma is correlated with lymph node metastasis and tumor recurrence. Thyroid : official journal of the American
Thyroid Association 19 (2009) 21-25. [22] J.P. Thiery, H. Acloque, R.Y. Huang, M.A. Nieto, Epithelial-mesenchymal transitions in development and disease. Cell 139 (2009) 871-890. [23] J.P. Thiery, J.P. Sleeman, Complex networks orchestrate epithelial-mesenchymal transitions. Nature reviews. Molecular cell biology 7 (2006) 131-142. [24] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012. CA Cancer J Clin 65 (2015) 87-108. [25] A.D. Wagner, W. Grothe, J. Haerting, G. Kleber, A. Grothey, W.E. Fleig, Chemotherapy in advanced gastric cancer: a systematic review and meta-analysis based on aggregate data. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 24 (2006) 2903-2909. [26] H. Yoshidome, H. Kohno, T. Shida, F. Kimura, H. Shimizu, M. Ohtsuka, Y. Nakatani, M. Miyazaki, Significance of monocyte chemoattractant protein-1 in angiogenesis and survival in colorectal liver metastases. International journal of oncology 34 (2009) 923-930. [27] T. Yoshimura, E.A. Robinson, S. Tanaka, E. Appella, J. Kuratsu, E.J. Leonard, Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants. The Journal of experimental medicine 169 (1989) 1449-1459. [28] G. Yu, J. Wang, Y. Chen, X. Wang, J. Pan, G. Li, Z. Jia, Q. Li, J.C. Yao, K. Xie, Overexpression of phosphorylated mammalian target of rapamycin predicts lymph node metastasis and prognosis of chinese patients with gastric cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 15 (2009) 1821-1829. [29] J. Zhang, L. Patel, K.J. Pienta, CC chemokine ligand 2 (CCL2) promotes prostate cancer
tumorigenesis and metastasis. Cytokine & growth factor reviews 21 (2010) 41-48. [30] J. Zhang, Y. Yan, Y. Yang, L. Wang, M. Li, J. Wang, X. Liu, X. Duan, J. Wang, High Infiltration of Tumor-Associated Macrophages Influences Poor Prognosis in Human Gastric Cancer Patients, Associates With the Phenomenon of EMT. Medicine 95 (2016) e2636. [31] H.J. Zijlmans, G.J. Fleuren, H.J. Baelde, P.H. Eilers, G.G. Kenter, A. Gorter, The absence of CCL2 expression in cervical carcinoma is associated with increased survival and loss of heterozygosity at 17q11.2. The Journal of pathology 208 (2006) 507-517.
Fig.1 Immunohistochemical staining of CCL2 and Snail level in GC tissue. A High CCL2 staining in GC tissue. B High Snail staining in GC tissue. C Low CCL2 staining in GC tissue. D Low Snail staining in GC tissue. Original magnifications, ×400.
Fig.2 Expression of CCL2 and Snail associated predict poor prognosis of GC. A Patients that high expression CCL2 demonstrated shorter OS than those in the low expression group (P <0.001). B Patients that high expression Snail demonstrated shorter OS than those in the low expression group (P <0.001).
Table 1 Correlation of CCL2 and Snail expression with clinicopathologic characteristics of gastric cancer Variables
CCL2 N
high
Snail low
P
high
low
0.115
Gender
0.053
male
125
75
50
67
58
female
53
25
28
20
33
0.584
Age
0.074
≤60
94
51
43
40
54
＞60
84
49
35
47
37
<0.001
TNM
<0.001
Ⅰ
21
6
15
0
21
Ⅱ
34
10
24
5
29
Ⅲ
83
53
30
53
30
Ⅳ
40
31
9
29
11
<0.001
Depth of invasion
<0.001
T1
16
5
11
0
16
T2
18
7
11
1
17
T3
67
30
37
25
42
T4
77
58
19
61
16
<0.001
Nodal involvement
<0.001
N0
49
14
35
7
42
N1
36
25
11
22
14
N2
35
22
13
20
15
N3
58
39
19
38
20
0.002
Metastasis M0
138
69
69
P
0.001 58
80
M1
40
31
29
9
11
0.197
Borrmann type
0.016
Ⅰ
22
8
14
6
16
Ⅱ
55
30
25
22
33
Ⅲ
62
38
24
34
28
Ⅳ
39
24
15
25
14
0.109
Differentiation
0.037
well
12
4
8
3
9
moderate
57
29
28
23
34
poor
109
67
42
61
48
0.001
Tumor diameter
<0.001
＜3cm
44
14
30
7
37
3cm-5cm
54
36
18
32
22
＞5cm
80
50
30
48
32
Table 2 The association between CCL2 and Snail CCL2
Pearson Χ2
high
low
high
70
17
low
30
61
P
Snail 40.751
<0.001
Table 3 Univariate Analysis for Overall Survival UNIVARIATE Variables
HR
95%CI
P
CCL2
3.309
2.273-4.817
<0.001
Snail
3.586
2.497-5.150
<0.001
Gender
1.070
0.735-1.557
0.725
Age
1.279
0.906-1.806
0.161
TNM
1.751
1.415-2.168
<0.001
Depth of invasion
1.452
1.184-1.782
<0.001
Nodal involvement
1.469
1.263-1.708
<0.001
Metastasis
2.594
1.761-3.820
<0.001
Borrmann type
1.167
0.971-1.403
0.100
Differentiation
1.350
1.009-1.807
0.043
Tumor diameter
1.369
1.101-1.702
0.005
Table 4 Multivariate Cox Proportional Hazards Analysis for Overall Survival Overall survival Variables
HR
95%CI
P
CCL2
2.279
1.522-3.411
<0.001
Snail
2.270
1.529-3.369
<0.001
Nodal involvement
1.323
1.118-1.566
0.001