Abstract
Improving the early detection rate and prediction of bladder cancer remains a great challenge in management of this disease. To examine the value of urinary orosomucoid 1 (ORM1) for the early detection and surveillance of bladder cancer, two-dimensional differential gel electrophoresis (2-DE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOFMS) were applied to identify the differently expressed proteins in urine between bladder cancer and healthy controls. Thirteen different proteins including ORM1 were identified. After verification by western blotting, the ORM1 expressions were quantified in 186 urine samples by enzyme-linked immunosorbent assay (ELISA) correcting for creatinine expression. ELISA quantification showed the urinary ORM1-Cr was found to be higher in bladder cancer patients compared to controls and benign cases (7172.23±3049.67 versus 2243.16±969.01, 2493.48±830.37 ng/ml, respectively, P<0.0001). Furthermore, the pearson correlation analysis indicated that urinary ORM1 had high positive correlation with the pathology classification of bladder cancer. Receiver operating characteristic (ROC) analysis was used to calculate the cut-off value for early diagnosis of bladder cancer, and rendered an optimum cut-off value of 3912.97 ng/mg corresponding to 91.96% sensitivity and 94.34% specificity. Moreover, a cut-off value with 7351.28 ng/mg was utilized to distinguish infiltrating urothelial carcinoma from bladder cancer patients corresponding to 91.89% sensitivity and 90.67% specificity. In conclusion, our findings suggested the elevated urinary ORM1 could be a useful biomarker for bladder cancer. Further research is warranted to elucidate the pathogenic mechanisms of elevated ORM1.
Keywords: Orosomucoid 1, bladder cancer, urine, diagnosis, excretion
Introduction
Bladder cancer, one of the most common cancers, remains a major threat to public health all over the world [1]. Clinically, it is characterized by high recurrent rates and poor prognosis once tumors invade deeper layers. Great progress has been made in diagnostic intensity and treatment, but early diagnosis and effective surveillance of bladder cancer progression remain a substantial challenge. Cystoscopy and bladder biopsy are currently considered as the gold standard for diagnosis of bladder cancer [2-4]. Thereof, the development of a reliable, non-invasive biomarker is of considerable clinical importance, aiming at increasing the early detection rate of bladder cancer and predicting the progression of superficial tumors in time [5].
Human body fluids play a vital role in searching biomarkers for early detection of cancer. Amongst them, urine samples are the most easily obtained compared with serum or plasma specimens in clinical analysis [6]. Furthermore, urine is directly exposed to the bladder epithelium reflecting the risk of bladder cancer at some extent.
ORM1 (Orosomucoid-1, α1-acid glycoprotein), mainly functions as a transport protein in the bloodstream. ORM1 is normally produced by human liver cells, but it can also be produced in endothelial cells and some tumor cells [7]. Briefly, ORM1 plays important roles in modulating the activity of the immune system during the acute-phase reaction [8,9]. However, the association of urinary ORM1 expression with the disease of bladder cancer is unknown.
In this study, urinary ORM1 levels were identified and significantly elevated in bladder cancer than healthy controls by proteomic methods. Furthermore, after verification of ORM1 expression by western blotting, the ORM1 expressions were quantified by enzyme-linked immunosorbent assay (ELISA) correcting for creatinine expression, aiming to further investigate the association of ORM1 with bladder cancer.
Material and methods
Urinary samples collection and preparation
All subjects in the present study were recruited from the Chinese Han population at Nanfang Hospital (Guangzhou, China) from May 2011 to May 2015. The study protocol was approved by the Nanfang Hospital Ethics Committee. Informed consent was obtained from all donors prior to the study. 112 patients with bladder cancer and 21 cases with benign bladder damage were selected. All patients had their upper tracts cleared via examinations. 53 healthy volunteers had no evidence of disease and were used as control. Pre-cystoscopy voided urine specimens were collected from patients presenting positive findings under suspicion of bladder cancer. The bladder cancer was confirmed by cystoscopy coupled with histopathological information after surgical operations. The diagnosis was conducted by two pathologists in Nanfang hospital of Southern Medical University according to the criteria of World Health Organization classification of tumours [10].
The second voided clean-catch urine samples from subjects were collected in the early morning. Each urine sample (20 ml) was collected into a sterile plastic tube and then immediately centrifuged at 1500×g for 5 min at 4°C to remove cell debris and particulate matter. The supernatant was stored at -80°C for further analysis. All the clinical data of those subjects were collected as well.
2-Dimensional electrophoresis (2-DE) analysis
Equal volume urine specimens from 15 bladder cancer patients and 15 controls were pooled respectively for 2-DE analysis. Of these bladder cancer patients, there were ten cases of non-invasive papillary urothelial carcinoma (five were low grade and five were high) and five cases of infiltrating urothelial carcinoma. The pooled samples were centrifuged using Centricon Plus-20, 10,000 MWCO devices (Millipore, Bedford, MA, USA), and measured by using the Bradford method. 100 μg of isolated urinary protein were separated by 24 cm Immobiline™ DryStrip 3-10 non-linear on an IPGphor apparatus (GE Healthcare) in the first dimension. After equilibration, IPG gel strips were transferred onto a homogeneous SDS-PAGE (12%) using an Ettan DALT twelve system (GE Healthcare) for the second dimension. After fixing for 5 hours, all gels were visualized by a silver staining technique. Protein spots were quantified with ImageMaster 2D Elite software (Amersham Biosciences). All experiments were repeated for three times. Differences in protein spot intensities were analyzed by using quantitative analysis sets after scanning. Protein spots whose intensities either increased or decreased by 2-fold or greater were marked for identification.
Mass spectrometric analysis of proteins
The differentially expressed protein spots were cut from gels, and then identified by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS) according to previously described procedures [5]. Briefly, Protein spots from gels were digested with trypsin solution, and the peptide mixtures was analyzed by using an ABI Voyager DE-STR mass spectrometer (Applied Biosystems, Foster City, CA, USA). A trypsin-fragment peak was served as internal standard for mass calibration. A list of the corrected mass peaks was the peptide mass fingerprinting (PMF). Monoisotopic peptide masses were assigned and used for database searches with the MASCOT search engine (Matrix Science, London, United Kingdom).
Western blotting analysis
The samples involved four low grade and four high grade non-invasive papillary urothelial carcinoma, eight infiltrating urothelial carcinoma and twelve healthy controls. The extracted proteins from equal 20 ml urine were centrifuged using Centricon Plus-20, 10,000 MWCO devices (Millipore, Bedford, MA, USA) to be concentrated. The prepared urinary proteins were separated by 12% SDS-PAGE respectively and then transferred onto Polyvinylidene Xuoride (PVDF) (Immobilon P, Millipore, Bedford, Massachusetts) membranes. The membranes were blocked in TBS solution containing 5% nonfat milk powder and 0.1% Tween-20 for 1 h at 37°C and then incubated overnight at 4°C with the monoclonal antibody against the human ORM1 protein (diluted 1:1000; Abcam, UK). After three times washes in TBS-T for 10-mins each, the membranes were incubated with horseradish peroxidase horseradish (HRP) conjugate of goat anti-rabbit IgG (Bioworld Technology, Louis Park, MN, USA) at a 1:6000 dilution at 37°C for 1 h. The proteins were detected using an enhanced chemiluminescence (ECL, Pierce, Rockford, IL, USA) detection system. Relative intensities were documented and analyzed by densitometry. Triplicate gel images of identical samples were used for analysis. It was quantified by strip densitometry.
Enzyme-linked immunosorbent assay (ELISA) analysis
The expression level of urinary ORM1 protein was measured by a commercially available ELISA kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. The assay has a minimum detectable dose of ORM1 at 0.538 ng/ml, and the linearity was ranged from 3.12 to 200 ng/ml [9]. A standard curve was made with the suppliers’ lyophilized human ORM1. The concentrations of ORM1 in urine samples were calculated by adjusting with urinary creatinine (Cr), and expressed as ORM1 -to-Cr ratio (ORM1/Cr nanograms per miligrams of creatinine). Urine creatinine (Cr) levels were measured at the Department of Clinical Laboratory of Nanfang Hospital (Guangzhou, China).
Statistical methods
All data were collected and used for statistical analyses with SPSS software 13.0 (SPSS, Chicago, IL, USA). Values were presented as mean ± standard deviation (SD). Student’s t test and one-way ANOVA were used for comparisons between two and three groups respectively. A Spearman analysis was performed to explore the correlations between urinary ORM1-Cr and the clinical features of bladder cancer subjects where appropriate. Univariate (nonparametric rank sum test) and multivariate (logistic regression) analyses were conducted to evaluate the relationship between urinary ORM1 expression and clinical parameters of bladder cancer. Receiving operating curve (ROC) analysis was applied to define the most optimal diagnostic cutoff and the diagnostic performance given by the area under the curve (AUC), evaluating the sensitivity versus its false-positive rate at an optimal cutoff. The results were considered statistically significant at P-value <0.05.
Results
Clinical characteristics of subjects
Table 1 has shown the clinical characteristics of all subjects in our study. There were no statistically significant differences in clinical characteristics (e.g. age, sex, serum creatinine and urinary creatinine). Furthermore, there were 112 bladder cancer patients, 21 cases with benign bladder damage and 53 healthy volunteers recruited for ELISA quantitative analysis. Of these 112 subjects with bladder cancer, 75 cases were non-invasive papillary urothelial carcinoma (39 were low grade and 36 were high) and 37 were infiltrating urothelial carcinoma.
Table 1.
Urine samples in the study
| Samples for 2-DE | Samples for Western blotting | Samples for ELISA | |||||
|---|---|---|---|---|---|---|---|
|
|
|||||||
| Healthy Control | Bladder Cancer | Healthy Control | Bladder Cancer | Healthy Control | Benign Damage | Bladder Cancer | |
| Number | 15 | 15 | 12 | 16 | 53 | 21 | 112 |
| Gender (Male/Female) | 7/8 | 7/8 | 6/6 | 8/8 | 28/25 | 11/10 | 69/43 |
| Age (years) | 67.64±8.34 | 69.23±14.11 | 63.12±7.54 | 67.89±17.21 | 65.33±10.57 | 61.27±9.77 | 64.85±18.85 |
| Serum creatinine (umol/L) | 103.34±29.89 | 119.35±33.78 | 95.62±33.85 | 113.47±36.45 | 99.54±36.93 | 101.65±29.84 | 117.74±49.81 |
| Urine creatinine (mg/dl) | 107.57±57.45 | 115.46±61.68 | 100.79±45.14 | 109.58±57.38 | 97.91±46.03 | 103.37±40.75 | 113.29±63.08 |
Data are expressed as mean ± SD.
Identification of urinary ORM1 by proteomic methods
After 2-DE analysis, we systematically analyzed the differently expressed urinary proteins from patients with bladder cancer and controls. 15 protein spots changed by or over 2-fold were identified by MALDI-FOF/TOF-MS. A total of 12 different proteins were identified (Figure 1; Table 2). The seven proteins which were significantly up-regulated in bladder cancer included Cadherin-1 (CDH1), Uromodulin (UMOD), Pancreatic alpha-amylase (AMY2A), Apolipoprotein A-I (APOA1), Alpha-1-acid glycoprotein 1 (ORM1) (Figure 2A), Retinol-binding protein 4 (RBP4) and Cystatin D (CST5). The other five down-regulated proteins were Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Kininogen-1 (KNG1), Serum albumin (ALB), Leukocyte elastase inhibitor (SERPINB1) and Ig lambda-1 chain C regions (IGLC1).
Figure 1.
Distribution of all 15 differentially expressed protein spots from urine samples between patients with bladder cancer and healthy controls in two-dimensional difference gel electrophoresis gels.
Table 2.
Differentially urinary proteins expressed between bladder cancer and healthy control
| Master number | Accession No. | Gene Name | Protein molecular weight | Protein PI | Pep. Counta | Total ion score | Fold changesb | Overall trendc |
|---|---|---|---|---|---|---|---|---|
| 1 | Q14624 | ITIH4 | 103521.1 | 6.51 | 5 | 251 | -4.96±2.32 | down |
| 2 | P12830 | CDH1 | 97852 | 4.58 | 4 | 225 | 7.15±1.29 | up |
| 3 | P07911 | UMOD | 72451.4 | 5.05 | 9 | 308 | 8.85±2.47 | up |
| 4 | P04746 | AMY2A | 58354.3 | 6.6 | 8 | 290 | 5.39±2.31 | up |
| 5 | P04746 | AMY2A | 58354.3 | 6.6 | 8 | 307 | 4.81±2.32 | up |
| 6 | P01042 | KNG1 | 72995.6 | 6.34 | 7 | 269 | -3.74 ±1.39 | down |
| 7 | P02768 | ALB | 71317.2 | 5.92 | 9 | 259 | -8.31±1.71 | down |
| 8 | P30740 | SERPINB1 | 42828.7 | 5.9 | 5 | 273 | -5.96±2.78 | down |
| 9 | P01842 | IGLC1 | 11400.6 | 6.92 | 3 | 132 | -4.77±1.51 | down |
| 10 | P02647 | APOA1 | 30758.9 | 5.56 | 5 | 238 | 4.56±1.79 | up |
| 11 | P02763 | ORM1 | 23724.8 | 4.93 | 3 | 282 | 6.81±2.56 | up |
| 12 | P02763 | ORM1 | 23724.8 | 4.93 | 3 | 298 | 7.78±3.12 | up |
| 13 | P01842 | IGLC1 | 11400.6 | 6.92 | 3 | 142 | -8.04±3.89 | down |
| 14 | P02753 | RBP4 | 23337.4 | 5.76 | 2 | 267 | 5.61±2.81 | up |
| 15 | P28325 | CST5 | 16080.5 | 6.71 | 2 | 157 | 2.78±1.23 | up |
Calculated by amino acid count.
Calculated based on the spot intensity analysis, represented as mean ± SD.
up: up-regulated in the bladder cancer group. down: down-regulated in the bladder cancer group.
Figure 2.
ORM1 expression levels were verified in the urine samples. A. MS of in-gel trypsin digests of the protein and analysis of the depicted peptide spectrum resulted in the identification of ORM1. B. Verification of ORM1 expression in individual urine samples of patients with bladder cancer, bladder benign cases and healthy controls by western blotting. C. The ORM1 expression of western blotting was calculated according to the immunosignals quantified by densitometric scanning. *P<0.05 compared with that of the control group; **P<0.05 compared with that of the LNIPUC group and ***P<0.05 compared with that of the HNIPUC group. LNIPUC: Low grade non-invasive papillary urothelial carcinoma; HNIPUC: High grade non-invasive papillary urothelial carcinoma; IPUC: Invasive papillary urothelial carcinoma.
Analysis of urinary ORM1 by western blotting
To examine the expression of urinary ORM1 in individual samples from patients with bladder cancer and control groups. The results clearly demonstrated that urinary ORM1 was significantly up-regulated in bladder cancer cases that that of the control group. Among the bladder cancer group, the urinary ORM1 expressions in cases of infiltrating urothelial carcinoma were significantly higher than cases of low or high grade non-invasive papillary urothelial carcinoma (Figure 2B, 2C).
Detection of urinary ORM1 expression by ELISA
ELISA was used to quantify urinary ORM1 expression in our analysis After normalization by creatinine level, the urinary ORM1 was markedly elevated in bladder cancer patients than in controls and benign cases (7172.23±3049.67 versus 2243.16±969.01, 2493.48±830.37 ng/ml, respectively, P<0.0001) (Figure 3A). After division of the patients into groups by tumour classification, the urinary ORM1 concentrations were 5313.35±1341.39, 5892.76±1943.94 and 10376.49±2677.76 ng/mg for bladder cancer with low grade non-invasive papillary urothelial carcinoma, high grade non-invasive papillary urothelial carcinoma and infiltrating urothelial carcinoma respectively (Figure 3C). The relationship between urinary ORM1 levels and the clinical characteristics of bladder cancer patients is presented in Table 3. There was no significant association between the urinary ORM1 expression and other clinical features of bladder cancer such as age, gender, diabetes, hypertension, hematuria, recurrence and metastasis, except for classification of pathology and urinary inflammation (Table 3).
Figure 3.
Urinary ORM1 as a useful biomarker for Bladder cancer. A. ELISA was used to quantify urinary ORM1 levels in 112 bladder cancer patients, 21 cases with benign bladder damage and 53 healthy volunteers. *P<0.05 compared with that of the control group; **P<0.05 compared with that of the bladder benign group. B. ROC curve of urinary ORM1 as a marker for detecting bladder cancer was based on a series of 185 urine samples. The optimal cut off was 3912.97 ng/mg, and the area under the ROC curve (AUC) was 0.965 (95% confidence interval (CI) 0.923-0.987). C. The expression level of ORM1 in different pathology classification of bladder cancer. *P<0.05 compared with that of the LNIPUC group and **P<0.05 compared with that of the HNIPUC group. HNIPUC: High grade non-invasive papillary urothelial carcinoma; IPUC: Invasive papillary urothelial carcinoma. D. ROC curve of urinary ORM1 as a marker for distinguish infiltrating urothelial carcinoma from bladder cancer patients. The optimal cut off was 7351.28 ng/mg, and the area under the ROC curve (AUC) was 0.921 (95% confidence interval (CI) 0.853-0.963).
Table 3.
Associations of urinary ORM1/Cr level with bladder cancer
| Clinical feature | Number | Urinary GC levels (ng/mg) | P |
|---|---|---|---|
| Age ≥65 years | 52 | 6988.25±3107.29 | 0.256 |
| Age <65 years | 60 | 7401.97±4451.57 | |
| Male | 97 | 7257.12±4297.54 | 0.548 |
| Female | 15 | 6806.27±3317.95 | |
| With diabetes | 34 | 6884.79±4187.56 | 0.741 |
| Without diabetes | 78 | 7647.29±3533.47 | |
| With hypertension | 45 | 7579.46±3557.75 | 0.642 |
| Without hypertension | 67 | 6952.42±3972.41 | |
| With hematuria | 61 | 7502.19±3788.85 | 0.756 |
| Without hematuria | 51 | 7009.85±4387.69 | |
| With urinary inflammation | 40 | 6652.58±4477.84 | <0.0001* |
| Without urinary inflammation | 72 | 7290.58±4156.75 | |
| New cases | 66 | 7467.87±3719.97 | 0.256 |
| Recurrences | 46 | 6647.45±3859.74 | |
| No metastasis | 91 | 7002.36±2998.69 | 0.113 |
| Metastasis | 21 | 8045.45±4841.74 | |
| Low grade non-invasive papillary urothelial carcinoma | 39 | 5313.35±1341.39 | <0.0001* |
| High grade non-invasive papillary urothelial carcinoma | 36 | 5892.76±1943.94 | |
| Infiltrating urothelial carcinoma | 37 | 10376.49±2677.76 |
Data are expressed as mean ± SD. *Significant *P<0.05.
Correlation and multivariate logistical regression analyses
The Spearman rank correlation was 0.712, which indicated that the levels of urinary ORM1 had a strong positive correlation with the pathologic stage of bladder cancer (P<0.0001). Furthermore, the multivariate analysis suggested that the urinary ORM1 content was associated with the urinary inflammation (OR: 1.841, 95% CI: 1.123-2.565, P=0.024) and the classification of tumor pathology in bladder cancer (OR: 2.432, 95% CI: 1.253-3.589, P<0.001) (Table 4).
Table 4.
The multivariate analysis of urinary ORM1/Cr level and clinical features
| Feature | p-value (uni) | p-value (multi) | OR | 95% CI |
|---|---|---|---|---|
| Age | 0.723 | 0.297 | 1.121 | 0.487-1.521 |
| Sex | 0.412 | 0.215 | 1.215 | 0.321-1.715 |
| Diabetes | 0.442 | 0.311 | 1.745 | 0.714-1.962 |
| Hypertension | 0.741 | 0.485 | 2.312 | 0.505-3.547 |
| Hematuria | 0.951 | 0.412 | 1.451 | 0.325-2.032 |
| Urinary inflammation | <0.001 | 0.024 | 1.841 | 1.123-2.565 |
| Recurrences | 0.685 | 0.314 | 0.954 | 0.856-1.285 |
| Metastasis | 0.315 | 0.278 | 3.089 | 0.523-3.542 |
| Pathology classification | <0.001 | <0.001 | 2.432 | 1.253-3.589 |
Urinary ORM1 as a useful biomarker for bladder cancer
After quantitative measurement of ORM1 in 186 urine samples by ELISA, ROC curves were applied to assess the potential utility of urinary ORM1 in early diagnosis and surveillance of bladder cancer, ROC analyses rendered an optimum cut-off value of 3912.97 ng/mg corresponding to 91.96% sensitivity and 94.34% specificity. The area under the ROC curve (AUC) of ORM1-Cr for diagnosis of bladder cancer was 0.965 (95% confidence interval (CI) 0.923-0.987) (Figure 3B). Furthermore, we tested whether ORM1-Cr could be utilized to distinguish infiltrating urothelial carcinoma from bladder cancer patients. ROC analyses indicated a cut-off value with 7351.28 ng/mg corresponding to 91.89% sensitivity and 90.67% specificity. The AUC was 0.921 (95% CI 0.853-0.963) (Figure 3D).
Discussion
The incidence and prevalence of bladder cancer remain increasing globally. The early detection and long term prognosis of this disease are still challenge, because cystoscopy is an invasive procedure for patients, and the commonly used cytology has low sensitivity and high variability [11-13]. However, easily applicable screening markers for the early detection of the clinical course of bladder cancer are currently lacking. Therefore, to search for a better noninvasive method for reliable detection of new or recurrent bladder cancer is needed. Proteomic method is a useful tool to find novel biomarker or key proteins in disease of cancer.
In this present study, we have identified thirteen differently expressed proteins in urine from the patients of bladder cancer and normal control by the means of 2-DE analysis and MALDI-FOF/TOF-MS. Of these identified proteins, the association of urinary ORM1 quantitation with bladder cancer has not been examined before, though ORM is found to be significantly increased in urine samples of patients with bladder cancer [14]. Of the protein ORM1 expression was confirmed by Western blot analysis. 186 urine samples were examined by ELISA to correlate urinary ORM1 levels with clinical significance. Our findings have shown that urinary ORM1 could be a potential tumor marker in early diagnosis and surveillance of bladder cancer.
Orosomucoid (ORM) is a polymorphic acute-phase reactant with immunosuppressive properties [7,15]. Previous investigations have suggested that ORM and other acute-phase reactants may act as blocking factors protecting tumor cells against immunological attack, thereby contributing to the ‘immune escape’ of the tumor [15,16]. Many previous studies have demonstrated that serum level of ORM is elevated in inflammatory and lymphoproliferative disorders and cancer, such as breast, lung and ovary cancer [17,18]. Only recently have some studies begun to look at changes in ORM levels in the urine of patients with certain diseases. Christiansen et al. [19] discovered that the ORM expressions in urine were significantly increased in patients with type 2 diabetes, even in normal buminuric patients [20]. The finding was in line with the enhanced urinary ORM concentrations in pregnant women with pregestational type 1 diabetes to predict preeclampsia [21].
Different ORM types have different roles in the progression of cancers [17]. ORM1 (Orosomucoid-1, α1-acid glycoprotein 1) mainly functions as a transport protein in the bloodstream, which is normally produced by human liver cells, and can also be produced in endothelial cells and some tumor cells as well [7,8,22]. Some studies have demonstrated ORM1 has a regulatory dampening effect on the inflammatory cascade, thereby protecting against tissue damage from excessive inflammation [23,24]. The exaggerated excretions of urinary ORM were also detected in hypertensive subjects and in chronic heart failure patients [22]. However, the association of urinary ORM1 levels with bladder cancer is unclear.
In this analysis, our finding showed that the expression of ORM1 was significantly increased in the bladder cancer group compared with healthy controls in individuals through the method of western blotting. Following quantitative measurements of 186 urine samples by ELISA analysis, it was identified that the levels of urinary ORM1 were significantly higher in the urine samples from patients with bladder cancer than in the urine from controls and benign cases. Furthermore, the Spearman analysis indicated that urinary ORM1 had a strong positive correlation with the tumor pathology classification of bladder cancer, meaning that the elevated urinary ORM1 expression was associated with invasive nature of bladder cancer, Furthermore, the multivariate analysis indicated that the urinary ORM1 expression correlated with urinary inflammation (P=0.024) and pathology classification of bladder cancer (P<0.001). In addition, ROC curves suggested at an optimum cut-off value of 3912.97 ng/mg with 91.96% sensitivity and 94.34% specificity respectively is appropriate for diagnosis of bladder cancer patients, and a cut-off value of 7351.28 ng/mg with 91.89% sensitivity and 90.67% specificity could be used to detect the infiltrating urothelial carcinoma.
However, the mechanisms underlying the elevated ORM1 expression in urine from bladder cancer patients are unclear. One possible explanation could be that the increased ORM1 may be associated with the inflammatory activation in bladder cancer patients. Inflammation has been proved to play a pivotal role in the development of bladder cancer through both animal and human research [14,25-27]. Another possibility may be regarding the ORM1 produced by vascular endothelial cells, especially angiogenically activated blood vessels [14]. One characteristic phenomenon of angiogenically activated blood vessels is the abnormal vascular leakage or permeability in the development of cancer disease [28,29]. This would explain the highest increase in mean protein amount of ORM in bladder cancer with infiltrating urothelial carcinoma than cases of low or high grade non-invasive papillary urothelial carcinoma. Therefore, further studies are needed to confirm the reasons leading to the significantly elevated urinary ORM1 in bladder cancer patients.
There are several limitations in the current study that needed to be considered when contemplating the potential value of urinary ORM1 in early detection and surveillance of bladder cancer. Firstly, one of the most important points that needed to be addressed is about the about the specificity of this biomarker. Though our findings were measured in 112 bladder cancer patients, 21 cases with benign bladder damage and 53 healthy controls, the control groups were limited probably resulting in an overestimation of the specificity of ORM1. Furthermore, we did not detect an association between urinary ORM1 levels and other clinical characteristics of bladder cancer such as age, gender, diabetes, hypertension, hematuria, recurrence and metastasis, but we could not exclude other known factors that may affect the urinary ORM1 expression. Thereby, further validation of the urinary marker in bladder cancer is necessary to be considered through larger-scale studies with adequate control groups and adjusted factors.
Conclusions
To sum up, our findings have shown that the ORM1 expression levels in urine are significantly increased in patients with bladder cancer compared to controls. Furthermore, the expression level of urinary ORM1 is positively associated with the pathological classification of bladder cancer. Thus, our study indicates that the urinary ORM1 levels may be a potential biomarker for the early diagnosis and surveillance of bladder cancer. Further research is warranted to elucidate the pathogenic mechanisms of elevated ORM1 and its putative mechanistic role in the pathogenesis of bladder cancer.
Acknowledgements
This work was supported by Dean’s research fund of Nanfang Hospital, the Southern Medical University (2013C022) (F.L.), the Natural Science Foundation of Guangdong Province of China (2014A030310424) (F.L.), Southern medical university scientific research plan (201311) (F.L.), Guangdong Provincial science and technology innovation project of education (2013KJCX0039) (W.L.T.), Guangdong Provincial Science and technology projects (2013B022000067) (W.L.T.), the Natural Science Foundation of Guangdong Province of China (2015A030313289) (W.L.T.) and the National Natural Science Foundation of China (No. 81272844) (W.L.T.).
Disclosure of conflict of interest
None.
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