Renal cell carcinoma (RCC) is a common tissue tumor that occurs across all age groups and has become one of the types of cancer with the fastest increasing incidence. Due to the resistance of RCC chemo- and radiotherapy, surgery is the only currently effective treatment. Therefore, specific markers for the diagnosis and prognosis of RCC are expected to result in novel methods of treatment. Ecto-5′-nucleotidase, also termed cluster of differentiation (CD)73, is a protein that is activated in several types of aggressive cancer and may promote cancer progression. CD73 was examined in the present study to determine the association between the protein and RCC. The expression levels of CD73 in 159 RCC tissue sections and 30 paratumorous normal renal tissue samples obtained from 235 patients that underwent nephrectomy were examined by immunohistochemical staining. By contrast, the expression level of P-glycoprotein (P-gp), a potential prognostic factor in RCC, was also examined in 85 RCC and 13 normal tissue samples. Intense CD73 expression was identified in 75 out of 159 RCC cell membranes compared with normal renal tissues. In contrast, there was high P-gp expression in the blood vessels of 42 out of 85 RCC tissues and there was no significant difference between the P-gp expression identified in RCC cells (34 out of 85) and the cell membrane of normal renal cells (2 out of 13). The expression level of CD73 in RCC cells was significantly associated with tumor type, tumor node metastasis (TNM) stage, and tumor grade. However, the expression of P-gp in RCC cells was only associated with the TNM stage and tumor grade. Using a multivariable Cox regression analysis, it was found that the median survival rate of RCC patients with intense CD73 expression in RCC cells was 62.06±5.35 months, which was drastically shorter compared with rare CD73 expression (103.72±3.67 months). In conclusion, the expression level of CD73 is significantly associated with RCC tumor progression and may serve as a favorable marker for the diagnosis and prognosis of RCC, in addition to being a therapeutic target for the treatment of RCC.
Renal cell carcinoma (RCC) is the most common malignant kidney neoplasm that occurs across all age groups, and the disease accounts for ~3% of all cancers worldwide (
P-glycoprotein (P-gp) is a drug efflux pump that is widely associated with the chemoresistance of a variety of tumors (
Ecto-5′-nucleotidase, also termed cluster of differentiation (CD)73, is a glycosyl phosphatidylinositol-linked membrane protein found on the surface of a variety of cell types (
Pathological tissues were collected from patients with RCC who had undergone surgery between January 2004 and July 2012 at the Second Hospital of Lanzhou University (Lanzhou, China). A total of 235 patients aged between 35 and 87 years of age (median, 58 years) that were diagnosed with RCC and 30 transitional-normal tissues collected from certain patients were selected for the present retrospective study. Pathological confirmation was conducted on standard sections stained with hematoxylin and eosin according to the World Health Organization guidelines (
The expression levels of CD73 and P-gp were determined using a two-step immunohistochemical assay procedure. Briefly, P-gp was detected using a PV-6000 Polymer Detection System (Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China). Surgically resected specimens were fixed in 10% formalin and cut into 4-µm thick slices. The sections were subsequently mounted on Superfrost Plus slides (Zhongshan Golden Bridge Biotechnology, Co., Ltd.), de-waxed with xylene and gradually hydrated. Antigen retrieval was then achieved by pressure-cooking the samples in 0.01 M citrate buffer (pH 6.0) for 2 min, cooling to room temperature, washing with PBS and incubating in 3% hydrogen peroxide for 10 min. Monoclonal rabbit anti-human CD73 (cat. no. ab115289; Abcam, Cambridge, MA, USA) and monoclonal mouse anti-human P-gp (cat. no. zm0189; Zhongshan Golden Bridge Biotechnology, Co., Ltd.) antibodies were used as primary antibodies at a 1:200 dilution, with 50 µl of solution being used for each section. Mouse IgG (Abcam) was used as a control. The sections were incubated with the primary antibodies for 2 h at 37°C, washed with PBS, incubated with horseradish peroxidase-conjugated polyclonal sheep anti-rabbit (cat. no. pv6000) or anti-mouse (cat. no. pv6000) IgG secondary antibodies (Zhongshan Golden Bridge Biotechnology, Co., Ltd.) for 30 min at 37°C and then washed three times in 0.1% Tween-20 (Zhongshan Golden Bridge Biotechnology, Co., Ltd.). Subsequently, the sections were developed with diaminobenzidine tetrahydrochloride (DAB) and selected samples were counterstained with hematoxylin. The immunohistochemical assays were performed within seven days of section preparation. To prevent antigen degradation, the sections were stored at 4°C prior to analysis.
The results from the immunohistochemical staining of tissue slides were independently evaluated by two pathologists in a double-blind process. Immunohistochemical staining of the tissue slides indicated whether the stained CD73 and P-gp proteins were located in the cytoplasm, cell membrane or vascular wall. A semi-quantitative scoring system was developed (
The data were analyzed using SPSS software, version 19.0 (IBM, Armonk, NY, USA). Categorical data were reported as counts and percentages. Fisher's exact test and the χ2 test for trends were used to assess the significance of associations between the expression of CD73 or P-gp. Associations between clinicopathological parameters were tested using the Mann-Whitney
A total of 235 patients were included in the present study, which used the intact data from 205 RCC tissues, comprising 157 clear cell cancer and 48 urothelial carcinoma tissues, and 30 normal renal tissues. The patients ranged in age between 35 and 87 years (58.1±5.2 years) and the male to female ratio was 137:98. The mean tumor size was 7.6±3.2 cm. A total of 89 RCC patients possessed lesions classified as stage I, 78 were classified as stage II, 33 were classified as stage III and 5 were classified as stage IV. The median follow-up time was 78 months (range, 1–118 months) and a total of 14 patients (6.83%) succumbed to the disease.
The analysis of CD73 expression was performed in 159 tumor samples, consisting of 124 clear cell RCC and 35 uroepithelium cell carcinoma tissues, and 30 paratumorous normal renal tissue samples (
Cells were classified into three levels according to the percentage and intensity of CD73 staining (
The present analyses revealed that clinicopathological parameters, including the tumor type and an advanced tumor stage, were significantly associated with CD73 expression in RCC tissue cells. CD73 expression was also associated with an increased tumor grade (
Based on analysis of the TNM stage, P-gp expression in RCC tissue cells was also found to be associated with tumor differentiation (
At the end of the five-year follow-up, 14 out of 189 patients had succumbed to RCC. Therefore, the overall five-year survival rate of RCC patients was 80.60%. The five-year survival rate of patients with intense CD73 expression in RCC tissue was 67.6%, whereas the rate was 91.7% in patients with low CD73 expression (P<0.001). The median survival of RCC patients with intense CD73 expression was 78.00 months (range, 37.63–118.37 months;
The five-year survival rate of patients with intense P-gp expression was 44.1%, which was not significantly increased compared with the five-year survival rate of 27.1% in patients with low P-gp expression (Log-rank analysis, P=0.957; Breslow test, P=0.795). Patients with RCC that exhibited intense P-gp expression demonstrated a median survival of 47.00 months (range, 39.83–54.17 months;
In addition, multivariable Cox regression analysis, including gender, age, tumor type, TNM, tumor stage and grade, indicated that CD73 expression and histological grade, but not P-gp expression, were strongly associated with RCC prognosis (
In the present study, the expression levels of CD73 and P-gp were examined in RCC tumor and surrounding normal kidney tissues. An association was identified between intense CD73 expression and clear cell RCC, high TNM stage, high tumor grade and a low five-year survival rate, which indicates that CD73 is involved in the progression of clear cell RCC and may be used as a biomarker for the diagnosis and prognosis of RCC.
Previous studies have revealed that CD73 is applicable as a cell-surface marker of malignant tumors, including those in bladder cancer (
An increasing quantity of novel evidence has revealed that cancer stem cells (CSCs) may exist in RCC (
A high level of CD73 expression is associated with a poor prognosis in colorectal cancer (
Inflammatory hypoxia (
Although the vascular expression of CD73 in RCC samples was not significantly different from that of normal renal cells, CD73 may play other roles in RCC progression. CD73 is known to participate in leukocyte extravasation from blood in endothelial cells and lymphatics (
RCC is resistant to antitumor drugs, implying high expression levels of membrane transport proteins that inhibit the cellular influx and increase the efflux of chemotherapeutic drugs. P-gp is a well-known plasma membrane drug efflux pump involved in the chemo resistance of numerous types of tumors (
In the present study, it was found that the expression level of CD73 in RCC tumors was associated with the tumor stage, tumor grade and patient survival. The expression of CD73 in clear cell RCC tumor tissues was associated with a high tumor stage and tumor grade, but was associated with low patient survival, indicating the potential application of CD73 as a novel diagnostic and prognostic marker of clear cell RCC.
This study was supported by funding from the National Science Foundation of China (grant no., 30571860).
The low expression of CD73 and P-gp processed by immunohistochemistry in normal renal tissue. (A) CD73 (10×20); (B) P-gp (10×20).
Expression of cluster of differentiation 73 in clear cell renal cell carcinoma. (A) Level 1 (magnification, x100); (B) Level 2 (magnification, x100); (C) Level 2 (magnification, x200); and (D) Level 3 (magnification, x100).
Cluster of differentiation 73 expression in vascular smooth muscle and leukomonocytes in renal cell carcinoma tissue (magnification, x200).
P-glycoprotein staining in RCC tissues using immunohistochemical staining. (A) Level 1 clear cell RCC tissue (magnification, x100). (B) Level 2 clear cell RCC tissue (magnification, x100). (C) Level 3 clear cell RCC tissues (magnification, x100). (D) Level 1 uroepithelial carcinoma tissue (magnification, x200). (E) Level 2 uroepithelial carcinoma tissues (magnification, x200). (F) Level 3 urothelial carcinoma tissue (magnification, x100).
Immunohistochemical staining of P-glycoprotein in (A) glomeruli, tubules and (B) lymphocytes in renal cell carcinoma tissues (magnification, x200).
Percentage and intensity grade of staining.
Level | Percentage of intensively stained cells, % | Intensity of staining |
---|---|---|
0 | 0 | Low |
1 | <25 | Weak |
2 | 25≤ |
Moderate |
3 | 50≤ |
Strong |
Immunohistochemical staining properties of CD73 and P-gp for two kinds of kidney cancer and transitional-normal tissues.
A, CD73 expression and location in different renal cell carcinoma | ||||||
---|---|---|---|---|---|---|
Location | Expression level | Normal renal tissue, n (%) | Clear cell RCC, n (%) | Uroepithelial carcinoma, n (%) | χ2 | P-value |
Cell membrane | Rare | 30 (100.00) | 64 (51.61) | 20 (57.14) | 22.568 | 0.000 |
Intensive | 0 (0.00) | 60 (48.39) |
15 (42.86) |
|||
Vascular wall | Rare | 26 (86.67) | 110 (88.71) | 35 (100.00) | 4.989 | 0.074 |
Intensive | 4 (13.33) | 14 (11.29) | 0 (0.00) | |||
B, P-gp expression and location | ||||||
Location | Expression level | Normal renal tissue, n (%) | Clear cell RCC, n (%) | Uroepithelial carcinoma, n (%) | χ2 | P-value |
Cell membrane | Absent | 11 (84.6) | 39 (65.0) | 12 (48.0) | 4.72 | 0.095 |
Intensive | 2 (15.4) | 21 (35.0) | 13 (52.0) | |||
Vascular wall | Absent | 13 (100.0) | 27 (45.0) | 16 (64.0) | 16.30 | 0.000 |
Intensive | 0 (0.0) | 33 (55.0) | 9 (36.0) |
P<0.05 between normal renal cells and different RCC
P<0.05 between normal renal cells and different RCC. CD73, cluster of differentiation 73; P-gp, P-glycoprotein; RCC, renal cell carcinoma.
CD73 expression in RCC and its association with clinicopathological factors.
Intensive expression, n (%) | |||||||
---|---|---|---|---|---|---|---|
Variable | Number of samples, n | Low expression, n (%) | Level 1 | Level 2 | Level 3 | χ2 | P-value |
Gender | |||||||
Male | 119 | 73 (61.3) | 42 (35.3) | 3 (2.5) | 1 (0.8) | 0.677 | 0.498 |
Female | 70 | 41 (58.6) | 21 (30.0) | 6 (8.6) | 2 (2.9) | ||
Tumor type | |||||||
Normal | 30 | 30 (100.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 30.184 | 0.000 |
Clear cell RCC | 157 | 76 (48.4) | 68 (43.3) | 7 (4.5) | 6 (3.8) | ||
Urothelium carcinoma | 48 | 24 (50.0) | 17 (35.4) | 4 (8.3) | 3 (6.3) | ||
TNM stage | |||||||
T1N0M0 | 73 | 35 (47.9) | 35 (47.9) | 2 (2.7) | 1 (1.4) | 12.186 | 0.002 |
T2N0M0 | 70 | 45 (64.3) | 21 (30.0) | 1 (1.4) | 3 (4.3) | ||
T3N0M0 | 28 | 8 (28.6) | 14 (50.0) | 5 (17.9) | 1 (3.6) | ||
T4N0M0 N+M+ | 2 | 0 (0.0) | 1 (50.0) | 1 (50.0) | 0 (0.0) | ||
Tumor grade | |||||||
1 | 49 | 33 (67.3) | 12 (24.5) | 4 (8.2) | 0 (0.0) | 12.821 | 0.002 |
2 | 95 | 48 (50.5) | 43 (45.3) | 1 (1.1) | 3 (3.2) | ||
3 | 53 | 17 (32.1) | 28 (52.8) | 5 (9.4) | 3 (5.7) |
RCC, renal cell carcinoma; TNM, tumor-node-metastasis; N+, lymph node involvement; M+, presence of metastasis.
P-glycoprotein expression in RCC and its association with clinicopathological factors.
Intensive expression, n (%) | |||||||
---|---|---|---|---|---|---|---|
Variable | Number of samples, n | Low expression, n (%) | Level 1 | Level 2 | Level 3 | χ2 | P-value |
Gender | |||||||
Male | 63 | 35 (55.6) | 20 (31.7) | 7 (11.1) | 1 (1.6) | 0.232 | 0.816 |
Female | 42 | 25 (59.5) | 11 (26.2) | 4 (9.5) | 2 (4.8) | ||
Tumor type | |||||||
Normal | 13 | 11 (84.6) | 0 (0.0) | 2 (15.4) | 0 (0.0) | 8.616 | 0.196 |
Clear cell RCC | 75 | 45 (60.0) | 19 (25.3) | 8 (10.7) | 3 (4.0) | ||
Urothelium carcinoma | 29 | 15 (51.7) | 11 (37.9) | 3 (10.3) | 0 (0.0) | ||
TNM stage | |||||||
T1N0M0 | 42 | 29 (69.0) | 10 (23.8) | 3 (7.1) | 0 (0.0) | 8.463 | 0.015 |
T2N0M0 | 33 | 19 (57.6) | 11 (33.3) | 1 (3.0) | 2 (6.1) | ||
T3N0M0 | 18 | 6 (33.3) | 6 (33.3) | 5 (27.8) | 1 (5.6) | ||
T4N0M0 N+M+ | 2 | 2 (100.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | ||
Tumor grade | |||||||
1 | 25 | 20 (80.0) | 4 (16.0) | 1 (4.0) | 0 (0.0) | 7.282 | 0.026 |
2 | 38 | 20 (52.6) | 12 (31.6) | 5 (13.2) | 1 (2.6) | ||
3 | 42 | 20 (47.6) | 15 (35.7) | 5 (11.9) | 2 (4.8) |
RCC, renal cell carcinoma; TNM, tumor-node-metastasis; N+, lymph node involvement; M+, presence of metastasis.
Association of tumor specific characteristics with CD73 and P-gp expression in tumor blood vessel.
CD73, n (%) | P-gp, n (%) | |||||||
---|---|---|---|---|---|---|---|---|
Variable | Low | Intensive | χ2 | P-value | Low | Intensive | χ2 | P-value |
Gender | ||||||||
Male | 113 (90.4) | 12 (9.6) | 1.323 | 0.250 | 34 (53.13) | 30 (46.88) | 0.000 | 0.994 |
Female | 74 (94.9) | 4 (5.1) | 23 (54.76) | 19 (45.24) | ||||
Tumor type | ||||||||
Normal | 26 (86.7) | 4 (13.3) | 4.365 | 0.113 | 13 (100.00) | 0 (0.00) | 14.590 | 0.001 |
Clear cell RCC | 141 (89.81) | 16 (10.19) | 36 (48.00) | 39 (52.00) | ||||
Urothelium carcinoma | 45 (97.8) | 1 (2.2) | 20 (66.67) | 10 (33.33) | ||||
Stage | ||||||||
T1N0M0 | 61 (85.9) | 10 (14.1) | 4.261 | 0.235 | 19 (45.24) | 23 (54.76) | 3.226 | 0.358 |
T2N0M0 | 67 (95.7) | 3 (4.3) | 18 (52.94) | 16 (47.06) | ||||
T3N0M0 | 25 (89.3) | 3 (10.7) | 11 (61.11) | 7 (38.90) | ||||
T4N0M0-N+M+ | 2 (100.0) | 0 (0.0) | 2 (100.00) | 0 (0.00) | ||||
Grade | ||||||||
1 | 43 (87.8) | 6 (12.2) | 2.310 | 0.315 | 7 (28.00) | 18 (72.00) | 11.948 | 0.003 |
2 | 87 (91.6) | 8 (8.4) | 21 (53.85) | 18 (46.15) | ||||
3 | 49 (96.1) | 2 (3.9) | 30 (71.43) | 12 (28.57) |
CD73, cluster of differentiation 73; P-gp, P-glycoprotein; RCC, renal cell carcinoma; TNM, tumor-node-metastasis; N+, lymph node involvement; M+, presence of metastasis.
Multivariate Cox proportional hazards model of RCC.
Variable | HR | 95% CI | P-value |
---|---|---|---|
Gender | 3.061 | 0.888–10.552 | 0.076 |
Age | 0.974 | 0.909–1.043 | 0.453 |
Type of tumor | 1.524 | 0.265–8.776 | 0.637 |
Pathological stage | 3.537 | 0.952–13.139 | 0.059 |
Histological grade | 3.929 | 1.098–14.062 | 0.035 |
Tumor size | 0.870 | 0.696–1.089 | 0.224 |
CD73 intensive | 3.989 | 1.089–14.611 | 0.037 |
P-gp intensive | 0.703 | 0.184–2.679 | 0.605 |
CD73, cluster of differentiation 73; P-gp, P-glycoprotein; HR, hazard ratio; CI, cluster of differentiation.
Means and medians of survival time of renal cell carcinoma patients with rare and intensive CD73 expression.
Mean |
Median | |||||||
---|---|---|---|---|---|---|---|---|
95% confidence interval | 95% confidence interval | |||||||
CD73 | Estimated | SE | Lower bound | Upper bound | Estimated | SE | Lower bound | Upper bound |
Rare | 103.719 | 3.667 | 96.532 | 110.907 | ||||
Intensive | 62.058 | 5.354 | 51.564 | 72.553 | 78.000 | 20.597 | 37.630 | 118.370 |
Overall | 89.460 | 4.021 | 81.579 | 97.342 |
Estimation is limited to the largest survival time if patients succumbed prior to reaching the mean survival time. CD73, cluster of differentiation 73; SE, standard error.
Means and medians of survival time of RCC patients with rare and intensive P-gp expression.
Mean |
Median | |||||||
---|---|---|---|---|---|---|---|---|
95% confidence interval | 95% confidence interval | |||||||
P.gp | Estimated | SE | Lower bound | Upper bound | Estimated | SE | Lower bound | Upper bound |
Rare | 46.608 | 4.352 | 38.079 | 55.137 | 45.000 | 4.734 | 35.722 | 54.278 |
Intensive | 47.408 | 5.582 | 36.468 | 58.348 | 47.000 | 3.656 | 39.834 | 54.166 |
Overall | 46.918 | 3.414 | 40.227 | 53.610 | 47.000 | 2.381 | 42.333 | 51.667 |
Estimation is limited to the largest survival time if patients succumbed prior to reaching the mean survival time. P-gp, P-glycoprotein; SE, standard error.