Prognostic significance of adhesion molecules (sICAM-1, sVCAM-1) and VEGF in colorectal cancer patients

Thrombosis Research 129 (2012) e47–e50

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Regular Article

Prognostic significance of adhesion molecules (sICAM-1, sVCAM-1) and VEGF in colorectal cancer patients Violetta Dymicka-Piekarska a,⁎, Katarzyna Guzinska-Ustymowicz b, Adam Kuklinski c, Halina Kemona a a b c

Department of Clinical Laboratory Diagnostics, Medical University of Bialystok, Poland Department of General Pathomorphology, Medical University of Bialystok, Poland Department of General and Gastroenterological Surgery, Medical University of Bialystok, Poland

a r t i c l e

i n f o

Article history: Received 16 September 2011 Received in revised form 9 November 2011 Accepted 1 December 2011 Available online 31 December 2011 Keywords: adhesion molecules angiogenesis colorectal cancer

a b s t r a c t Introduction: Adhesion molecules take part in the interaction between host cells and cancer cells. In the current study the relationship between the soluble adhesion molecules sICAM-1 and sVCAM-1 and proangiogenic factor VEGF in colorectal cancer progression were measured. Materials and methods: The study group consisted of 46 patients with colorectal carcinoma (classified due to TNM classification) and 40 controls. sICAM-1, sVCAM-1 and VEGF plasma concentration were measured by enzyme-linked immunosorbent assay (ELISA). Results: All measured parameters levels were increased significantly in patients with colorectal cancer in comparison to controls (p b 0.001). sICAM-1, sVCAM-1 and VEGF increased significantly due to colorectal cancer progression. There was a positive correlation between sICAM-1 and sVCAM-1 in all study groups. Conclusions: Our results demonstrated in CRC patients significantly increased levels of soluble adhesion molecules (VCAM-1 and sICAM-1) and angiogenic factor (VEGF) as compared to control group. The dynamics of these molecules showed the growing tendency along with tumor size and metastasis formation. © 2011 Elsevier Ltd. All rights reserved.

Introduction Tumor growth and development of metastases are a multi-stage process that occurs with the involvement of various cells, e.g. endothelial cells, leukocytes or thrombocytes. Upon the cell activation, adhesion molecules and ligands appear on their surface, enabling a direct contact with cancer cells. On the other hand, these cells are a source of numerous chemokines, cytokines and growth factors promoting tumor growth. Interactions between host cells and cancer cells involve active participation of cell adhesion molecules (CAM) [1–3,7]. They allow mutual adherence and contact of cells with e.g. extracellular cell matrix (ECM). The CAM family includes immunoglobulin-like molecules intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) [3]. ICAM-1 occurs on the surface of leukocytes or endothelial cells [3]. The expression of ICAM and VCAM is regulated by various cytokines, such as TNF-α, INF-γ, IL-2 and IL-6, which may

Abbreviations: CAM, cell adhesion molecules; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; CRC, colorectal cancer. ⁎ Corresponding author at: Department of Clinical Laboratory Diagnostics, Medical University of Bialystok, Waszyngtona 15A, 15–276 Bialystok, Poland. Tel./Fax: + 48 85 745 85 84. E-mail address: [email protected] (V. Dymicka-Piekarska). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.12.004

be beneficial for anticancer response [4]. VCAM is an adhesion molecule on the vascular endothelium, supporting leukocyte adhesion to vascular endothelial cells, which is indispensable for their migration outside the vascular system [5]. Adhesion molecules are shed from the cell surface to the blood, where they can be determined as soluble molecules (sICAM-1, sVCAM-1) [6]. Their elevated plasma levels have been observed in inflammations, infections and many neoplasms, including gastric, pancreatic, prostate, colon and other cancers [3,6–8]. Tumor development is possible due to formation of new blood vessels. VEGF (vascular endothelial growth factor) is one of the major factor that stimulates angiogenesis and its concentration could be a prognostic marker in solid tumors [9,10]. VEGF expression has been described in a large variety of human malignancies e.g. lung, breast, colon [11–13]. Some studies suggested that expression of VEGF correlates with poor prognosis and metastasis [13–15]. Furthermore, also soluble forms of adhesion molecules sE-selectin, sICAM and sVCAM were shown to possess angiogenic activity [16]. In recent years, the involvement of adhesion molecules in tumor development and formation of metastatic foci has become an object of research. Scientists have been trying to determine the significance of their expression in early diagnostics, in the assessment of cancer stage and metastatic development. Their role in local tumor growth, in the formation of distant metastases and involvement in angiogenesis has been investigated. The objective of the current study was to assess the relationship between the adhesion molecules ICAM-1, VCAM-1 and the main

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Table 1 Clinical and demographic features of CRC patients.

Age (years) Sex (male/female) Primary site Colon Rectum Grade G2 G3

group 1

group 2

group 3

(n = 22)

(n = 14)

(n = 10)

68.8 14/8

66.3 6/8

64.6 6/4

Assays

16 6

10 4

7 3

20 2

11 3

8 2

Venous blood was collected to S-Monovette sample tubes (Saerstedt) from the ulnar vein without stasis, in the morning one day before surgery. In the control group, the blood was collected once in the same conditions as in the study group. The levels of soluble adhesion molecules (CAMs) and VEGF were determined in plasma using the immunoenzymatic method. Venous blood samples (3 ml) were collected to test-tubes containing anticoagulant (sodium citrate). Within 30 min. after collection, the blood was centrifuged at 1000 rpm for 15 min and plasma samples were stored at −70 C until analysis. Before the assay, the samples were slowly thawed and centrifuged again. The following kits were used to determine the levels of the investigated parameters: sICAM-1: sHuman soluble ICAM-1/CD54 Immunoassay, sVCAM-1: Quantikine Human sVCAM-1 Immunoassay and VEGF: Quantikine Human VEGF Immunoassay, R&D Systems (Abingdon, UK). The assays were performed according to the manufacturer's instructions. The samples were examined in duplicates and no statistically significant differences were found between the assays.

Table 2 Mean values ± SD of sICAM-1, VCAM-1 and VEGF in the study patients and controls. Parameters

sICAM-1 sVCAM-1 VEGF

The patients enrolled in the study gave written informed consent for participation. The study was approved by the Ethical Committee, Medical University of Bialystok according to the Guidelines for Good Clinical Practice.

CRC group

Control group

(n = 46)

(n = 40)

228.02 ± 52.59 524.43 ± 162.49 501.69 ± 60.07

201.69 ± 24.69 435.92 ± 58.05 83.93 ± 49.50

p

0.02 0.03 0.001

CRC-colorectal cancer.

proangiogenic factor VEGF, and their involvement in colorectal cancer progression. The levels of sICAM-1, sVCAM-1 and VEGF were evaluated in colorectal cancer patients depending on clinical advancement according to TNM, i.e. tumor size and the presence or lack of metastases to lymph nodes and liver as compared to healthy subjects matched by age and sex. Materials and Methods Patients The study involved 46 patients (20 women and 26 men; mean age 66 years) with histopathologically diagnosed primary colorectal carcinoma (Adenocarcinoma) and 40 healthy subjects - control group (15 women and 24 men, mean age 64 years) without any systemic disease. The patients were diagnosed and treated in II Department of General and Gastroenterological Surgery, University Hospital in Bialystok. The diagnosis of colorectal cancer (CRC) was based on clinical symptoms and imaging investigations (ultrasonography, radiology and computed tomography), as well as endoscopy (colonoscopy). None of the patients recruited to the study received chemotherapy or radiotherapy. All of them were waiting for tumor resection. The patients with colorectal cancer were divided into groups depending on clinical advancement according to TNM classification: group 1 (22 patients, stage I and II; T1-T2N0M0), group 2 (14 patients, stage III - lymph node involvement; TxN + M0), group 3 (10 patients, stage IV - distant metastases to the liver; TxNxM+).

Statistical analysis The results were subjected to statistical analysis, in which the arithmetic mean and standard deviation were calculated. The Shapiro-Wilk test was used for normally distributed data, the t-Student test to make a comparison between two groups and the univariate analysis of variance with Bonferroni post-hoc test to compare the data between multiple groups. The Mann–Whitney test and Kruskal-Wallis test with Dunn's analysis of contrasts were applied for features inconsistent with this distribution. Correlation coefficients were calculated depending on the Pearson's or Spearman's distribution. The ROC curves were also analyzed. The differences were considered statistically significant for p b 0.05. The calculations were performed using the statistical package SPSS and NCSS. Results Table 1 presents demographic and clinical data of patients. Among the colorectal cancer patients there were more men than women (56.5% and 43.5%, respectively). The means ± SD of the study parameters in both groups are presented in Table 2. The mean levels of sICAM-1 and sVCAM-1 in the entire CRC group were 228.02 ± 52.59 and 524.43± 162.49 ng/ml, respectively. The differences between the groups were statistically significant (pb 0.02 and

Table 3 Plasma concentrations of soluble adhesion molecules (sICAM-1 and sVCAM-1) and VEGF in controls and in CRC patients according to TNM classification. Parameters

stage I/II

stage III

stage IV

Controls

(n = 22 )

(n = 14 )

(n = 10)

(n = 40)

sICAM-1

213,86 ± 49,38

216,14 ± 29,10

275,80 ± 60,5*

201,69 ± 24,69

sVCAM-1

455,06 ± 147,67

567,69 ± 165,77*

612,12 ± 133,74**

435,92 ± 58,05

VEGF

423,79 ± 203,71**

474,74 ± 145,28**

692,51 ± 198,53**

83,93 ± 49,50

*p b 0.05 vs K, ** p b 0.001 vs K.

p

I/II vs. IV 0.003 III vs. IV 0.01 I/II vs. III 0.04 I/II vs. IV 0.009 NS

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1,0

900

Source of the Curve ICAM VCAM VEGF Reference Line

r=0,309 p=0,037

800 0,8

Sensitivity

sVCAM

700

600

500

400

0,6

0,4

0,2

300 0,0 200 120

0,0 140

160

180

200

220

240

260

280

300

320

340

360

0,2

0,4

0,6

0,8

1,0

1 - Specificity

sICAM Fig. 1. Correlation between sICAM-1 and sVCAM-1 in all colorectal cancer patients.

p b 0.03). The level of VEGF was statistically significantly higher in the group of patients as compared to the control subjects (501.69± 212.21 vs. 83.93 ± 49.50 ng/ml; p b 0.001). Table 3 shows the values of the study parameters according to the CRC stage. The mean level of sICAM-1 in stage IV patients was statistically significantly higher than in patients with stage I /II or III (p b 0.003 i p b 0.01) and controls (p b 0.001). Also the level of sVCAM-1 was found to correlate with clinical advancement, and the differences between stage IV and stage I/II patients as well as between stage III and stage I / II patients were highly statistically significant (p b 0.001; p b 0.02, respectively). Statistically significant differences in the level of sVCAM-1 were also noted between patients with stage III/IV and control subjects (p b 0.04 and p b 0.009). However, no statistically significant differences were observed in the level of VEGF depending on TNM classification, although such correlations were found compared to the control group (p b 0.001) (Table 3). The statistical analysis showed a poor correlation between the level of sICAM-1 and sVCAM in the entire group of CRC patients (r = 0.309, p b 0.37) (Fig. 1) and in the stage-related subgroups (Table 4). The relationship between diagnostic sensitivity and specificity of the study parameters was investigated using ROC function (Receiver Operating Characteristics). VEGF was found to exhibit the highest diagnostic power (AUC = 0.991, SE = 0.009) (Fig. 2). Discussion Endothelium and cell adhesion molecules are engaged in tumor invasion. They take part in the adhesion of the cancer cell-leukocyteplatelet complexes to the stimulated endothelium and then in their migration beyond the circulation. It has been shown that increased expression of the cell adhesion molecules in neoplastic disease is associated with cancer progression and poor prognosis [7,6]. Numerous studies have reported a significant relationship between the levels of sICAM-1 i

Table 4 Correlations between sICAM-1 and sVCAM-1 in CRC patients. CRC groups

r

p

CRC patients (n = 46) Stage I/II (n = 22) Stage III (n = 14) Stage IV (n = 10)

0.309 0.453 0.608 0.759

0.037 0.034 0.021 0.01

Test Result Variable(s) ICAM VCAM VEGF

AUC ,661 ,650 ,991

Std. Error(a) ,064 ,067 ,009

AUC- Area Under the Curve Fig. 2. ROC curve for investigated parameters.

sVCAM-1 and the disease stage [17,18]. Our earlier study on colorectal cancer revealed the highest levels of sICAM-1 and sE-selectin, present on the surface of active endothelial cells in patients with distant metastases to the liver, showing at the same time that these molecules can serve as a marker of cancer progression [18]. A more aggressive form of tumor and formation of metastases lead to increased mortality. The formation of metastases and their development in a new site is a complex process, with mechanisms that are not completely clear. Our findings revealed a significant increase in the circulating adhesion molecules sICAM-1 and sVCAM-1 in patients with colorectal cancer as compared to healthy subjects. Their levels were statistically significantly correlated with clinical advancement of colorectal cancer. The increase was proportional to the tumor size and the presence of distant metastases to the liver. This seems to indicate that the levels were the highest in stage IV patients, being statistically significantly higher especially when compared to stages I and II, that is when the cancer was limited to the colon. As shown by literature data, the adhesion molecules play a major role in cancer proliferation and invasion as well as metastasizing [19]. De Cicco et al. observed that patients suffering from prostate cancer, with VCAM level above 633 ng/ml were at a higher risk of metastases or death than those with lower level of VCAM. They suggest that the elevated level of this adhesion molecule may reflect more aggressive phenotype of prostate cancer [17]. Also ICAM is considered to be a marker of metastasizing, which has been reported by Santarosa et al. who found a correlation between the expression of ICAM-1 and renal cancer relapse [20]. The expressions of ICAM and VCAM are induced by various cytokines, such as TNF-α, secreted by cancer cells, known to stimulate strongly their expressions and involvement in the metastasizing process [6]. However, other authors claim that the increased expression of sICAM-1 on the cell surface promotes recognition and destruction of cancer cells by the immune cells of the host [4]. Likewise, Coskun et al. found that the increase in the level of adhesion molecules might reflect the elevated immunity against tumor cells [21]. The significant increase in the levels of sICAM-1 and sVCAM-1 observed in our current study indicates stimulation of endothelial cells, which conditions the initiation of the inflammatory process and further development of cancer associated with the formation of metastases, i.e. adhesion to endothelial cells and then extravasation of tumor

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cells beyond the vascular wall to a new site, in which new blood vessels have to be formed to provide growth factors to cancer cells. As shown by our study, angiogenesis is stimulated in CRC patients, which is reflected in a highly statistically significant increase in VEGF level, being the main stimulator of this process, as compared to healthy subjects. This cytokine is considered to have a predictive value for cancer progression, formation of metastases and the disease relapse [13,14]. Our findings confirmed this correlation, as the level of VEGF was found to increase proportionally to the stage of advancement, tumor size and the disease extension. The analysis of this correlation allows an assumption that the more advanced the neoplastic process, the greater need of the tumor for nutrients and oxygen. Hence, angiogenesis is enhanced and the level of VEGF, as the main stimulator of this process, is significantly elevated as compared to control. However, despite considerable numerical differences between the groups of patients, they were not statistically significantly correlated with colorectal cancer advancement stage. The discrepancy between the presence or lack of correlation of VEGF with the advancement stage of colorectal cancer, also observed by others, can be due to high standard deviations and medians. Similar conclusions have been presented by e.g. Kaya et al. [22]. This can be a considerable limitation to the use of VEGF as a biomarker and independent prognostic factor in colorectal cancer patients, despite its very high diagnostic power (according to the calculated ROC curve), and requires further research. A significant positive correlation noted between the transmembrane molecules, sICAM-1 and sVCAM-1, in all the study groups is an interesting observation. In the group of patients with liver metastasis (stage IV) the correlations were characterized by the highest statistical significance, as well as a similar diagnostic value (ROC curve) could imply their connection with the disease progression. However, we did not observe any such correlations between the molecules and VEGF, nor did De Cicco et al. [17]. Yet, the dynamics of the increase in the expression of VEGF, like the adhesion molecules in CRC patients may indirectly suggest their co-involvement in neoplastic progression and metastasis formation. Nevertheless, these correlations require further observations and research. In conclusion, our results demonstrated highly statistically increased levels of soluble adhesion molecules, VCAM-1 and ICAM-1, and the main neoangiogenesis stimulating factor, VEGF, in patients with colorectal cancer as compared to healthy subjects. As the highest level of these molecules was observed in patients with the most advanced cancer (stage IV), it can be assumed that their level could be associated with tumor growth, and with regional and distant metastases. Thus, our findings may encourage further studies on the use of adhesion molecules as biomarkers of advancement and progression of colorectal cancer.

Conflict of interest There is no any potential conflict of interest of this paper.

References [1] Vanky F, Wang P, Patarroyo M, Klein E. Expression of the adhesion molecule ICAm-1 and major histocompatibility complex class I antigens on human tumor cells is required for their interaction with autologous lymphocytes in vitro. Cancer Immunol Immunother 1990;31:19–27. [2] Blann AD, Herrick A, Jayson MIV. Altered levels of soluble adhesion molecules in rheumatoid arthritis, vasculitis and systemic sclerosis. Br J Rheumatol 1995;34: 814–9. [3] Sawada T, Kimura K, Nishihara T, Onoda N, Teraoka H, Yamashita Y, et al. TGF-β1 down-regulates ICAM-1 expression and enhances liver metastasis of pancreatic cancer. Adv Med Sci 2006;51:60–5. [4] Schwaeble W, Kerlin M, Meyer KH, Dippold W. De Novo expression of intercellular adhesion molecule 1 (ICAM-1, CD 54) in pancreatic cancer. Int J Cancer 1993;53:328–33. [5] Sawa Y, Ueki T, Hata M, Iwasawa K, Tsuruga E, Kojima H, et al. LPS-induces IL-6, IL8, VCAM-1 and ICAM-1 expression in human lymphatic endothelium. J Histochem Cytochem 2008;56:97–109. [6] Lieuw-a-Fa M, Schalkwijk C, van Hinsbergh VWM. Distinct accumulation patterns of soluble forms of E-selectin, VCAM-1 and ICAM-1 upon infusion of TNFα in tumor patients. Thromb Haemost 2003;89:1052–7. [7] Alexandrakis MG, Passam FH, Sfiridaki K, Kafarakis P, Karydi E, Liapi D, et al. Clinical significance of circulating endothelial adhesion molecules (sE-selectin and sICAM-1) in treated multiple myeloma patients. Clin Chem Acta 2004;349:39–43. [8] Ten Kate M, Hofland LJ, van Grevenstein WMU, van Koetsveld PV, Jeekel J, van Eijck CHJ. Influence of proinflammatory cytokines on the adhesion of human colon carcinoma cells to lung microvascular endothelium. Int J Cancer 2004;112:943–50. [9] Chin KF, Greenman J, Gardiner E, Kumar H, Topping K, Monson J. Pre-operative serum vascular endothelial growth factor can select patients for adjuvant treatment after curative resection in colorectal cancer. Br J Cancer 2000;83:1425–31. [10] Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int 1999;56:794–814. [11] Caine GJ, Lip GYH, Stonelake PS, Ryan P, Blann AD. Platelet activation, coagulation and angiogenesis in breast and prostate carcinoma. Thromb Haemost 2004;92: 185–90. [12] Rasheed S, McDonald PJ, Northover JM, Guenther T. Angiogenesis and hypoxic factors in colorectal cancer. Pathol Res Pract 2008;204:501–10. [13] Ferroni P, Spila A, Martini F, D'Alessandro R, Mariotti S, Del Monte G, et al. Prognostic value of vascular endothelial growth factor tumor tissue content of colorectal cancer. Oncology 2005;69:145–53. [14] Seo HY, Park JM, Park KH, Kim SJ, Oh SCh, Kim BS, et al. Prognostic significance of serum vascular endothelial growth factor per platelet count in unresectable advanced gastric cancer patients. Jpn J Clin Oncol 2010;40:1147–53. [15] Volm M, Koomagi R, Mattern J. PD-ECGF, bFGF, and VEGFexpression in non-small cell lung carcinomas and their association with lymph node metastasis. Anticancer Res 1999;19:651–6. [16] Koch AE, Halloran MM, Haskel CJ, Shah MR, Polverini PJ. Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1. Nature 1995;376:517–9. [17] De Cicco C, Ravasi L, Zorzino L, Sandri MT, Botteri E, Verweij F, et al. Circulating levels of VCAM and MMP-2 may help identify patients with more aggressive prostate cancer. Curr Cancer Drug Targets 2008;8:199–206. [18] Dymicka-Piekarska V, Kemona H. Does colorectal cancer clinical advancement affect adhesion molecules (sP-selectin, sE-selectin and sICAM-1) concentration? Thromb Res 2009;124:80–3. [19] Banks RE, Gearing AJH, Hemingway IK, Norfolk DR, Perren TJ, Selby PJ. Circulating intercellular adhesion molecule-1 (ICAM-1). E-selectin and vascular cell adhesion molecule − 1 (VCAM-1)in human malignancies. Br J Cancer 1993;68:122–4. [20] Santarosa M, Favaro D, Quaida M, Spada A, Sacco C, Galliogioni E. Expression and release of intercellular adhesion molecule-1 in renal cancer patients. Int J Cancer 1995;62:271–5. [21] Coskun U, Sancak B, Sen I, Bukan N, Tufan MA, Gulbahar O, et al. Serum P-selectin, soluble vascular cell adhesion molekule-1 (s-VCAM-1) and soluble intercellular adhesion molecule-1 (s-ICAM-1) levels in bladder carcinoma patients with different stages. Int Immunopharmacol 2006;6:672–7. [22] Kaya A, Ciledag A, Gulbay BE, Poyraz BM, Celik G, Sen E, et al. Savas I (2004) The prognostic significance of vascular endothelial growth factor levels in sera of nonsmall cell lung cancer patients. Respir Med 2004;98:632–6.