Colorectal cancer (CRC) is the fourth most common cancer in men and the third most common cancer in women worldwide. However, in recent years, high colorectal cancer rates have been reported in newly developed countries around the globe in which the risk was once low (Center et al., 2009). In USA, CRC constitutes 9.5% of all cancers, whereas in Egypt, it contributes 6.5% of all cancers (EL-Bolkainy et al., 2006).
Although the 5-year survival rate for patients with local CRC approaches 90%, the spread of disease to distant sites decreases the 5-year survival rate to 19% (Jemal et al., 2004). Metastasis in CRC is not a random but an organ-selective process with an increased and early metastatic propensity to abdominal lymph nodes, peritoneum, and liver (Kim et al., 2005). The precise mechanisms determining the directional migration and invasion of CRC cells into specific organs remain to be established. However, new evidence suggested that a number of molecules have been implicated in the metastasis of cancer cells (Schimanski et al., 2005). Therefore, it is necessary to evaluate whether some metastasis-related molecules can be used as prognostic markers for metastasis of colon cancer.
Chemokines are a superfamily of proinflammatory polypeptide cytokines that selectively attract and activate different cell types. Chemokines are characterized by their ability to induce directional migration and activation of leukocytes. They regulate leukocyte adhesion, trafficking, homing, and angiogenesis, and contribute to lymphopoiesis and hematopoiesis (Le et al., 2000).
Chemokine receptor may direct lymphatic and hematogenous spread and may additionally influence the sites of metastatic growth of different tumors. Originally, chemokines and their G protein coupled receptor were reported to mediate different proinflammatory and anti-inflammatory responses. The chemokine receptor CXCR4 was initially described to regulate the homing of lymphocytes in inflammatory tissues. Its natural ligand, the stromal cell-derived factor 1a (SDF-1a), is highly expressed in tissues of metastatic growth, such as lung, liver, and lymph nodes, and attracts lymphocytes to these organs (Schimanski et al., 2005). Several studies have found a correlation between angiogenesis measured as microvessel density (MVD) and distant metastasis or prognosis in malignant tumors (Kreuter et al., 2004).
In this study, we aimed to investigate the expression of CXCR4 chemokine receptor in human colon cancer specimens using immunohistochemistry and study its relationship with clinical, pathological factors, and prognosis of colon cancer. We also aimed to study MVD to clarify the angiogenic properties of the CXCR4 chemokine receptor.
Materials and methods
This retrospective study was conducted on archival material of 54 cases with primary diagnosis of colorectal adenocarcinoma in the Gastroentrology centre, Mansoura University, Egypt in the period from January 2006 to January 2008 with follow-up period until January 2010 (minimal 24 months follow-up). None of the patients received any neoadjuvant chemotherapy or radiotherapy. Complete clinicopathological information was obtained including patient's age, sex, and the complete information of the patients' follow-up visits to evaluate the response and prognosis. The available follow-up period in the data sheet is 24 months.
The paraffin blocks were retrieved and 4-μm thick sections were prepared for routine hematoxylin and eosin. Other sections were prepared on coated slides for immunohistochemistry. Examination of the slides was carried out by two examiners for histopathologic diagnosis and tumor node metastasis staging.
Deparaffinized sections of all cases were incubated for 30 min with 0.3% hydrogen peroxide in methanol and were microwave heated in EDTA buffer. Subsequently, an indirect immunoperoxidase technique was applied using monoclonal antibodies for anti-CXCR4 (Thermo Scientific Clone 44716.111 Cat.♯ MA1-20157) and anti-CD34 (Thermo Scientific Clone SPM123 Cat.♯ MA1-37328). It is performed using ImmunoPure Ultra-Sensitive ABC Peroxidase (Thermo Scientific Cat. ♯ 32052), with diaminobenzene as chromogen.
CXCR4 chemokine receptor expression in the tumor cells was scored. Cases were considered positive when there was nuclear staining in more than 10% tumor cells with or without cytoplasmic staining and were considered negative when nuclear staining was less than or equal to 10%. For CD34 expression, MVD was assessed without knowledge of the patient's outcome; areas of invasive tumor containing the most capillaries and small venules (i.e. areas of most intense neovascularization) were examined by light microscopy. Tumors were frequently heterogeneous in their MVD, but the areas of highest neovascularization were found by scanning the tumor sections at low power (×40 and ×100) and identifying the areas of invasive carcinoma with the highest number of discrete microvessel staining for CD34 (Weidner et al., 1991). In each section slide, microvessels were counted in at least three independent hot spots per section (range, 3–4 hot spots per section) and in 2–3 sections stained with anti-CD34 antibody. The mean value of all independent readings of the tumor biopsy specimen of a single patient was calculated, and MVD was defined as the mean count of microvessels per 0.26 mm2 field area (i.e. ×400 field). The median MVD of the entire group was predetermined to classify patients into two groups with high (median) and low (median) MVD according to an international consensus report (Vermeulen et al., 2002).
All statistical analyses were conducted using the Statistical Program for Social Sciences (SPSS 10.0, SPSS Inc., Chicago, Illinois, USA). Qualitative data were presented as number and percentage. Comparison between groups was made by χ2 test. P value of less than 0.05 was considered as a significant difference. Overall survival time was counted (months) from the date of diagnosis to the date of death or last follow-up before study closure. The Kaplan–Meier method was used to estimate the overall survival.
Fifty-four patients colorectal carcinoma were studied retrospectively. They were admitted to the Gastroentrology centre, Mansoura University for primary surgical treatment between January 2006 and January 2008. Characteristics of all patients are summarized in Table 1. The age of the patients ranged from 28 to 73 years, with a mean age of 50.61±13.46 years. Of 54 patients, 31 (57.4%) were men and 23 (42.4%) were women. Thirty-three (61.1%) patients had cancer of the colon and 21 (38.9%) had rectal cancer.
Histopathologically, all the 54 tumors were adenocarcinomas. Eleven tumors were adenocarcinoma grade I (20.4%), 37 (68.5%) were grade II, and six (11.1%) were grade III. Staging was done according to the 6th edition of American Joint Cancer Council 2002 staging system (Greene et al., 2002). Fourteen patients (25.9%) were of stage I, 19 (35.2%) had stage II, 18 (33.3%) had stage III, and three (5.6%) had stage IV.
Evaluation of immunohistochemical expression of CXCR4 chemokine receptor depends on the nuclear staining of tumor cells. Twenty-three adenocarcinomas specimens (42.6%) were classified as CXCR4 chemokine receptor protein positive (Figs 1–3). In contrast, 31 adenocarcinomas specimens (57.4%) were CXCR4 chemokine receptor protein negative (Fig. 4).
Table 1 demonstrates immunohistochemical CXCR4 chemokine receptor expression in relation to different clinicopathologic data of the studied cases. CXCR4 expression varies in different tumor grades. The higher grades were associated with increased immunopositivity to CXCR4 chemokine receptor; it was expressed in 83.3% of GIII adenocarcinomas (five cases), 40.5% of GII adenocarcinomas (15 cases), and in 27.3% of GI adenocarcinomas (three cases). However, this relationship did not reach a statistically significant level (P=0.075).
Invasion of the stromal lymphatics and vascular channels was studied as a significant prognostic indicator for aggressive behavior. Tumors with lymphovascular invasion had significant increase in expression of CXCR4 chemokine receptor (P=0.000). CXCR4 chemokine receptor was expressed in 18 (78.3%) of 23 tumors with lymphovascular invasion compared with five (16.1%) of 31 tumors without lymphovascular invasion. Expression of CXCR4 chemokine receptor in the studied colorectal carcinomas showed a statistically significant increase in cases with nodal involvement (P=0.003). It was expressed in 13 (31.7%) of 41 specimens in cases with free lymph nodes (N0), whereas it was expressed in 10 (76.9%) of 13 specimens in cases with N1 and N2.
Similarly, positive expression of CXCR4 chemokine receptor had a statistically significant association with increase in the clinical stage of studied colorectal carcinomas (P=0.018). All stage IV tumors (100%) expressed nuclear CXCR4 chemokine receptor, whereas it was expressed in 11 (61.1%) stage III tumors, six (31.6%) stage II tumors, and three (21.4%) stage I tumors.
The relationship between positive CXCR4 chemokine receptor expression and age, sex, tumor location (colon vs. rectum), and T stage was statistically insignificant (P>0.05).
MVD of the tumor specimens was evaluated. Tumor specimens having values lower than the median were placed in the low MVD (Fig. 5) group, whereas those having higher values were labeled as tumor with high MVD (Fig. 6). Microvessel count of 25 was considered as cut-off value of high versus low MVD. Low MVD group accounted for 50.7% (28 of 54) of cases, whereas high MVD group accounted for 48% of tumor specimens (26 of 54).
The analytical study showed that out of 28 tumor specimens having low MVD, three had positive expression of CXCR4 chemokine receptor receptor in CRC accounting for 10.7%, which was much lower than the high MVD group, which showed positive CXCR4 chemokine receptor expression in 76.9% of cases (20 of 26) (P=0.000).
During the follow-up period, three patients developed metastasis. Specimens of both showed positive expression of CXCR4 chemokine receptor. Similarly, all the six cases suffered recurrence (11.1%) and showed CXCR4 chemokine receptor expression in their tumor tissue.
A statistically significant decrease in patient's disease-free survival was associated with CXCR4 chemokine receptor positivity. The mean survival was 17.2±4.7 in CXCR4 chemokine receptor-negative cases (Fig. 4), whereas it was 11±4.2 in CXCR4 chemokine receptor-positive cases (Table 2 and Fig. 7).
More than 100 years after postulation of the ‘seed and soil’ theory, the precise mechanisms determining the directional migration and invasion of diminished cancer cells into specific organs remain to be established. This theory was hypothesized by Paget (1989). Through his experience with breast cancer patients, Paget theorized that cancers metastasize preferentially to organs or sites that support and/or nurture the growth of cancer cells. Later, Ewing (1928) countered that the patterns of blood flow from the primary tumor could entirely account for the patterns of metastasis. His ‘mechanical’ hypothesis predicted that the circulation would deliver the greatest metastatic tumor burden to the first organ encountered. Neither model is absolute, as clinical examples of exceptions to both exist (Kim et al., 2006). Recent studies increasingly show that chemokines and their receptor were an important factor in this process of organ-selective metastasis (Ruffini et al., 2007; Speetjens et al., 2009).
CXCR4 chemokine receptor expression in different types of tumors (breast cancer, nasopharyngeal carcinoma, lung cancer, and hepatocellular carcinoma) and in some of these nuclear staining was correlated with clinical outcome, although with discrepant results (Kang et al., 2005). There was evidence of CXCR4 chemokine receptor expression simultaneous with other chemokine receptor in brain metastasis (Salmaggi et al., 2009). CXCR4 chemokine receptor was also expressed and functional on melanoma cells (Kim et al., 2006). Different cancers express different chemokine receptor, and the corresponding ligands were sometimes expressed at sites of tumor spread. There is one chemokine receptor, however, that seems to be expressed by a majority of cancer types and that is CXCR4 chemokine receptor (Zhang et al., 2007).
In the last few years, CXCR4 chemokine receptor was studied in colorectal carcinoma. Researchers suggest that the expression of CXCR4 chemokine receptor depends on the nuclear staining of tumor cells, whereas cytoplasmic expression may reflect an active form of the receptor that after its binding with the ligand enters into the cell by endocytosis (Salvucci et al., 2006). No explanation about nuclear CXCR4 chemokine receptor localization is available until now (Salmaggi et al. 2009).
This retrospective study was conducted on 54 cases with primary diagnosis of colorectal adenocarcinoma. We are aiming to investigate the expression of CXCR4 chemokine receptor in human colon cancer specimens using immunohistochemistry and study the relationship with clinical, pathologic factors, and prognosis of colon cancer. We also studied MVD to clarify the angiogenic properties of CXCR4 chemokine receptor.
Studying CXCR4 chemokine receptor immunohistochemically, we found positive expression in 23 (42.6%) adenocarcinoma specimens, whereas 31 (57.4%) specimens were CXCR4 chemokine receptor protein negative. The interpretation of CXCR4 chemokine receptor expression depended on the nuclear staining of tumor cells.
In this study, the relationship between positive nuclear CXCR4 chemokine receptor expression and age, sex, tumor location (colon vs. rectum), and T stage was statistically insignificant (P>0.05). This come also in agreement with the findings by Ottaiano et al. (2006) who found insignificant relationship between positive nuclear CXCR4 chemokine receptor expression and age, sex, tumor location as well as with T stage.
In this study, we analyzed the immunohistochemical CXCR4 chemokine receptor expression in relation to tumor grades. The higher grades were associated with increased immunopositivity to CXCR4 chemokine receptor. It was expressed in 83.3% of GIII adenocarcinomas (five cases), 40.5% of GII adenocarcinomas (15 cases), and in 27.3% of GI adenocarcinomas (three cases). However, this relationship did not reach a statistically significant level (P=0.075); this comes in agreement with a study done by Ottaiano et al. (2006).
In this study, tumors with lymphovascular invasion had significant increase in expression of CXCR4 chemokine receptor (P=0.000). CXCR4 chemokine receptor was expressed in 18 (78.3%) of 23 tumors with lymphovascular invasion compared with five (16.1%) of 31 tumors without lymphovascular invasion. Expression of CXCR4 chemokine receptor in the studied colorectal carcinomas showed a statistically significant increase in cases with nodal involvement (P=0.003). Similarly, positive expression of CXCR4 chemokine receptor had a statistically significant association with increase in the clinical stage of studied colorectal carcinomas (P=0.018). Ottaiano et al. (2006) studied 72 patients of stage II and stage III CRCs and discovered that 77.3% of CXCR4 chemokine receptor-positive patients had lymph node metastasis. In addition, Yoshitake et al. (2008) documented that CXCR4 chemokine receptor expression was significantly positive in CRCs with a high tumor stage and lymph node metastasis. Comparison of nuclear-type and cytomembrane-type CXCR4 chemokine receptor-positive CRCs revealed that the nuclear-type CRCs were significantly more likely to show poor differentiation and lymphatic invasion.
Related to this study, the low MVD group accounted for 50.7% (28 of 54) of cases, whereas the high MVD group accounted for 48% of tumor specimens (26 of 54). The analytical study showed that of 28 tumor specimens having low MVD, three had positive expression of CXCR4 chemokine receptor in CRC accounting for 10.7%, which was much lower than the high MVD group, which showed positive CXCR4 chemokine receptor expression in 76.9% (20 of 26) (P=0.000) of cases. This is an agreement with Zeelenberg et al. (2003) who reported in their study that the CXCR4 chemokine receptor is upregulated by the microenvironment and that isolated metastatic cells are likely to require CXCR4 chemokine receptor signals to initiate proliferation. They also suggested that CXCR4 chemokine receptor inhibitors have potential as anticancer agents to suppress outgrowth of micrometastases. Another study conducted by Guleng et al. (2005) stated that the SDF-1/CXCR4 axis plays a pivotal role in tumor progression through promoting tumor neovascularization. The SDF-1/CXCR4 axis does not always regulate tumor angiogenesis in a vascular endothelial growth factor-dependent manner. For example, the SDF-1/CXCR4 axis might contribute to functional vascular establishment by the regulation of endothelial tube formation.
Expression of CXCR4 chemokine receptor is a strong and independent predictor of early distant relapse in CRC. CXCR4 triggers a plethora of phenomena, including stimulation of clonogenic growth, induction of vascular endothelial growth factor release, and ICAM-1 upregulation. These data support the inhibition of CXCR4 chemokine receptor to prevent the development of CRC metastasis (Ottaiano et al., 2006).
In our study, the three cases showing metastasis as well as all the six (11.1%) cases suffered recurrence showed CXCR4 chemokine receptor expression in their tumor tissue. Statistically significant decrease in patient's disease-free survival was associated with CXCR4 chemokine receptor positivity. The mean survival was 17.2±4.7 in CXCR4 chemokine receptor-negative cases, whereas it was 11±4.2 in CXCR4 chemokine receptor-positive cases. This comes in agreement with the study by Speetjens et al. (2009) who showed that nuclear distribution of CXCR4 chemokine receptor in the tumor cells was inversely associated with disease-free and overall survival, whereas expression in the cytoplasm was not associated with prognosis. Even they documented that a high expression of nuclear-localized CXCR4 chemokine receptor in tumor cells is an independent predictor for poor survival for patients with CRC.
CXCR4 chemokine receptor was significantly upregulated in colorectal liver metastasis compared with the tumor neighboring liver tissues. These results are in line with recent findings that CXCR4 gene expression in primary CRC demonstrated significant associations with recurrence and survival suggesting CXCR4 chemokine receptor as a prognostic factor for poor disease outcome (Rubie et al., 2006). There was a significant association between high CXCR4 gene expression of primary CRC tumors and poor clinical outcomes. This finding was concordant with reports in other cancers identifying a correlation of CXCR4 chemokine receptor expression with disease outcome (Kim et al., 2006).
In conclusion, positive nuclear CXCR4 chemokine receptor expression associated with poor prognosis and could be a potential predictive factor for recurrence or metastasis of CRC patients. Thus, CXCR4 chemokine receptor may be a potential target for specific therapeutic interventions in the future.
1. Center MM, Jemal A, Smith RA, Ward E. Worldwide variations in colorectal cancer. Ca Cancer J Clin. 2009;59:366–378
2. El-Bolkainy TN, Sakr MA, Nouh AA, El-Din NH. A comparative study of rectal and colonic carcinoma: demographic, pathologic and TNM staging analysis. Journal of the Egyptian Nat Cancer Inst. 2006;18:258–263
3. Ewing J Neoplastic diseases. 19286th ed Philadelphia Saunders
4. Greene FL, Page DL, Fleming ID, Fritz A, Balch CM, Haller DG, Morrow M, editors . AJCC cancer staging manual. 20026th ed NewYork, NY Springer
5. Guleng B, Tateishi K, Ohta M, Kanai F, Jazag A, Ijichi H, et al. Blockade of the stromal cell derived Factor-1/CXCR4 axis attenuates in vivo tumor growth by inhibiting angiogenesis in a vascular endothelial growth factor − independent manner. Cancer Res. 2005;65:5864–5871
6. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, et al. Cancer statistics, 2004. CA Cancer J Clin. 2004;54:8–29
7. Kang H, Watkins G, Douglas-Jones A, Mansel RE, Jiang WG. The elevated level of CXCR4 is correlated with nodal metastasis of human breast cancer. Breast. 2005;14:360–367
8. Kim J, Mori T, Chen SL, Amersi FF, Martinez SR, Kuo C, et al. Chemokine receptor CXCR4 expression in patients with melanoma and colorectal cancer liver metastases and the association with disease outcome. Ann Surg. 2006;244:113–120
9. Kim J, Takeuchi H, Lam ST, Turner RR, Wang HJ, Kuo C, et al. Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival. J Clin Oncol. 2005;23:2744–2753
10. Kreuter M, Bieker R, Bielack SS, Auras T, Buerger H, Gosheger G, et al. Prognostic relevance of increased angiogenesis in osteosarcoma. Clin Cancer Res. 2004;10:8531–8537
11. Le Y, Gong W, Shen W, Li B, Dunlop NM, Wang JM. A burgeoning family of biological mediators: chemokines and chemokine receptors review. Archivum Immunologiae et Therapiae Experimentalis. 2000;48:143–150
12. Ottaiano A, Franco R, Talamanca AA, Liguori G, Tatangelo F, Delrio P, et al. Overexpression of both CXC chemokine receptor 4 and vascular endothelial growth factor proteins predicts early distant relapse in stage II-III colorectal cancer patients. Clin Cancer Res. 2006;12:2795–2803
13. Paget S. The distribution of secondary growths in cancer of the breast. Cancer Metastasis Rev. 1989;8:98–101
14. Rubie C, Frick VO, Wagner M, Weber C, Kruse B, Kempf K, et al. Chemokine expression in hepatocellular carcinoma versus colorectal liver metastases. World J Gastroenterol. 2006;12:6627–6633
15. Ruffini PA, Morandi P, Cabioglu N, Altundag K, Cristofanilli M. Manipulating the chemokine-chemokine receptor network to treat cancer. Cancer. 2007;109:2392–2404
16. Salmaggi A, Maderna E, Calatozzolo C, Gaviani P, Canazza A, Milanesi I, et al. Clinical study CXCL12, CXCR4 and CXCR7 expression in brain metastases. Cancer Biology&Therapy. 2009;8:1608–1614
17. Salvucci O, Bouchard A, Baccarelli A, Deschênes J, Sauter G, Simon R, et al. The role of CXCR4 receptor expression in breast cancer: a large tissue microarray study. Breast Cancer Res Treat. 2006;97:275–283
18. Schimanski CC, Schwald S, Simiantonaki N, Jayasinghe C, Gönner U, Wilsberg V, et al. Effect of chemokine receptors CXCR4 and CCR7 on the metastatic behaviour of human colorectal cancer. Clin Cancer Res. 2005;11:1743–1750
19. Speetjens FM, Liefers GJ, Korbee CJ, Mesker WE, van de Velde CJ, van Vlierberghe RL, et al. Nuclear localization of CXCR4 determines prognosis for colorectal cancer patients. Cancer Microenvironment. 2009;2:1–7
20. Vermeulen PB, Gasparini G, Fox SB, Colpaert C, Marson LP, Gion M, et al. Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours. Eur J Cancer. 2002;38:1564–1579
21. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1–8
22. Yoshitake N, Fukui H, Yamagishi H, Sekikawa A, Fujii S, Tomita S, et al. Expression of SDF-1a and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer. British Journal of Cancer. 2008;98:1682–1689
23. Zeelenberg IS, Ruuls-Van Stalle L, Roos E. The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases. Cancer Res. 2003;63:3833–3839
24. Zhang M, Iqbal K, Yu S. Positive expression and correlation of chemokine receptor CXCR4 with nodal metastasis and prognosis in colorectal cancer. Chinese-German Journal of Clinical Oncology. 2007;6:552–556
Keywords:©2011Egyptian Journal of Pathology
colorectal carcinoma; CXCR4; lymph node metastasis; microvessel density; prognosis