The Immunohistochemical Staining And Evaluation Biology Essay

To clarify the function of CXCR4 in vesica transitional cell carcinoma, analyzing CXCR4 look in BTCC tissue and the relationship of CXCR4 with clinicopathological characteristics. [ Methods ] The look of CXCR4 was assessed in 18 instances of normal vesica tissues and 70 instances of BTCC tissues by immunohistochemistry and the association between CXCR4 look and lymph node metastasis, as evaluated by lymphatic vas denseness ( LVD ) , was examined. [ Results ] The over-expression of CXCR4 was detected in 58/70 ( 82.9 % ) BTCC instances, whereas merely 3/18 ( 16.7 % ) instances in normal vesica. The look was significantly higher in BTCC than that in normal vesica tissues ( P & A ; lt ; 0.

01 ) . The CXCR4 look was significantly associated with tumour size, pathological class, clinical phase, and pelvic lymph node metastasis ( P & A ; lt ; 0.05 ) . Multivariate analysis showed the CXCR4 look and lymph node metastasis were the independent factors for disease-free endurance ( P & A ; lt ; 0.

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05 ) . Meanwhile, the disease-free endurance rate of the patients with higher CXCR4 look was markedly lower than those with no or low CXCR4 look ( P & A ; lt ; 0.01 ) . [ Conclusion ] CXCR4 were often expressed in BTCC. The over look of CXCR4 may play a critical function in the invasion and metastasis of BTCC and scrutiny of CXCR4 look in biopsy specimens might be good in foretelling pelvic lymph node metastasis.

Key words bladder transitional cell carcinoma ( BTCC ) , CXCR4, lymph node metastasis, forecast ;


Bladder transitional cell carcinoma ( BTCC ) is the 9th most common malignant neoplastic disease in worlds, with a about three times higher incidence in work forces than in adult females [ 1 ] . Extremist cystectomy is the standard intervention for muscle-invasive vesica malignant neoplastic disease. However, for patients with metastatic disease, chemotherapy is the first pick alternatively of surgical mode. Although BTCC is chemosensitive, response continuances are non excessively long and the average endurance is still low [ 2 ] . Therefore, curative invention for handling patients with metastatic BTCC is urgently needed.It has been reported that malignant cells can show chemokine receptors and respond to chemokine gradients and these may hold something to make with the malignant neoplastic disease metastasis [ 3,4 ] . CXCR4, as one chemokine receptor, appears to be expressed by a bulk of malignant neoplastic disease types. Meanwhile, its ligand CXCL12 are besides expressed in sorts of malignant neoplastic diseases and play an of import function in the growing and migration of malignant neoplastic disease cells [ 5 ] .

At present, CXCR4 and CXCL12 have been pulling people ‘s attending. Andreas et al [ 6 ] have demonstrated the high look of the CXCR4 receptor on vesica malignant neoplastic disease cells and shown that the agonist CXCL12 stimulates signal transduction tracts which play an indispensable function in malignant neoplastic disease metastasis. Possibly encirclement of the CXCR4 receptor is an interesting intervention mode to detain disease patterned advance in vivo in the hereafter.

However, there are few studies about the relationship of the CXCR4 look and the clinicopathological characteristics. Therefore, we examined the CXCR4 look and clinical significance in BTCC.

Patients and Methods

Patients and Samples

We obtained blessing for this survey from The Ethics Committee of Xiangya Hospital, Central South University. Written informed consent was obtained from all patients before enrolled into the survey. The survey included 70 patients showing with BTCC in 2004-2006 at the writers ‘ infirmary. No patients received chemotherapy or radiation therapy before surgery.

Normal urothelium tissues were obtained from 18 patients undergoing unfastened vesica surgery due to diseases other than vesica carcinoma, such as BPH, injury and so on. Patients ‘ clinical informations had been collected before the survey ( Table 1 ) . All biopsy specimens were obtained before get downing intervention ; each specimen was divided into halves and the two specimens fixed in 10 % formaldehyde solution for histopathological scrutiny. All the patients showing with BTCC were received extremist cystectomy included vesica and environing fat tissue and distant terminal of ureteral and pelvic lymphadenectomy. The cranial boundary line of the lymphadenectomy was the degree of the inferior mesenteric arteria, the sidelong boundary line was the genitofemoral nervus and the caudal boundary line was pelvic floor. Histological cell type of the BTCC specimen was proposed by the WHO 1973 categorization: 18 were classified as class I, 23 were grade II and 29 as class III. The clinical phase was reviewed based on the UICC-TNM theatrical production system: 9, 22, 31 and 8 were phase T1, T2, T3 and T4, severally. The median ( scope ) age of the patients at the clip of intervention was 61 ( 41-75 ) old ages.

The immunohistochemical staining and rating for CXCR4 and LVD

Immunohistochemical staining for CXCR4 protein was carried out by utilizing streptavidin-peroxidase conjugated method ( SP Ready-To-Use kit ; Beijing Zhongshan Biotechnology, Beijing, China ) . Deparaffinized and rehydrated were been done before paraffin-embedded subdivisions ( 3 ?m midst ) were immersed in 0.01M citric buffer ( PH 6.0 ) . The slides were incubated with 3 % H peroxide for 10 min and so incubated in normal caprine animal 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 harmonizing to the maker ‘s instructions. Sections were so incubated with caprine animal anti-polyvalent antibody for 10 min and later with streptavidin peroxidase for 10 min. The slides were visualized utilizing diaminobenzidine and counterstained with haematoxylin before observation. The add-on of the first antibody, rabbit anti-human CXCR4, was omitted in the protocols for negative controls.10 different countries incorporating tumour cells were evaluated indiscriminately for each slide under a low power microscopy field by two independent experienced research workers without anterior cognition of the patient disease informations. ?100 tumour cells were examined per field.

Two hiting systems including staining strength and per centum of stained cells were used ; the staining strength was scored on a semi-quantitative 4-point graduated table: 0, tantamount to the negative control ; 1, weak cytoplasmic and atomic discoloration somewhat darker than the negative control ; 2, moderate discoloration, defined as an strength of mark 1-3 ; 3, intense discoloration, tantamount to or darker than the positive control. The per centum of stained cells was besides scored on a semi-quantitative 4-point graduated table: 0, & A ; lt ; 10 % ; 1, 10-25 % ; 2, 25-50 % ; 3 & A ; gt ; 50 % . Finally, uniting the mark of staining strength and per centum of stained cells: 0-1 was – , 2 was + , 3-4 was ++ , 5-6 was +++ .For immunohistochemical staining for lymphatic vas denseness ( LVD ) , subdivisions were examined by two independent experienced research workers without anterior cognition of the patient information as described by Weidner et al [ 7 ] . Regions with the most intense lymphangiogenesis ( hot topographic point ) were selected under low power magnification ( -100 ) . Lymphatic vass were counted at high magnification ( -200 ) for each hot topographic point after 5 hot musca volitanss were determined following understanding of the two research workers. For each instance, the average count of the 5 Fieldss was calculated as LVD.

CT rating for Lymph Node Metastasis of BTCC

All patients were received CT scans of venters and pelvic girdle at least 5 yearss earlier surgery.

Philips CT-TWIN ( Philips, Andover, MA ) was used and its piece thickness and bed are both 10 millimeter. Ultravist ( 80 milliliter, Bayer Healthcare Pharmaceuticals, Wayne, NJ ) was used as the contrast agent. The CT images were reevaluated by the urologists and uroradiologists without cognition of the concluding pathological consequences. The criterion for positive lymph node metastases was any pelvic lymph node & A ; gt ; 10 millimeter.

Statistical Analysis

Statistical analyses were carried out by utilizing SPSS, version 16.0 ( SPSS Inc, Chicago, IL, USA ) . The association between the variables was tested by utilizing the chi-square trial, Fisher ‘s exact chance trial, Mann-Whitney U-test or stepwise logistic arrested development analysis.

Survival curves were plotted by the Kaplan-Meier method and differences were examined by the log-rank trial. Cox relative jeopardy theoretical account was used as a multivariate analysis to measure the consequence of tumour variables on forecast. P values & A ; lt ; 0.

05 were considered statistically important in all trials


CXCR4 look degrees in BTCC Patients and Normal Controls

CXCR4 was expressed in the cytol and atomic of tumour cells, which was rather small in the normal transitional epithelial tissue. The frequence of noticeable CXCR4 in BTCC was significantly higher than that in normal urothelium tissue ( P & A ; lt ; 0.01 ) ( Fig. 1 ) ; CXCR4 look significantly correlated with tumour size, pathological class, clinical phase, lymph vas engagement, pelvic lymph node metastasis and return ( all P & A ; lt ; 0.01 ) except the age ( P & A ; gt ; 0.05 ) ( Table 1 ) . The mean ( SD ) LVD in BTCC with CXCR4 look were 14.33±3.

263, which were 8.76±2.897 in BTCC without CXCR4 look and they were important different ( P & A ; lt ; 0.

01 ) ( Fig. 2 ) .

Correlation between Lymph node metastasis and clinicopathological characteristics

Pelvic lymph node metastasis was significantly associated with some clinicopathological characteristics such as tumour size, pathological class, clinical phase, lymph vas engagement and CXCR4 look. CXCR4 look was shown to be the independent forecaster for lymph node metastasis in multivariate analysis based on a Cox relative jeopardy theoretical account ( Odds ratio ( 95 % CI ) was 39.23 ( 3.98-109.4 ) , P & A ; lt ; 0.01 ) .

On the other manus, sensitiveness, specificity and truth of CXCR4 look and CT scan in the rating of lymph node metastasis were 91.3 % , 33.3 % , 71.4 % ( Table 1 ) and 41.

2 % , 90.7 % , 67.2 % ( informations were non shown ) , severally.

Significance of CXCR4 Expression in Predicting the Prognosis

During the follow-up period, 27 patients died of the tumour metastasis, including local invasion and distant metastasis while 3 patients died of other causes. The disease-free endurance rate of patients with high CXCR4 look ( ++ , +++ ) was important lower than those with no or low CXCR4 look ( – , + ) ( P & A ; lt ; 0.

01 ) ( Fig. 3 ) . Meanwhile, tumour size, pathological class, clinical phase, lymph vas engagement, pelvic lymph node metastasis and CXCR4 look were important for disease-free endurance.

Patients with negative CXCR4 look had markedly better forecasts than those with the positive CXCR4 look in the univariate analysis ( P & A ; lt ; 0.05 ) . CXCR4 look ( P=0.017, with ? , SEM, Wald value and Exp ( B ) of 1.328, 0.402, 5.

721 and 0.247 ) and lymph node metastasis ( P=0.011, with? , SEM, Wald value and Exp ( B ) of 1.443, 0.622, 6.

441 and 0.178 ) were revealed as independent forecasters of disease-free endurance by multivariate analysis based on a Cox relative jeopardy theoretical account.


Chemokines are little secreted proteins ( 8-11 kDa ) that may play a important function in the ordinance of leukocyte attachment to endothelial cells, leukocyte migration through transendothelial membranes, and tissue invasion [ 8 ] . It besides has been reported that the development of hematopoetic cells and the homing of hematopoetic cells to cram marrow were regulated by them [ 9,10 ] . Furthermore, more and more researches have proposed that the yoke of chemokines to their specific receptors were involved in the activation of legion signaling tracts, which were associated with the growing and metastasis of tumour cells.

.CXCR4, as a G protein-coupled receptor, comprises seven transmembrane spheres and this protein has been revealed as a co-receptor for T-trophic HIV [ 11 ] . CXCL12, as its specific ligand, is widely expressed in normal tissues. They play a cardinal function in fetal development, mobilisation of haemopoietics stem cells, and trafficking of naive lymph cells [ 9 ] . Recently, Some researches found that CXCR4 was over expressed on the surface of tumour cells than normal tissue cells, which might advance the metastasis into some mark variety meats [ 12,13 ] . CXCR4/CXCL12 receptor/ligand interaction, as one of chemokine and cytokine receptors interactions, has been shown to positive modulate the growing and invasive ability of sorts of tumour cells, such as nephritic malignant neoplastic disease, chest malignant neoplastic disease, neuroblastoma and non-small cell lung malignant neoplastic disease [ 14-17 ] . It besides has been demonstrated that CXCR4 mRNA look degree in BTCC cell lines was higher than that in normal human urothelium.

The add-on of CXCL12 was found to increase Matrigel invasion and cell growing, but was non effectual in increasing intracellular Ca in BTCC cell lines. These effects could be reversed by a little peptide adversary AMD3100 which was a extremely selective adversary of the CXCR4 receptor. Furthermore, the look degree of CXCR4 in BTCC cells was claimed to be correlated with the promotion of pathological tumour grade [ 6,18,19 ] . To day of the month, about all researches informations about the CXCR4 look are some experiments in vitro and there are few studies about the association of CXCR4 look degree and the clinicopathological characteristics in BTCC.In the present survey, we foremost proved that the CXCR4 look degree was higher in BTCC cells than that in normal human urothlium, which was consistent with old informations [ 6,19 ] .

In add-on, CXCR4 look was further examined to find whether it was associated with some clinicopathological characteristics. The consequences revealed that the frequence of noticeable CXCR4 look was notably higher in patients with larger tumour size, higher pathological class, subsequently clinical phase, lymph vas engagement and pelvic lymph node metastasis. These findings indicated that CXCR4 look was associated with lymphatic invasion and lymph node metastasis.On the other manus, it remains a difficult inquiry that how to measure lymph node engagement and forecast earlier. Lymphangiography, computed imaging ( CT ) or magnetic resonance imagination ( MRI ) are used to name lymph node metastasis in BTCC patients. All three modes provide similar consequences in observing lymph node metastasis. CT is normally used to measure BTCC in our infirmary.

However, the sensitiveness and truth of CT is still unsatisfactory until now [ 20 ] . Furthermore, merely metastatic lymph nodes of important size ( at least 0.5 centimeter, stated by some research workers ) can be detected by CT, but the clinical phase was frequently rather late and the forecast was really hapless at that clip. Thus a more sensitive tool to observe micro lymph node metastasis earlier is urgently needed. In the present survey, we considered the CXCR4 look degree in BTCC cells could be used to measure micro lymph node metastasis and forecast because the sensitiveness 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 % ) , therefore the consequences showed that a combination of CT and CXCR4 could supply more helpful information about the early diagnosing of lymph node metastasis of BTCC.

It has reached a consensus that lymph node metastasis mode is the primary metastasis mode of BTCC cells. Some old informations have shown a important association between LVD and lymph node metastasis in sorts of tumour cells including BTCC [ 21 ] , stomachic carcinoma [ 22 ] and prostate glandular cancer [ 23 ] . Therefore, we examined the correlativity between CXCR4 look and LVD in BTCC. Obviously, we observed that LVD positive correlated with CXCR4 look significantly. These consequences farther implicated that CXCR4 look were associated with lymph node metastasis.Finally, the disease-free endurance was analyzed.

Patients with high CXCR4 look had a markedly poorer forecast than those with no or low CXCR4 look and the information showed that CXCR4 look was the independent forecaster of forecast. Inhibition of CXCR4 tract may stand for a fresh curative scheme and farther probes are needed.


CXCR4 were often expressed in BTCC, which positive correlated with some clinicopathological characteristics, including tumour size, pathological class, clinical phase, lymph vas engagement, pelvic lymph node metastasis and return. The over look of CXCR4 may play a critical function in the invasion and metastasis of BTCC and the scrutiny of CXCR4 look in biopsy specimens might be good in foretelling pelvic lymph node metastasis.

Conflict of involvement

None declared.


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