Bladder cancer is one of the most common urinary tumors, and the incidence is increasing with industrial development, increased smoking, and population aging [
With developments in molecular biology and immunology, numerous bladder cancer markers have emerged. Bladder cancer–specific nuclear matrix proteins (BLCAs) are specifically expressed in cancer tissue and can be released into the body during cell lyses [
The role of serum or urine BLCA-4 in NMIBC detection has not been described yet. Therefore, in this study, we assessed the levels of BLCA-4 in both serum and urine from NMIBC patients, benign prostate hyperplasia (BPH) patients, and healthy volunteers to evaluate its diagnostic value.
Among inpatients treated in our hospital’s Department of Urology from December 2012 to May 2013, 60 patients were randomly selected. Twenty subjects with primary NMIBC were included in the experimental group (14 men, 6 women; mean age 59.10 ± 12.72 years, range 35–79 years). According to the 2004 World Health Organization grading method, 2 subjects had papillary urothelial neoplasm of low malignant potential, 13 had low-grade tumors, and 5 had high-grade tumors. According to the 2009 tumor node metastasis staging standard, 14 subjects were in stage Ta and 6 were in stage T1. Blood and urine samples were collected from all patients after admission, while cancer and adjacent tissue samples were collected during transurethral or open resection. Control group A was the normal control group (20 men with routine physical examination in the outpatient clinic, mean age 37.60 ± 13.51 years, range 21–67 years). Blood and urine samples were collected from all patients. Control group B comprised 20 patients with benign prostatic hyperplasia (BPH) (mean age, 67.75 ± 5.57 years, range 58–79 years). Blood and urine samples were collected preoperatively, while normal bladder wall tissue samples were collected intraoperatively. Patients were excluded if they met any of the following criteria: recurrent bladder cancer; cystoscopy, urinary catheterization, or urethral dilatation within 1 week; concurrent urinary stones, urinary tract infection, other urinary tract inflammatory stricture, or neoplastic disease; other severe disease and inability to tolerate surgery; or autoimmune disease, such as systemic lupus erythematosus or rheumatism arthritis. The study was approved by the Medical Research Ethics Committee, and all subjects provided written informed consent.
On the morning after admission, 10 mL of midstream urine was collected, and a non-anticoagulant tube was used to collect 5 mL of cubital venous blood from each patient. Patients in the experimental group underwent transurethral resection of the bladder tumor or open resection at 2 cm from the tumor for complete resection, during which cancer and adjacent tissue samples were collected for pathologic and IHC testing. Patients in control group B underwent transurethral resection of the prostate or open resection. Full-thickness mucosal tissue samples from the posterior bladder wall, which were smooth and had no inflammatory congestion, ulcer, or other abnormity, were simultaneously collected for IHC testing.
Products were from the following vendors: human BLCA-4 kit (CSB-E14959h; Wuhan Cusabio Biotech, China); BLCA-4 antigen (Lot No. CPAH260174; Wuhan Cusabio Biotech, China); blocking solution, secondary antibody, horseradish peroxidase (HRP) streptavidin, and 3,3
In a 96-microwell plate coated with anti-BLCA-4 antibodies, 50
Bladder tissue was embedded in paraffin, cut, deparaffinized, and hydrated, and the treated samples were placed in 3% H2O2 solution to incubate at 37°C for 30 min. Then, endogenous peroxidase of the tissue was inactivated, followed by washing with PBS for 5 min, 3 times. Goat serum working solution was added dropwise to the block at 37°C for 30 min. After the blocking solution was disposed, 1 : 100 diluted primary antibody was added dropwise, and the reaction could proceed overnight at 4°C. The next day, the insulated box was rewarmed at 37°C for 30 min, followed by washing with PBS for 5 min, 3 times. Biotinylated secondary antibody was added dropwise, and incubation was conducted at 37°C for 30 min, followed by washing thrice with PBS for 5 min. Then, HRP streptavidin was added dropwise, and incubation was conducted at 37°C for 30 min, followed by washing thrice with PBS for 5 min. After color development using DAB color reagent, a microscope was used for observation. A positive result was defined as BLCA-4 diffusely distributed in the nucleus with a yellowish-brown color. Image results were recorded for analysis.
Intergroup comparisons of measurement data were conducted using the rank-transformed nonparametric test, analysis of variance, or independent-sample
In the present study, a total of 60 participants, 20 patients with NMIBC, 20 with benign prostatic hyperplasia (BPH), and 20 normal controls, were included. Patients’ characteristics are shown in Table
Characteristics of the participants.
Characteristics | Groups ( | ||
---|---|---|---|
NMIBC | BPH | Normal | |
Age, y (mean, range) | 59.1, 35–79 | 67.7, 58–79 | 37.6, 21–67 |
Gender (male, %) | 14, 70% | 11 | 20, 100% |
NMIBC: non-muscle-invasive bladder cancer; BPH: benign prostatic hyperplasia.
Expression of BLCA-4 in bladder cancer tissues, corresponding adjacent normal tissues, and normal bladder tissues from normal people was detected using the immunohistochemistry method. In general, the BLCA-4 staining intensity gradually increased from the normal tissue from normal people and benign adjacent tissue from the patient to the cancer tissue, as shown in Figure
Immunohistochemistry images of BLCA-4 expression in the bladder cancer tissue (a), the adjacent normal tissue (b), and the normal tissue (c).
First, we detected if BLCA-4 levels differ in the urine of bladder cancer patients compared to BPH patients and healthy individuals. We found that the median urine BLCA-4 level was 0.759 ng/mL and significantly increased in the cancer patients compared with BPH patients (
The diagnosis effect of urine BLCA-4 level on the bladder cancer. (a) BLCA-4 urine levels (ng/mL) of non-muscle-invasive bladder cancer (NMIBC) patients compared with benign prostatic hyperplasia (BPH) and normal individuals. (b) Receiver operating characteristic (ROC) curves for BLCA-4 distinguishing between NMIBC and non-NMIBC. The area under the curve (AUC) was 0.986 (95% confidence interval (CI) 0.963–1.000,
Comparison of BLCA-4 expression in non-muscle-invasive bladder cancer tissue with different clinical and pathologic features.
Parameter | No. of subjects | BLCA-4 expression, INT |
||
---|---|---|---|---|
Tumor size, cm | ≥2 | 9 | 295.42 ± 15.37 | 0.436 |
<2 | 11 | 305.46 ± 25.64 | ||
Grade | Papilloma | 2 | 297.53 ± 9.82 | 0.268 |
Low | 13 | 301.56 ± 18.62 | ||
High | 5 | 314.27 ± 19.53 | ||
Stage | Ta | 14 | 304.26 ± 18.64 | 0.501 |
T1 | 6 | 299.57 ± 16.25 |
All values are reported as mean ± SD.
Sensitivity and specificity of urinary BLCA-4 detection using a competitive enzyme-linked immunosorbent assay with different cutoff values.
Cutoff value (ng/mL) | Sensitivity | Specificity |
---|---|---|
0.483 | 1.000 | 0.900 |
0.589 | 0.950 | 0.925 |
0.620 | 0.950 | 0.975 |
0.637 | 0.900 | 0.975 |
0.730 | 0.700 | 1.000 |
The BLCA-4 serum levels (ng/mL) of non-muscle-invasive bladder cancer (NMIBC) patients compared with benign prostatic hyperplasia (BPH) and normal individuals.
Currently, there are numerous tumor markers for bladder cancer; however, none have a satisfactory sensitivity or specificity. BLCA-4 is a nuclear matrix protein specifically expressed in bladder tumor tissue, discovered by Getzenberg et al. [
The cells in bladder cancer tissue grow and metabolize quickly, and the nuclear matrix protein can be released into the blood and urine during apoptosis, forming the molecular basis on which immunologic methods can be used to detect bladder cancer–specific nuclear matrix proteins. In this study, competitive ELISA was used to determine the urinary BLCA-4 level in patients with NMIBC, patients with BPH, and subjects with a healthy urinary tract. The results indicated that the urinary BLCA-4 level was significantly elevated in the NMIBC group compared with the other 2 groups. According to the ROC curve, when the cutoff value for the urinary BLCA-4 level was set at 0.620 ng/mL, the sensitivity and specificity for NMIBC diagnosis were 95% (19/20) and 97.5% (39/40), respectively. Besides, expression of urinary BLCA-4 in patients with NMIBC showed no correlation with age, sex, number of tumors, or tumor size, stage, or grade, which is consistent with the results of a study conducted by Getzenberg et al. [
In the present study, the urinary BLCA-4 level was significantly elevated in the BPH group compared with the normal control group, which is inconsistent with the results of Konety et al. [
In summary, we show that BLCA-4 was significantly increased in the urine of the NMIBC and a little increased in BPH patients compared to those bladder- or prostate-disease-free individuals. The results of our study demonstrated that urine BLCA-4 might serve as a diagnostic marker to distinguish NMIBC. Finally, there was an obvious limitation in this study, which was the 20-sample size of each group. Future studies with a larger number of samples are needed to confirm these findings further.
The original data used to support the findings of this study are available from the corresponding author upon request.
The authors have declared that no competing interests exist.
Special thanks are due to our colleagues, Qian XU and Shu-Bin MI, for their technical help. This work was supported by the Medical Application Tracking Project in Hebei Province (grant number GL200928).