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Chemokine Receptor CCR6 Expression Level and Liver Metastases in Colorectal Cancer

Pirus Ghadjar, Sarah Ellen Coupland, Il-Kang Na, Michel Noutsias, Anne Letsch, Andrea Stroux, Sandra Bauer, Heinz J. Buhr, Eckhard Thiel, Carmen Scheibenbogen, Ulrich Keilholz

From the Departments of Hematology, Medical Oncology, and Transfusion Medicine, Pathology, Cardiology and Pneumonology, and Surgery, Institute for Medical Informatics, Biometry, and Clinical Epidemiology, Charité, Campus Benjamin Franklin, Berlin, Germany.

Address reprint requests to Ulrich Keilholz, MD, Charité, Campus Benjamin Franklin, Medizinische Klinik III, Hämatologie, Onkologie und Transfusionsmedizin, Hindenburgdamm 30, 12200 Berlin, Germany; e-mail: ulrich.keilholz{at}charite.de

	ABSTRACT

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PURPOSE: The liver is the primary organ of metastasis in colorectal cancer (CRC). Chemokine receptor CCR6 is expressed on a subset of T cells and is associated with their migration into the liver. This study was performed to analyze a possible association between CCR6 expressed by primary CRC and liver metastases.

PATIENTS AND METHODS: CCR6 expression levels were evaluated by immunohistology in 64 CRC primary tumor specimens. Twenty-four of 64 patients had synchronous liver metastases. Evaluation of immunostaining was performed semiquantitatively by visual assessment and quantitatively by digital image analysis (DIA). Multiple logistic regression analysis was performed to assess relevant parameters for liver metastases.

RESULTS: CCR6 expression was verified in all 64 primary tumor specimens with considerable variations in intensity; 21 tumors (33%) demonstrated weak CCR6 staining, 32 (50%) demonstrated intermediate staining, and 11 (17%) demonstrated strong staining. Quantitative assessment by DIA showed an up to 5-log difference in CCR6 values. CCR6 staining was significantly stronger in tumor cells compared with adjacent colon epithelial cells (P < .0005). Multiple logistic regression analysis, controlling for age, sex, tumor stage, nodal status, pathologic grade, and preoperative carcinoembryonic antigen levels, revealed that CCR6 staining in the primary tumor was independently associated with the presence of liver metastases (odds ratio = 2.1; P = .002).

CONCLUSION: The association between expression level of CCR6 in primary CRC and synchronous liver metastases suggests that CCR6 and its ligand may be involved in the metastatic spread to the liver. Therefore, CCR6 may be a potential target for specific therapeutic interventions.

	INTRODUCTION

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The liver is the primary site of hematogenous metastases in colorectal cancer (CRC). Already at the time of diagnosis of the primary tumor, approximately 20% of patients present with synchronous liver metastases, and another 20% to 30% of patients will develop liver metastases after resection of the primary tumor. The presence of liver metastases at the time of initial staging is the most important prognostic factor for survival. The 5-year survival rate of 90% in patients with tumor restricted to the colon decreases to 10% in the presence of distant metastases.¹

Metastasis in CRC and many other neoplasias is not random but an organ-selective process. Lymphoid cells are well known to differentially express a broad array of chemokine receptors, serving as key regulators of migration into specific compartments and tissues. Studies by the group at Zlotnik² were the first to suggest that aberrant expression of chemokine receptors on tumor cells is involved in organ-selective tumor metastasis, and this concept has been supported by several subsequent investigations.^3-8 Clinical studies have shown an association between increased expression of the lymph node–homing chemokine receptor CCR7 and lymph node metastases in patients with gastric, esophageal, lung, and breast cancer.^3-6 Recent studies in CRC have revealed an important role for CXCR4 in CRC metastasis; high CXCR4 expression in the primary tumor was correlated with increased risk for recurrence and shorter survival.^7,8 However, no association of CXCR4 expression with liver metastasis was observed in the study by Kim et al.⁷

The chemokine receptor CCR6 is of particular interest in CRC metastasizing to the liver. CCL20, which was originally identified in the liver and called liver- and activation-related chemokine, is the only chemokine known to interact with CCR6.^9,10 CCL20 is expressed mainly in mucosa-associated and lymphoid tissues and the liver.¹⁰ The receptor-ligand pair CCR6-CCL20 plays an important role for the chemoattraction of T cells to the liver.^11,12 CCR6 is physiologically expressed only in a small subset of 10% to 20% of CD4⁺ T cells, but the majority of intrahepatic CD4⁺ T cells in patients with various liver diseases are CCR6 positive.^10,11 In major histocompatibility complex class II–mismatch recipients of CD4⁺ T cells from wild-type mice, a more than 10-fold increase in CD4⁺ T-cell liver infiltrates was observed 3 weeks after cell transfer in contrast to T cells from CCR6-deficient mice, which did not accumulate in the liver.¹² Normal colon epithelial cells and colon cancer cells were also shown to bear CCR6.¹³ Stimulation of colon cancer cells by CCL20 has been shown to result in signaling events associated with cell adhesion and migration.¹³ In a murine model, stimulation of CCR6-expressing plasmocytoma cells with CCL20 before intravenous application resulted in a marked increase in their homing to the liver.¹⁴

The current study was undertaken to analyze a potential association between CCR6 expression in primary CRC and liver metastasis. All CRC samples revealed CCR6 expression in the tumor cells, but the intensity varied considerably. There was a strong association between CCR6 staining intensity and synchronous liver metastases, suggesting a role of CCR6 in the process of metastasis of CRC to the liver.

	PATIENTS AND METHODS

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Patient Selection and Tissue Specimens
A series of 24 patients with primary CRC of various stages and synchronous liver metastases (stage M1) were randomly retrieved from the institutional database in the General Pathology Department of Charité, Campus Benjamin Franklin, Berlin, Germany. In addition, 40 patients with primary CRC without liver metastases (stage M0) were randomly selected. All patients had undergone surgery for American Joint Committee on Cancer stages I to IV CRC in the Department of Surgery of Charité, Campus Benjamin Franklin. From all 64 patients, primary tumor specimens were available. Liver metastasis specimens were available from 16 of the 24 patients with synchronous liver metastasis for analysis. The investigation was approved by the institutional ethics committee.

Immunohistochemical Staining
All specimens had been fixed in 4% buffered formalin and embedded in paraffin. For immunostaining of paraffin-embedded tissues, tissue sections (4 µm) were cut from routine blocks, de-waxed, rehydrated, and subjected to heat-induced epitope retrieval methods before incubation with the appropriate antibodies. Sections were immersed in sodium citrate buffer solution at pH 6.0 and were subsequently heated in a pressure cooker. After rinsing in running water and Tris-buffered saline, the sections were incubated with the primary antibody mouse antihuman CCR6 monoclonal antibody (dilution of 1:200; Becton Dickinson, Heidelberg, Germany), which was used according to the manufacturer's guidelines. This dilution was considered optimal after antibody titration using human tonsils. For detection of CCR6, the alkaline phosphatase–antialkaline phosphatase complex was used. Alkaline phosphatase was revealed by Fast Red (Sigma, Taufkirchen, Germany) as chromogen. Negative controls were performed by replacing the primary antibody with mouse isotype control antibody. Normal human tonsils and lymph nodes were used as positive control tissue for the immunostaining experiments showing expression of CCR6 on lymphocytes. Tonsillar epithelium provided an additional negative control. Furthermore, the intramural ganglion cells of the colon served as an intrinsic positive control for CCR6, whereas the intramural muscle was an intrinsic negative control.

Evaluation of Immunostaining
Evaluation of immunostaining was performed semiquantitatively by visual assessment and quantitatively by digital image analysis (DIA). For visual assessment, specimens were considered immunopositive for CCR6 if 1% of the tumor cells had clear evidence of immunostaining. As previously performed by others,⁴ we attempted to create an immunoreactive score by multiplying the score of percent positive cells and the score of staining intensity. However, because more than 95% of tumor cells stained positively for CCR6 in all specimens, scoring for the immunostaining intensity only was undertaken. This was rated as follows: 0, none; 1, weak; 2, intermediate; and 3, intense. More than one plain of section of all tumors was stained and evaluated. Wherever possible, CCR6 expression of adjacent normal colon tissue was assessed and compared with tumor expression. The assessment of the staining was conducted by two authors independently (S.E.C. and P.G.) who were not aware of the patients' clinical data and presence of liver metastases. Less than 10% of cases were rated differently and were re-evaluated, and score assessment was made by joint decision. Furthermore, one third of the carcinomas were randomly restained and evaluated a second time to test the reproducibility of staining and evaluation. No discordant results were obtained.

Quantification of the CCR6 immunostaining was performed using a macro programmed on the platform of LUCIA G (Version 3.52ab; Nikon, Düsseldorf, Germany), as described in detail elsewhere.^15,16 In brief, all available fields from tumor tissue areas were grabbed by a Leica DMRD microscope at 100-fold magnification (Leica, Bensheim, Germany) and were transferred to a Sony 3CCD color (red-green-blue [RGB]) video camera (Sony, Tokyo, Japan) using a Leica C-mount adapter (0.35 magnification; Leica) under constant light conditions. The measurement frame of each evaluated field was restricted to the tumor tissue area. After digital sharpening and contrasting of the captured image in a standardized manner, a threshold was set to detect the artifactual areas not covered by tissue (minimum RGB values: 0, 0, 0; maximum RGB values: 13, 21, 17). Subsequently, a second threshold step detected specifically the immunohistologically stained area within the fields (minimum RGB values: 75, 0, 60; maximum RGB values: 255, 0, 176). The CCR6 area fraction (AF) was calculated as the ratio of immunohistologically stained area to the net tissue area and was expressed in percent AF values (multiplied by 100).

Statistical Analysis
For statistical analyses, age and preoperative serum carcinoembryonic antigen (CEA) were dichotomized (ie, < v 70 years old and normal or < 5 µg/L v elevated or 5 µg/L CEA). Thus, descriptive statistics for sample characteristics include absolute and relative numbers for each demographic and clinical factor (see Table 1). Fisher's exact test was used for confirmatory analyses with regard to potential associations between semiquantitative CCR6 expression or the presence of liver metastases and the other factors (see Tables 2 and 3). For DIA outcomes of CCR6 expression, mean ± standard deviation, median, and range are presented for the entire study population as well as for the demographic and clinical factor subgroups separately. For comparisons concerning DIA between these subgroups, the Mann-Whitney U test or Kruskal-Wallis test for more than two groups was performed (see Table 2). Comparisons between CCR6 expression in the primary tumor and CCR6 expression in adjacent mucosa were performed using Wilcoxon's signed rank test. For multivariate analysis with presence of liver metastases as the dependent variable, binary logistic regression analysis was performed (see Table 3), with forward and backward selection for model identification. Diagnostic accuracy of CCR6 expression with regard to the presence of liver metastases was quantified via receiver operating characteristic (ROC) analysis.

	RESULTS

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View larger version (92K): [in this window] [in a new window]   Fig 1. CCR6 expression in primary colorectal cancer representing examples of strong, intermediate, and weak staining intensity (original magnification, x100). Values from digital image analysis for strong, intermediate, and weak staining intensity were 90.1%, 18.9%, and 0.02% CCR6 area fraction, respectively.

View larger version (13K): [in this window] [in a new window]   Fig 2. Correlation of visual assessment of CCR6 staining intensity of primary colorectal cancer tissues rated into three groups of weak (n = 21), intermediate (n = 32), and strong (n = 11), with corresponding quantitative CCR6 area fraction (CCR6-AF) values as assessed by digital image analysis (DIA; P < .0005). Circles indicate outliers, and asterisks indicate extreme values.

View larger version (14K): [in this window] [in a new window]   Fig 3. Comparison of CCR6 area fraction (CCR6-AF) values of (A) 53 paired mucosa/primary colorectal cancer (CRC) samples and (B) 16 paired primary CRC/liver metastasis samples. DIA, digital image analysis.

For the definitive confirmatory analysis, DIA-evaluated CCR6-AF values of the primary tumor were used. Patients were grouped according to presence of liver metastases, and univariate analyses as well as multiple logistic regression analysis were carried out to investigate whether CCR6-AF values would be an independent risk factor for liver metastases (Table 3). Univariate analyses again revealed the highly significant association between liver metastases and CCR6-AF values of the primary tumor (P < .0005) and also showed an association of liver metastases with nodal status (P = .008) and preoperative serum CEA (P = .055). Multiple logistic regression analysis, with presence of liver metastases as the dependent variable and all other clinical and pathologic parameters as independent variables, confirmed that the presence of liver metastases was strongly associated with the CCR6-AF values after forward and backward selection (odds ratio = 2.1 per 10-unit increase in DIA outcome; P = .002; Table 3). In the selected model, presence of liver metastases was further associated with preoperatively elevated serum CEA levels (odds ratio = 12.1; P = .027) and with nodal stage (odds ratio = 3.2; P = .032). Similar results were obtained when multiple logistic regression analysis was performed with CCR6 staining intensity classified as weak, intermediate, or strong as assessed visually (data not shown).

Further ROC analyses (ie, quantification of the accuracy of diagnostic parameters: sensitivity and specificity) revealed an area under the curve of 0.92 (95% CI, 0.83 to 1.0) for CCR6, nodal status, and preoperative CEA combined compared with 0.85 (95% CI, 0.75 to 0.94), 0.69 (95% CI, 0.56 to 0.82), and 0.67 (95% CI, 0.50 to 0.84) for CCR6, nodal status, and CEA alone, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 
This study revealed that high expression of CCR6 in primary CRC is strongly associated with synchronous liver metastases. In addition, in the univariate analysis, the known prognostic factors linked to the outcome (preoperative elevated CEA and nodal status) were associated with liver metastasis.¹⁷ The multivariate analysis showed that CCR6 is an independent risk factor for liver metastases. In a recent study by Kim et al,⁷ the prognostic role for strong expression of the chemokine receptor CXCR4 and survival was demonstrated in a multivariate analysis. However, in contrast to our findings for CCR6, no association of CXCR4 expression with liver metastasis was observed in the study by Kim et al.⁷

Our findings imply a possible pathogenetic role for CCR6 in the development of liver metastases. The CCR6 ligand CCL20 was found in liver tissue under constitutive and inflammatory conditions as well as in hepatocellular carcinoma.^9,11 By immunohistochemical staining, CCL20 was located in large periportal mononuclear cells, most likely macrophages or dendritic cells, at the presumed entry site of CRC cells into the liver.¹¹ Various in vitro and animal studies provide evidence that chemokines selectively expressed in certain tissues can promote metastasis by attracting specific CCR-expressing tumor cells and/or by providing growth-stimulatory signals.^2,18 On the basis of this concept, increased expression levels of CCR6 in CRC cells could facilitate chemoattraction of CRC cells by CCL20 expressed in the periportal area of the liver.¹¹ Our observation of lower expression of CCR6 in tumor cells in synchronous liver metastases might result from CCL20-induced downregulation because specific ligand binding generally enhances internalization and degradation of chemokine receptors.¹⁹ Similarly, CCR6 expression levels on T cells in the liver was noted to be lower than in peripheral blood, and in vitro studies showed that CCR6 expression is downregulated in T cells in response to CCL20.¹¹ On the basis of functional studies in lymphocytes, we do not consider the cytoplasmic CCR6 found in CRC cells to solely result from internalization of surface CCR6, but it is more likely to serve as a regulatory intracellular pool enabling rapid expression of CCR6 on the surface.²⁰

The identification of key targets promoting metastasis is of great interest for the development of specific treatment strategies. Attempts to inhibit metastasis by interfering with chemokine receptor/chemokine interactions is a promising new treatment strategy.²¹ Various small-molecule chemokine receptor antagonist compounds are currently undergoing development in phase I to III studies in infectious and autoimmune diseases and more recently also in cancer. Targeting of CCR6/CCL20 may represent a potential treatment strategy that could be developed for the prevention of liver recurrences.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 
The authors indicated no potential conflicts of interest.

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 

Conception and design: Pirus Ghadjar, Sarah Ellen Coupland, Carmen Scheibenbogen, Ulrich Keilholz Financial support: Carmen Scheibenbogen, Ulrich Keilholz Administrative support: Heinz J. Buhr, Eckhard Thiel Provision of study materials or patients: Sarah Ellen Coupland, Heinz J. Buhr, Ulrich Keilholz Collection and assembly of data: Pirus Ghadjar, Sarah Ellen Coupland, Il-Kang Na, Anne Letsch, Sandra Bauer, Ulrich Keilholz Data analysis and interpretation: Pirus Ghadjar, Sarah Ellen Coupland, Il-Kang Na, Michel Noutsias, Andrea Stroux, Eckhard Thiel, Carmen Scheibenbogen, Ulrich Keilholz Manuscript writing: Pirus Ghadjar, Sarah Ellen Coupland, Michel Noutsias, Andrea Stroux, Carmen Scheibenbogen, Ulrich Keilholz Final approval of manuscript: Pirus Ghadjar, Sarah Ellen Coupland, Michel Noutsias, Eckhard Thiel, Carmen Scheibenbogen, Ulrich Keilholz

 

	ACKNOWLEDGMENTS

 
We thank H. Protz for her excellent technical assistance.

	NOTES

 
Supported by a grant from the Charité.

P.G. and S.E.C. share first authorship; C.S. and U.K. share senior authorship.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

	REFERENCES

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4. Ding Y, Shimada Y, Maeda M, et al: Association of CC chemokine receptor 7 with lymph node metastasis of esophageal squamous cell carcinoma. Clin Cancer Res 9:3406-3412, 2003[Abstract/Free Full Text]

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6. Cabioglu N, Yazici MS, Arun B, et al: CCR7 and CXCR4 as novel biomarkers predicting axillary lymph node metastasis in T1 breast cancer. Clin Cancer Res 11:5686-5693, 2005[Abstract/Free Full Text]

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8. Schimanski CC, Schwald S, Simiantonaki N, et al: Effect of chemokine receptors CXCR4 and CCR7 on the metastatic behaviour of human colorectal cancer. Clin Cancer Res 11:1743-1750, 2005[Abstract/Free Full Text]

9. Hieshima K, Imai T, Opdenakker G, et al: Molecular cloning of a novel human CC chemokine liver and activation-regulated chemokine (LARC) expressed in liver: Chemotactic activity for lymphocytes and gene localization on chromosome 2. J Biol Chem 272:5846-5853, 1997[Abstract/Free Full Text]

10. Schutyser E, Struyf S, Van Damme J: The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev 14:409-426, 2003[CrossRef][Medline]

11. Shimizu Y, Murata H, Kashii Y, et al: CC-chemokine receptor 6 and its ligand macrophage inflammatory protein 3alpha might be involved in the amplification of local necroinflammatory response in the liver. Hepatology 34:311-319, 2001[CrossRef][Medline]

12. Varona R, Cadenas V, Gomez L, et al: CCR6 regulates CD4+ T-cell-mediated acute graft-versus-host disease responses. Blood 106:18-26, 2005[Abstract/Free Full Text]

13. Yang CC, Ogawa H, Dwinell MB, et al: Chemokine receptor CCR6 transduces signals that activate p130 Cas and alter cAMP-stimulated ion transport in human intestinal epithelial cells. Am J Physiol Cell Physiol 288:C321-C328, 2005[Abstract/Free Full Text]

14. Dellacasagrande J, Schreurs OJF, Hofgaard PO, et al: Liver metastasis of cancer facilitated by chemokine receptor CCR6. Scand J Immunol 57:534-544, 2003[CrossRef][Medline]

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16. Noutsias M, Fechner H, de Jonge H, et al: Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: Implications for cardiotropic viral infections. Circulation 104:275-280, 2001[Abstract/Free Full Text]

17. Compton CC, Fielding LP, Burgart LJ, et al: Prognostic factors in colorectal cancer: College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 124:979-994, 2000[Medline]

18. Zeelenberg IS, Ruuls-Van Stalle L, Roos E: The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases. Cancer Res 63:3833-3839, 2003[Abstract/Free Full Text]

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Submitted September 16, 2005; accepted February 14, 2006.

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