To elucidate the role of CXCR4 in bladder transitional cell carcinoma, examining CXCR4 expression in BTCC tissue and the relationship of CXCR4 with clinicopathological features. [Methods] The expression of CXCR4 was assessed in 18 cases of normal bladder tissues and 70 cases of BTCC tissues by immunohistochemistry and the association between CXCR4 expression and lymph node metastasis, as evaluated by lymphatic vessel density (LVD), was examined. [Results] The over-expression of CXCR4 was detected in 58/70 (82.9%) BTCC cases, whereas only 3/18 (16.7%) cases in normal bladder. The expression was significantly higher in BTCC than that in normal bladder tissues (P<0.01). The CXCR4 expression was significantly associated with tumor size, pathological grade, clinical stage, and pelvic lymph node metastasis (P<0.05). Multivariate analysis showed the CXCR4 expression and lymph node metastasis were the independent factors for disease-free survival (P<0.05). Meanwhile, the disease-free survival rate of the patients with higher CXCR4 expression was markedly lower than those with no or low CXCR4 expression (P<0.01). [Conclusion] CXCR4 were frequently expressed in BTCC. The over expression of CXCR4 may play a critical role in the invasion and metastasis of BTCC and examination of CXCR4 expression in biopsy specimens might be beneficial in predicting pelvic lymph node metastasis.
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Key words bladder transitional cell carcinoma (BTCC), CXCR4, lymph node metastasis, prognosis;
Bladder transitional cell carcinoma (BTCC) is the ninth most common cancer in humans, with a nearly three times higher incidence in men than in women. Radical cystectomy is the standard treatment for muscle-invasive bladder cancer. However, for patients with metastatic disease, chemotherapy is the first choice instead of surgical modality. Although BTCC is chemosensitive, response durations are not too long and the median survival is still low. Therefore, therapeutic innovation for treating patients with metastatic BTCC is desperately needed.
It has been reported that malignant cells can express chemokine receptors and respond to chemokine gradients and these may have something to do with the cancer metastasis[3,4]. CXCR4, as one chemokine receptor, appears to be expressed by a majority of cancer types. Meanwhile, its ligand CXCL12 are also expressed in kinds of cancers and play an important role in the growth and migration of cancer cells. At present, CXCR4 and CXCL12 have been attracting people's attention. Andreas et al have demonstrated the high expression of the CXCR4 receptor on bladder cancer cells and shown that the agonist CXCL12 stimulates signal transduction pathways which play an essential role in cancer metastasis. Maybe blockade of the CXCR4 receptor is an interesting treatment modality to delay disease progression in vivo in the future. However, there are few reports about the relationship of the CXCR4 expression and the clinicopathological features. Therefore, we examined the CXCR4 expression and clinical significance in BTCC.
Patients and Methods
Patients and Samples
We obtained approval for this study from The Ethics Committee of Xiangya Hospital, Central South University. Written informed consent was obtained from all patients before enrolled into the study. The study included 70 patients presenting with BTCC in 2004-2006 at the authors' hospital. No patients received chemotherapy or radiotherapy before surgery. Normal urothelium tissues were obtained from 18 patients undergoing open bladder surgery due to diseases other than bladder carcinoma, such as BPH, trauma and so on. Patients' clinical data had been collected before the study (Table 1). All biopsy specimens were obtained before starting treatment; each specimen was divided into halves and the two specimens fixed in 10% formaldehyde solution for histopathological examination. All the patients presenting with BTCC were received radical cystectomy included bladder and surrounding fat tissue and remote end of ureteral and pelvic lymphadenectomy. The cranial border of the lymphadenectomy was the level of the inferior mesenteric artery, the lateral border was the genitofemoral nerve and the caudal border was pelvic floor. Histological cell type of the BTCC specimen was proposed by the WHO 1973 classification: 18 were classified as grade I, 23 were grade II and 29 as grade III. The clinical stage was reviewed based on the UICC-TNM staging system: 9, 22, 31 and 8 were stage T1, T2, T3 and T4, respectively. The median (range) age of the patients at the time of treatment was 61 (41-75) years.
The immunohistochemical staining and evaluation for CXCR4 and LVD
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Immunohistochemical staining for CXCR4 protein was carried out by using streptavidin-peroxidase conjugated method (SP Ready-To-Use kit; Beijing Zhongshan Biotechnology, Beijing, China). Deparaffinized and rehydrated were been done before paraffin-embedded sections (3 μm thick) were immersed in 0.01M citric buffer (PH 6.0). The slides were incubated with 3% hydrogen peroxide for 10 min and then incubated in normal goat serum for 10 min at room temperature. Rabbit anti-human CXCR4 polyclonal antibody (Wuhan Boster Biological Technology, Wuhan, China) was used at a dilution of 1:50 as the first antibody according to the manufacturer's instructions. Sections were then incubated with goat anti-polyvalent antibody for 10 min and subsequently with streptavidin peroxidase for 10 min. The slides were visualized using diaminobenzidine and counterstained with haematoxylin before observation. The addition of the first antibody, rabbit anti-human CXCR4, was omitted in the protocols for negative controls.
10 different areas containing tumor cells were evaluated randomly for each slide under a low power microscopy field by two independent experienced investigators without prior knowledge of the patient disease data. ≥100 tumor cells were examined per field. Two scoring systems including staining intensity and percentage of stained cells were used; the staining intensity was scored on a semi-quantitative 4-point scale: 0, equivalent to the negative control; 1, weak cytoplasmic and nuclear stain slightly darker than the negative control; 2, moderate stain, defined as an intensity of score 1-3; 3, intense stain, equivalent to or darker than the positive control. The percentage of stained cells was also scored on a semi-quantitative 4-point scale: 0, <10%; 1, 10-25%; 2, 25-50%; 3 >50%. Finally, combining the score of staining intensity and percentage of stained cells: 0-1 was -, 2 was +, 3-4 was ++, 5-6 was +++.
For immunohistochemical staining for lymphatic vessel density (LVD), sections were examined by two independent experienced investigators without prior knowledge of the patient information as described by Weidner et al. Regions with the most intense lymphangiogenesis (hot spot) were selected under low power magnification (-100). Lymphatic vessels were counted at high magnification (-200) for each hot spot after 5 hot spots were determined following agreement of the two investigators. For each case, the mean count of the 5 fields was calculated as LVD.
CT evaluation for Lymph Node Metastasis of BTCC
All patients were received CT scans of abdomen and pelvis at least 5 days before surgery. Philips CT-TWIN (Philips, Andover, MA) was used and its slice thickness and layer are both 10 mm. Ultravist (80 ml, Bayer Healthcare Pharmaceuticals, Wayne, NJ) was used as the contrast agent. The CT images were reevaluated by the urologists and uroradiologists without knowledge of the final pathological results. The standard for positive lymph node metastases was any pelvic lymph node >10 mm.
Statistical analyses were carried out by using SPSS, version 16.0 (SPSS Inc, Chicago, IL, USA). The association between the variables was tested by using the chi-square test, Fisher's exact probability test, Mann-Whitney U-test or stepwise logistic regression analysis. Survival curves were plotted by the Kaplan-Meier method and differences were examined by the log-rank test. Cox proportional hazard model was used as a multivariate analysis to assess the effect of tumor variables on prognosis. P values <0.05 were considered statistically significant in all tests
CXCR4 expression levels in BTCC Patients and Normal Controls
CXCR4 was expressed in the cytoplasm and nuclear of tumor cells, which was quite little in the normal transitional epithelium. The frequency of detectable CXCR4 in BTCC was significantly higher than that in normal urothelium tissue (P<0.01) (Fig. 1); CXCR4 expression significantly correlated with tumor size, pathological grade, clinical stage, lymph vessel involvement, pelvic lymph node metastasis and recurrence(all P<0.01) except the age (P>0.05) (Table 1). The mean (SD) LVD in BTCC with CXCR4 expression were 14.33±3.263, which were 8.76±2.897 in BTCC without CXCR4 expression and they were significant different (P<0.01) (Fig . 2).
Correlation between Lymph node metastasis and clinicopathological features
Pelvic lymph node metastasis was significantly associated with some clinicopathological features such as tumor size, pathological grade, clinical stage, lymph vessel involvement and CXCR4 expression. CXCR4 expression was shown to be the independent predictor for lymph node metastasis in multivariate analysis based on a Cox proportional hazard model (Odds ratio (95% CI) was 39.23 (3.98-109.4), P<0.01). On the other hand, sensitivity, specificity and accuracy of CXCR4 expression and CT scan in the evaluation of lymph node metastasis were 91.3%, 33.3%, 71.4% (Table 1) and 41.2%, 90.7%, 67.2% (data were not shown), respectively.
Significance of CXCR4 Expression in Predicting the Prognosis
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During the follow-up period, 27 patients died of the tumor metastasis, including local invasion and distant metastasis while 3 patients died of other causes. The disease-free survival rate of patients with high CXCR4 expression (++, +++) was significant lower than those with no or low CXCR4 expression (-, +) (P<0.01) (Fig. 3). Meanwhile, tumor size, pathological grade, clinical stage, lymph vessel involvement, pelvic lymph node metastasis and CXCR4 expression were significant for disease-free survival. Patients with negative CXCR4 expression had markedly better prognoses than those with the positive CXCR4 expression in the univariate analysis (P<0.05). CXCR4 expression (P=0.017, with β, SEM, Wald value and Exp(B) of 1.328, 0.402, 5.721 and 0.247) and lymph node metastasis (P=0.011, withβ, SEM, Wald value and Exp(B) of 1.443, 0.622, 6.441 and 0.178) were revealed as independent predictors of disease-free survival by multivariate analysis based on a Cox proportional hazard model.
Chemokines are small secreted proteins (8-11 kDa) that may play a crucial role in the regulation of leukocyte adherence to endothelial cells, leukocyte migration through transendothelial membranes, and tissue invasion. It also has been reported that the development of hematopoetic cells and the homing of hematopoetic cells to bone marrow were regulated by them[9,10]. Moreover, more and more researches have proposed that the coupling of chemokines to their specific receptors were involved in the activation of numerous signaling pathways, which were associated with the growth and metastasis of tumor cells.
.CXCR4, as a G protein-coupled receptor, comprises seven transmembrane domains and this protein has been revealed as a co-receptor for T-trophic HIV. CXCL12, as its specific ligand, is widely expressed in normal tissues. They play a fundamental role in foetal development, mobilization of haemopoietics stem cells, and trafficking of naïve lymphocytes. Recently, Some researches found that CXCR4 was over expressed on the surface of tumor cells than normal tissue cells, which might promote the metastasis into some target organs[12,13]. CXCR4/CXCL12 receptor/ligand interaction, as one of chemokine and cytokine receptors interactions, has been shown to positive regulate the growth and invasive ability of kinds of tumor cells, such as renal cancer, breast cancer, neuroblastoma and non-small cell lung cancer[14-17]. It also has been demonstrated that CXCR4 mRNA expression level in BTCC cell lines was higher than that in normal human urothelium. The addition of CXCL12 was found to increase Matrigel invasion and cell growth, but was not effective in increasing intracellular calcium in BTCC cell lines. These effects could be reversed by a small peptide antagonist AMD3100 which was a highly selective antagonist of the CXCR4 receptor. Moreover, the expression level of CXCR4 in BTCC cells was claimed to be correlated with the advancement of pathological tumor grade[6,18,19]. To date, nearly all researches data about the CXCR4 expression are some experiments in vitro and there are few reports about the association of CXCR4 expression level and the clinicopathological features in BTCC.
In the present study, we first proved that the CXCR4 expression level was higher in BTCC cells than that in normal human urothlium, which was consistent with previous data[6,19]. In addition, CXCR4 expression was further examined to determine whether it was associated with some clinicopathological features. The results revealed that the frequency of detectable CXCR4 expression was notably higher in patients with larger tumor size, higher pathological grade, later clinical stage, lymph vessel involvement and pelvic lymph node metastasis. These findings indicated that CXCR4 expression was associated with lymphatic invasion and lymph node metastasis.
On the other hand, it remains a hard question that how to evaluate lymph node involvement and prognosis earlier. Lymphangiography, computed tomography (CT) or magnetic resonance imaging (MRI) are used to diagnose lymph node metastasis in BTCC patients. All three modalities provide similar results in detecting lymph node metastasis. CT is commonly used to evaluate BTCC in our hospital. However, the sensitivity and accuracy of CT is still unsatisfactory until now. Moreover, only metastatic lymph nodes of significant size (at least 0.5 cm, stated by some researchers) can be detected by CT, but the clinical stage was often quite late and the prognosis was very poor at that time. Thus a more sensitive tool to detect micro lymph node metastasis earlier is desperately needed. In the present study, we considered the CXCR4 expression level in BTCC cells could be used to evaluate micro lymph node metastasis and prognosis because the sensitivity of CT was lower than CXCR4 (41.2% vs. 91.3%). However, the specificity of CT was higher than CXCR4 (90.7% vs. 33.3%), thus the results showed that a combination of CT and CXCR4 could provide more helpful information about the early diagnosis of lymph node metastasis of BTCC.
It has reached a consensus that lymph node metastasis modality is the primary metastasis modality of BTCC cells. Some previous data have shown a significant association between LVD and lymph node metastasis in kinds of tumor cells including BTCC, gastric carcinoma and prostate adenocarcinoma. Therefore, we examined the correlation between CXCR4 expression and LVD in BTCC. Obviously, we observed that LVD positive correlated with CXCR4 expression significantly. These results further implicated that CXCR4 expression were associated with lymph node metastasis.
Finally, the disease-free survival was analyzed. Patients with high CXCR4 expression had a markedly poorer prognosis than those with no or low CXCR4 expression and the data showed that CXCR4 expression was the independent predictor of prognosis. Inhibition of CXCR4 pathway may represent a novel therapeutic strategy and further investigations are needed.
CXCR4 were frequently expressed in BTCC, which positive correlated with some clinicopathological features, including tumor size, pathological grade, clinical stage, lymph vessel involvement, pelvic lymph node metastasis and recurrence. The over expression of CXCR4 may play a critical role in the invasion and metastasis of BTCC and the examination of CXCR4 expression in biopsy specimens might be beneficial in predicting pelvic lymph node metastasis.
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