Annals of Oncology Advance Access originally published online on October 10, 2006
Annals of Oncology 2007 18(1):70-76; doi:10.1093/annonc/mdl342
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© 2006 European Society for Medical Oncology
gynecologic tumors |
Association of CXCR4 and CCR7 chemokine receptor expression and lymph node metastasis in human cervical cancer
1 Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2 Department of Obstetrics and Gynecology, Inner Mongolia Agriculture University Hospital, Huhehaote, China
* Correspondence to: Dr J. Kodama, Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: +81-862357320; Fax: +81-862259570; E-mail: kodama{at}cc.okayama-u.ac.jp
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Abstract |
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Background: The chemokine receptors CXCR4 and CCR7 have been suggested to play an important role in cancer invasion and metastasis. The expression of these receptors in human cervical cancer, however, has seldom been characterized.
Patients and methods: We investigated the expression of CXCR4 and CCR7 in cervical cancer specimens and determined the association between their expression and the clinicopathological features observed, including patient outcome.
Results: CXCR4 expression was significantly higher in elderly patients (P = 0.025); it was also significantly increased in patients with cancers displaying large tumor size (P = 0.010), deep stromal invasion (P = 0.0004), lymph–vascular space involvement (P = 0.0002), or lymph node metastasis (P < 0.0001). CCR7 expression was significantly higher in cases of squamous cell carcinomas (P = 0.010) and in patients with cancers showing large tumor size (P < 0.0001), deep stromal invasion (P < 0.0001), vaginal invasion (P = 0.047), lymph–vascular space involvement (P = 0.012), or lymph node metastasis (P < 0.0001). Logistic regression analysis revealed that deep stromal invasion (P = 0.017) and CXCR4 (P = 0.016) and CCR7 (P = 0.022) expression were independent factors that influenced pelvic lymph node metastasis. The disease-free survival and overall survival (OS) rates of patients exhibiting both CXCR4 and CCR7 expression were significantly reduced (P < 0.0001). In addition, the expression of both CXCR4 and CCR7 was an independent prognostic factor for OS (95% confidence interval = 1.03–17.86; P = 0.046).
Conclusions: CXCR4 and CCR7 expression may be associated with lymph node metastasis; moreover, the expression of these receptors can serve as an indicator of poor prognosis in patients with cervical cancer.
Key words: cervical cancer, CCR7, CXCR4, lymph node metastasis, prognosis
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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Tumor cell invasion and subsequent dissemination via the bloodstream and lymph vessels are critical steps in the progression of malignant tumors, including cervical cancer. The mechanisms involved in lymph node metastasis, however, are not fully understood. In a previous study, we demonstrated that the expression of vascular endothelial growth factor-C is involved in the promotion of lymph node metastasis in cervical cancer [1]. The determination of such a predictive molecular marker that is correlated with lymph node metastasis might be important in elucidating the mechanism of metastasis.
Chemokines belong to the small-molecule chemoattractive cytokine family and are categorized into four groups (CXC, CC, CX3C, and C) on the basis of the characteristic presence of four conserved N-terminal cysteines [2–4]. Chemokines mediate their chemical effect on target cells through G-protein-coupled heptahelical receptors, which are structurally characterized by seven transmembrane-spanning domains and are involved in the attraction of mononuclear and polymorphonuclear leukocytes. Recent data indicate that chemokine receptors may direct lymphatic and hematogenous spread and may additionally influence the sites of metastatic growth of different tumors [5]. Different cancers express varying combinations of CC and CXC chemokine receptors. The chemokine receptor CXCR4 appears to be expressed by a majority of cancer types [6]. CXCR4 was initially reported to regulate the homing of lymphocytes to inflammatory tissues [7]. The CXCR4/CXCL12 pathway is involved in stimulating the metastatic process of many different neoplasms, in which CXCR4 activates various phenomena, such as chemotaxis, invasion, angiogenesis, and proliferation [8]. Müller et al. [9] reported that neutralizing the interaction of CXCR4 and CXCL12 in vivo significantly impairs the metastasis of breast cancer cells to regional lymph nodes and the lungs. CCR7—the receptor for the two major chemokines CCL19 and CCL21—is expressed on naive T cells, memory T cells, B cells, and mature dendritic cells and is considered to play an important role in lymphocyte cell trafficking and homing to lymph nodes [10, 11]. It has been reported that CCR7 expression specifically enhances the metastatic ability of B16 melanoma cells through a lymph-mediated but not a blood-borne pathway [12].
Thus, the chemokine receptors CXCR4 and CCR7 may be important in lymph node metastasis; however, their expression in human cervical cancer has not been characterized. Therefore, we investigated the expression of CXCR4 and CCR7 in cervical cancer specimens by using immunohistochemistry. In addition, the association between CXCR4 and CCR7 expression and the clinicopathological features of cervical cancer, including patient prognosis, was determined.
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patients and methods |
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patients and tissue samples
In this study, we examined 174 patients presenting with the International Federation of Gynecology and Obstetrics (FIGO) stages IB–IIB cervical cancers. Each of these patients underwent radical hysterectomy and pelvic lymphadenectomy at the Department of Obstetrics and Gynecology of the Okayama University Medical School. Tumor specimens were obtained at the time of surgery and were immediately fixed in 10% neutral-buffered formalin and embedded in paraffin. Informed consent was obtained from each patient before sample collection. Histological cell typing was conducted according to the World Health Organisation classifications as follows: 102 were classified as squamous cell carcinomas, 49 as adenocarcinomas, and 23 as adenosquamous carcinomas. Clinical staging was assessed based on the FIGO staging system as follows: 92 were allocated to stage IB, four to stage IIA, and 78 to stage IIB. The median age at the time of surgery was 46 years (range 25–67 years). Patients with lymph node metastasis, parametrial involvement, deep stromal invasion, or marked lymph–vascular space involvement were treated with an adjuvant external whole-pelvic irradiation, combination chemotherapy, or chemoradiation. Disease-free survival (DFS) and overall survival (OS) rates were defined as the interval from the initial surgery to clinically or radiologically proven recurrence and death, respectively. The end date of the follow-up study for conducting the analysis was 31 March 2006, and the median duration of the follow-up was 56.5 months (range 1–131 months).
immunohistochemistry and staining evaluation
Sections of 4-µm thickness were obtained from several representative areas of each tumor specimen and were mounted on to glass slides for immunostaining according to the labeled streptavidin biotin procedure of the Dako LSAB kit (Dako North American Inc., Carpinteria, CA). Briefly, after the slides were dewaxed in xylene and rehydrated in an alcohol series, antigen retrieval was carried out in a microwave oven in 10 mM citric acid buffer (pH 6.0) for 3 x 10 min. The sections were then incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity, followed by incubation with normal horse serum for 5 min at room temperature. Immunostaining was then carried out by incubation with anti-CXCR4 mouse mAb (R&D systems Inc., Minneapolis, MN) and anti-CCR7 rabbit mAb (Epitomics Inc., Burlingame, CA) for 2 h at room temperature. The sections were subsequently incubated for 20 min with biotinylated anti-mouse and anti-rabbit immunoglobulin, followed by incubation with peroxidase-conjugated streptavidin for 20 min and 0.05% 3,3'-diaminobenzidine tetrahydrochloride solution (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing hydrogen peroxide for 10 min. Finally, the slides were counterstained with Mayer's hematoxylin and mounted in an aqueous mounting medium. At each step, the slides were washed carefully in phosphate-buffered saline (pH 7.4).
The specimen was considered as positive if the distinct cytoplasmic staining was observed in >50% cancer cells. In our study of endometrial cancer, CXCR4 and CCR7 protein levels were significantly correlated to messenger RNA levels on the basis of these criteria. Microscopic analyses were conducted independently by two of the authors who had no prior knowledge of the clinical data. The final evaluations of a few ambiguous cases were carried out using a conference microscope.
statistical analyses
The association between the variables was tested using the chi-square test or a stepwise logistic regression analysis. The survival rates were calculated by the Kaplan–Meier method and the differences between the survival curves were examined by the log-rank test. Factors found to be significant were then selected for a stepwise Cox multivariate proportional hazard model in order to determine their prognostic values. These analyses were carried out using the Stat-View 5.0 software (Abacus Concepts Inc., Berkley, CA). Probability values of <0.05 were considered statistically significant.
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results |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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CXCR4 protein expression in cervical cancer tissue
Figure 1A, B, and C shows the representative immunostaining profiles of CXCR4 in cervical cancer specimens. In normal cervical tissues, we found no distinct CXCR4 staining. Of the 174 tumor samples examined, 110 (63%) were positively stained and 64 (37%) were negatively stained. The association between CXCR4 expression and clinicopathological factors is shown in Table 1. CXCR4 expression was significantly higher in elderly patients (P = 0.025) and in FIGO stage II tumors (P = 0.024); it was also significantly elevated in patients with cancers showing large tumor size (P = 0.010), deep stromal invasion (P = 0.0004), lymph–vascular space involvement (P = 0.0002), or lymph node metastasis (P < 0.0001). Furthermore, no significant association was observed between CXCR4 immunoreactivity and histological type, vaginal invasion, parametrial invasion, or ovarian metastasis.
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CCR7 protein expression in cervical cancer tissue
Figure 1D and E shows the characteristic immunostaining profiles of CCR7 in cervical cancer specimens. In normal cervical tissues, we found no distinct CCR7 staining. Of the 174 tumor samples, 102 (59%) were positively stained and 72 (41%) were negatively stained. The association between CCR7 expression and clinicopathological factors is shown in Table 2. CCR7 expression was significantly higher in FIGO stage II cases (P < 0.0001) and squamous cell carcinomas (P = 0.010); it was also significantly increased in patients with cancers showing large tumor size (P < 0.0001), deep stromal invasion (P < 0.0001), vaginal invasion (P = 0.047), lymph–vascular space involvement (P = 0.012), or lymph node metastasis (P < 0.0001). Furthermore, no significant association was observed between CCR7 immunoreactivity and age, parametrial invasion, or ovarian metastasis.
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We noticed nuclear CCR7 staining in 24 samples (14%). Figure 1F shows the representative immunostaining profile of nuclear CCR7 in cervical cancer. The association between nuclear CCR7 expression and clinicopathological factors is shown in Table 3. Nuclear CCR7 expression was significantly lower in patients with cancers showing vascular space involvement (P = 0.039). There was no significant association between nuclear CCR7 immunoreactivity and any other clinicopathological factor.
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predictors of lymph node metastasis
The predictors of lymph node metastasis were determined by stepwise logistic regression analysis. Pelvic lymph node metastasis was correlated with tumor size, stromal invasion, parametrial invasion, lymph–vascular space involvement, ovarian metastasis, and CXCR4 and CCR7 expression status (Table 4). Multivariate analysis revealed that deep stromal invasion (P = 0.017) and CXCR4 (P = 0.016) and CCR7 (P = 0.022) expression were the independent factors that influenced pelvic lymph node metastasis.
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univariate survival analyses
Figure 2 presents the DFS curves of the 174 patients with cervical cancer on the basis of the CXCR4 expression status. Both the DFS and OS rates of patients exhibiting CXCR4 expression were significantly lower than those of patients lacking CXCR4 expression (P = 0.025 and P = 0.0001, respectively). Figure 3 presents the DFS curves of the 174 patients with cervical cancer on the basis of the CCR7 expression status. Both the DFS and OS rates of patients exhibiting CCR7 expression were significantly lower than those of patients demonstrating the lack of CCR7 expression (P = 0.006 and P = 0.038, respectively). Furthermore, the DFS and OS rates of patients exhibiting both CXCR4 and CCR7 expression were significantly poor (P < 0.0001) (Figure 4). There was no association between nuclear CCR7 expression and patient outcome. The results of the univariate survival analyses of other variables are shown in Table 5.
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multivariate survival analysis
We examined the prognostic value of both CXCR4 and CCR7 expression using the Cox proportional hazard model. Multivariate analysis showed that lymph node metastasis was the only independent prognostic factor for DFS [95% confidence interval (CI) 1.20–10.87; P = 0.022]. In addition, non-squamous cell carcinoma was the strongest independent prognostic factor for OS, followed by both CXCR4 and CCR7 expression (95% CI 1.09–10.20, P = 0.035, and 95% CI 1.03–17.86, P = 0.046, respectively).
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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This is the first study analyzing CXCR4 and CCR7 protein expressions in a large series of human cervical cancer specimens. We demonstrated that both CXCR4 and CCR7 expression is significantly higher in patients with cancers displaying large tumor sizes, deep stromal invasion, lymph–vascular space involvement, or lymph node metastasis. Furthermore, CXCR4 expression was significantly higher in elderly patients, while CCR7 expression was significantly greater in patients with cancers exhibiting vaginal invasion. The stepwise logistic regression analysis revealed that both CXCR4 and CCR7 expression influenced pelvic lymph node metastasis independently. This finding indicates that both CXCR4 and CCR7 expression is up-regulated in cases with positive nodes and may contribute to the lymph node metastasis occurring in cervical cancers. The present data are consistent with previous reports that describe a positive correlation between CXCR4 expression and lymph node metastasis in cases of non-small-cell lung cancer (NSCLC), nasopharyngeal cancer, colorectal cancer, and esophageal cancer [13–16]. Positive correlation has also been reported between CCR7 expression and lymph node metastasis in cases of NSCLC, breast cancer, gastric cancer, colorectal cancer, esophageal cancer, prostate cancer, and oral and oropharyngeal squamous cell carcinoma [13, 17–22]. Although the role of chemokines and their receptors in human cancers is complex, the chemokine receptors CXCR4 and/or CCR7 may have a critical role in determining lymph node metastasis in these kinds of tumors.
In concordance with previous studies conducted on other cancer types, the staining for CXCR4 and CCR7 was predominantly cytoplasmic. This might represent a functional status of the receptor because binding to a specific ligand induces receptor internalization [23]. The necessity of internalization for chemotaxis and signaling remains controversial. Endosomes are gaining considerable attention as scaffolds for signaling complexes. The assembly of signaling complexes on intracellular endosomal membranes indicates that the intracellular trafficking itinerary of chemokine receptors may have important implications for signaling [24]. We also showed that nuclear CCR7 expression was observed in some cases, although the nuclear staining of CXCR4 was not observed in any case. Nuclear CCR7 expression was exclusively observed in patients with cancers showing negative lymph–vascular space involvement. Cabioglu et al. [17] reported that the nuclear expression of CCR7 and CXCR4 was significantly higher in lymph node-negative breast cancers. They speculated that the different localization patterns of chemokine receptors, i.e. nuclear versus cytoplasmic, appear to have a different biological significance for the metastatic potential of cancer cells. Further studies are needed to elucidate the role of the nuclear localization of these chemokine receptors.
Thus far, no studies have reported the clinical outcome of cases with CXCR4 and CCR7 expression in cervical cancer. Our study is the first to demonstrate that the expression of CXCR4 and CCR7 is associated with reduced survival outcomes in patients with cervical cancer. Furthermore, the multivariate analysis in the present study showed that the expression of both CXCR4 and CCR7 is an independent prognostic factor for the OS of patients. An increased expression of CXCR4 has been reported to correlate with a poor prognosis in esophageal and nasopharyngeal carcinomas [16, 25], whereas increased expression of CCR7 has been reported to be correlated with a poor prognosis in gastric, colorectal, esophageal, prostate, and oral and oropharyngeal squamous cell carcinomas [18–22]. These studies indicate that CXCR4 and CCR7 may be promising prognostic factors for these types of cancer.
In conclusion, the expression of CXCR4 and CCR7 may be associated with lymph node metastasis in cervical cancer. Our findings also provide evidence that both CXCR4 and CCR7 expression can serve as an indicator of poor prognosis in patients with cervical cancer.
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Acknowledgements |
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We would like to Chisae Kodera for her help with histochemistry and in the preparation of sections.
Received for publication May 6, 2006. Revision received August 15, 2006. Accepted for publication August 16, 2006.
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References |
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1. Hashimoto I, Kodama J, Seki N, et al. (2001) Vascular endothelial growth factor-C expression and its relationship to pelvic lymph node status in invasive cervical cancer. Br J cancer 85:93–97.[CrossRef][ISI][Medline]
2. Baggiolini M, Dewald B, Moser B. (1997) Human chemokines: an update. Annu Rev Immunol 15:675–705.[CrossRef][ISI][Medline]
3. Rollins BJ. (1997) Chemokines. Blood 90:909–928.
4. Kelner GS, Kennedy J, Bacon KB, et al. (1994) Lymphotactin: a cytokine that represents a new class of chemokines. Science 266:1395–1399.
5. Arya M, Patel HR, Williamson M. (2003) Chemokines: key players in cancer. Curr Med Res Opin 19:557–564.[CrossRef][ISI][Medline]
6. Balkwill F. (2004) The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol 14:171–179.[CrossRef][ISI][Medline]
7. Murdoch C. (2000) CXCR4: chemokine receptors extraordinaire. Immunol Rev 177:175–184.[CrossRef][ISI][Medline]
8. Murphy PM. (2001) Chemokines and the molecular basis of cancer metastasis. N Engl J Med 345:833–835.
9. Müller A, Homey B, Soto H, et al. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410:50–56.[CrossRef][Medline]
10. Dieu MC, Vanbervliet B, Vicari A, et al. (1998) Selective recruitment of immature and mature dendritic cell by distinct chemokine expression in different anatomic sites. J Exp Med 188:373–386.
11. Hirano M, Onai N, Hiroishi K, et al. (2000) CC chemokine receptor-7 on dendritic cells in induced after interaction with apoptotic tumor cells: critical role in migration from the tumor site to draining lymph nodes. Cancer Res 60:2209–2217.
12. Wiley HE, Gonzalez EB, Maki W, et al. (2001) Expression of CC chemokine receptor-7 and regional lymph node metastasis of B16 murine melanoma. J Natl Cancer Inst 93:1638–1643.
13. Takanami I. (2003) Overexpression of CCR7 mRNA in nonsmall cell lung cancer: correlation with lymph node metastasis. Int J Cancer 105:186–189.[CrossRef][ISI][Medline]
14. Hu J, Deng X, Bian X, et al. (2005) The expression of functional chemokine receptor CXCR4 is associated with the metastatic potential of human nasopharyngeal carcinoma. Clin Cancer Res 11:4658–4665.
15. Schmanski CC, Schwald S, Simiantonaki N, et al. (2005) Effect of chemokine receptors CXCR4 and CCR7 on the metastatic behavior of human colorectal cancer. Clin Cancer Res 11:1743–1750.
16. Kaift JT, Yekebas EF, Schurr P, et al. (2005) Tumor-cell homing to lymph nodes and bone marrow and CXCR4 expression in esophageal cancer. J Natl Cancer Inst 97:1840–1847.
17. Cabioglu N, Yazici MS, Arun B, et al. (2005) CCR7 and CXCR4 as novel biomarkers predicting axillary lymph node metastasis in T1 breast cancer. Clin Cancer Res 11:5686–5693.
18. Mashino K, Sadanaga N, Yamaguchi H, et al. (2002) Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric cancer. Cancer Res 62:2937–2941.
19. Günther K, Leier J, Henning G, et al. (2005) Predictor of lymph node metastasis in colorectal carcinoma by expression of chemokine receptor CCR7. Int J Cancer 116:726–733.[CrossRef][ISI][Medline]
20. Ding Y, Shimada Y, Maeda M, et al. (2003) Association of CC chemokine receptor 7 with lymph node metastasis of esophageal squamous cell carcinoma. Clin Cancer Res 9:3406–3412.
21. Heresi GA, Wang J, Taichman R, et al. (2005) Expression of the chemokine receptor CCR7 in prostate cancer presenting with generalized lymphadenopathy: report of a case, review of the literature, and analysis of chemokine receptor expression. Urol Oncol 23:261–267.[ISI][Medline]
22. Tsuzuki H, Takahashi N, Kojima A, et al. (2006) Oral and oropharyngeal squamous cell carcinomas expressing CCR7 have poor prognoses. Auris Nasus Larynx 33:37–42.[CrossRef][ISI][Medline]
23. Roland J, Murphy BJ, Ahr B, et al. (2003) Role of the intracellular domains of CXCR4 in SDF-1 mediated signaling. Blood 101:399–406.
24. Neel NF, Schutyser E, Sai J, et al. (2005) Chemokine receptor internalization and intracellular trafficking. Cytokine Growth Factor Rev 16:637–658.[CrossRef][ISI][Medline]
25. Wang N, Wu QL, Fang Y, et al. (2005) Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma: pattern of expression and correlation with clinical outcome. J Trans Med 3:26.
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