Discovery of Potential Bladder Cancer Biomarkers by Comparative Urine Proteomics and Analysis

Original study

Discovery of Potential Bladder Cancer Biomarkers by Comparative Urine Proteomics and Analysis Ting Lei, Xuhong Zhao, Sheng Jin, Qian Meng, Hui Zhou, Man Zhang Abstract Our study focused on urine protein of patients with bladder cancer. By using 2-dimensional electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we identified 14 differentially expressed protein spots between 3 patients with bladder cancer and 3 normal controls. Apolipoprotein A-I is one of the proteins that was at increased levels in the bladder cancer urine specimens, and it was confirmed by using Western blot analysis. We concluded that 14 differential spots included apolipoprotein A-I and could be potential urinary biomarkers for bladder cancer. Objective: We searched for bladder tumor markers by analyzing urine samples from patients with bladder cancer and from normal controls. Methods: Proteins in urine samples of patients with bladder cancer and with normal controls were systematically examined by 2-dimensional electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The expression of the protein apolipoprotein A-I (apoA-I) was confirmed by Western blot analysis and further evaluated. Results: We successfully obtained the 2-dimensional electrophoresis gel maps of urinary proteins in patients with bladder cancer and in normal controls. Thirty differentially expressed protein spots were successfully matched by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Combined with the SWISS-PROT database, only 14 proteins (beta-2-microglobulin, fatty acid– binding protein adipocyte, gelsolin, isoform 1 of gelsolin, myoglobin, isoform 2 of fibrinogen alpha chain, apoA-I, prostaglandin D2 synthase 21 kDa [brain], protein AMBP, transthyretin, keratin type II cytoskeletal 1, type II cytoskeletal 8, putative uncharacterized protein ALB, putative uncharacterized protein MASP2 [fragment]) were identified, including 2 putative proteins. Furthermore, apoA-I was confirmed by Western blot analysis, and the high level of apoA-I was found in urine samples from patients with bladder tumors compared with normal controls. Conclusions: Analysis of urinary proteome may be a feasible, noninvasive, and efficient strategy for searching for potential bladder tumor biomarkers. A significant relationship of expressed apoA-I was established between bladder cancer and normal controls. We concluded that 14 differential spots included the apoA-I and would be potential urinary biomarkers for the diagnosis and surveillance of bladder cancer. Clinical Genitourinary Cancer, Vol. xx, No. x, xxx © 2012 Elsevier Inc. All rights reserved. Keywords: Apolipoprotein A-I, Biomarkers, Bladder cancer, Proteomics, Urine

Introduction Bladder cancer is the second most common malignancy that affects the urinary system. Among men, it is the fourth most common cancer and is the eighth leading cause of death from Department of Clinical Laboratory, Beijing Shijitan Hospital, Capital Medical University, China Submitted: Feb 29, 2012; Revised: May 17, 2012; Accepted: June 25, 2012 Address for correspondence: Man Zhang, PhD, Department of Clinical Laboratory, Beijing Shijitan Hospital, Capital Medical University, 10 Tieyi Rd, Haidian District, Beijing 100038, China Fax: 0086-010-63926283; e-mail contact: [email protected]

1558-7673/$ - see frontmatter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clgc.2012.06.003

cancer.1 It is estimated that bladder cancer will account for 70,530 new cases of cancer and 14,680 cancer-related deaths in the United States during 2010.2 Therefore, bladder cancer remains a focus in cancer research. Approximately 90% of bladder tumors are diagnosed as bladder transitional cell carcinoma, almost 75% of which are superficial tumors.3 The spectrum of bladder cancer includes nonmuscle-invasive, muscle-invasive, and metastatic disease, each with its own specific typical behavior, prognosis, and treatment. Generally, within 5 to 15 years after initial resection, the recurrence rate is as high as 60% to 85%.4 Moreover, approximately 20% of the recurred bladder cancer cases can develop into muscle-invasive tumors.5

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AQ: 1

Potential Bladder Cancer Biomarkers At present, direct cystoscopic visualization, voided urinary cytology, and imaging of the upper urinary tract are the traditional tools for the diagnosis and surveillance of patients with bladder carcinoma.6-9 Although considered as the criterion standard, cystoscopy still misses certain lesions, in particular, small areas of carcinoma in situ.10 Furthermore, cystoscopy is invasive for the patient and is an expensive procedure. Cytology, the second criterion standard, has a median sensitivity and specificity of 35% and 94%, respectively.11 The low sensitivity in low-grade cancer has limited the clinical relevance. Besides, inflammatory conditions of the bladder, such as infection or instillation, can confound the results of cytology. The limitations of cytology and the invasiveness of cystoscopy for detecting bladder cancer have generated interest in other noninvasive diagnostic tools. The development of new biomarkers has given us new tests in the detection and follow-up of bladder cancer.12-15 Currently, many bladder tumor markers are being researched, but only a few are commercially available US Food and Drug Administration approved products.1,16 Until now, none of the currently available urinary markers have met the standards in terms of sensitivity and specificity for replacing the combination of cystoscopy and cytology. Urine, as the terminal metabolic product of the blood, the changes of the composition, quantity, and quality reflect information that can represent the generation, development, and prognosis of the urinary diseases. Compared with the collection of plasma and other body fluid samples, the collection of urine is convenient and noninvasive. In this study, by using a comparative proteomic approach, we found the differentially expressed urinary proteins between the patients with bladder cancer and the normal controls. Furthermore, we identified and analyzed the proteins to find out the potential biomarkers that would be useful in diagnosis, surveillance, and clinical application for bladder cancer.

Patients and Methods Study Population This study was approved by the local research ethics boards. This study included 3 patients with bladder cancer and age-, sex-, and ethnicity-matched 3 healthy donors who were enrolled at Beijing Shijitan Hospital between 2008 and 2009. All the patients with bladder cancer had histopathologically confirmed tumors, and none had received chemotherapy or radiation before enrollment. Pathologic staging was done according to 2004 TNM staging, and the World Health Organization consensus classification was used to grade the tumors. The control subjects were healthy volunteers with no history of cancer who were recruited from the medical examination center of Beijing Shijitan Hospital. We also excluded control subjects with chronic urinary tract diseases.

Human Urine Sample Preparation Approximately 100 mL of clean-catch, the first morning midstream voided urine was collected in a sterile tube from healthy donors and patients with bladder cancer. Then, 50 mL of each sample from a total of 3 patients with cancer were pooled, centrifuged at 2000g, at 4°C for 15 minutes within 2 hours. The same process was used for the urine of normal volunteers. The supernatant was added to equal volumes of ice-cold acetone and kept for 15 minutes at 4°C. The sample was centrifuged at 12,000g, 4°C for 7 minutes. The supernatant was removed, and the pellet was air-dried. The pellet was

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then dissolved in lysate buffer (9 M urea, 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 65 mM dithiothreitol (DTT), 0.2% ampholytes) was passed through a pillar to remove the salt. The suspensions were lyophilized and resuspended in lysate buffer. The protein concentration was assessed with Bradford methods according to the kit (Sigma-Aldrich, St Louis, MO). Aliquots of he protein were take and stored at ⫺80°C until analyzed.

2-Dimensional Electrophoresis Analysis Urinary protein samples (100 ␮g each) were solubilized in isoelectric focusing (IEF) buffer that contained 4% CHAPS, 9 M urea, 65 mM DTT, 0.2% carrier ampholytes (pH 3-10), and 0.001% bromophenol blue. The terminal volume was approximately 450-500 ␮L. Solubilized samples were separated by 24-cm Immobiline DryStrip (Bio-Rad, CA) 3 to 10 linear on the immobilized pH gradient (IPG)phor IEF System (Bio-Rad, CA) for the first dimension. After equilibration, the IPG gel strips were transferred onto 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) vertical gels for the second dimension. Electrophoresed gels were fixed overnight and stained with Coomassie brilliant blue. Protein spots were quantified by using PDQuest analysis software (Bio-Rad, CA). The samples from patients with bladder cancer and normal controls were manipulated simultaneously. All experiments were repeated at least 3 times.

Mass Spectrometric Analysis of Urinary Proteins Coomassie brilliant blue–stained spots were excised and digested in gel with trypsin according to described procedures. Briefly, gels were destained by 1% potassium ferricyanide and 1.6% sodium thiosulfate (Sigma-Aldrich, USA), reduced with 25 mM NH4HCO3 that contained 10 mM dithiothreitol (Biosynth, Switzerland) at 60°C for 30 minutes and alkylated with 55 mM iodoacetamide (Sigma-Aldrich, USA) at room temperature for 30 minutes. After reduction and alkylation, the proteins were digested overnight with trypsin at 37°C. After digestion, tryptic peptides were acidified with 0.5% trifluoroacetic acid and loaded onto an MTP AnchorChip 600/384 TF (Bruker-Daltonik GmbH, Bremen, Germany). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed on the MALDI-TOF MS (Proteomics Discovery System 4700; ABI, USA). Monoisotopic peptide masses were assigned and used for database searches with the MASCOT (Matrix Science, USA) search engine.

Western Blotting Urinary proteins from patients with bladder cancer and from normal controls were lysed in sample buffer composed of 100 mM Tris (pH 6.8), 200 mM DTT, 20% glycerol, 4% sodium dodecyl sulfate (SDS), and 0.2% bromophenol blue. The mixtures were boiled and subsequently fractionated by 12% SDS-PAGE. Separated proteins in SDS-PAGE were electrotransferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was soaked in blocking solution that contained 5% (weight per volume) nonfat milk in Tris Buffered Saline, with Tween-20 (TBST) for 2 hours at room temperature. To assess apolipoprotein A-I (apoA-I) component levels, the soaked polyvinylidene difluoride membrane was incubated with monoclonal antibody (Abcam, Cambridge, UK) against apoA-I, diluted 1:500 in 5% (weight per volume) nonfat milk

Ting Lei et al Figure 1 (A) A Total of 100 ␮g Urine Protein From Normal Controls (left) or Patients With Bladder Cancer (right) Was Resolved by 2-Dimensional Electrophoresis, Which Included First Dimension With immobilized pH gradien (pH 3-10) and Second Dimension With 12% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; The Gels Were Stained by Using Coomassie Brilliant Blue. (B) Thirty Differentially Expressed Protein Spots Were Excised Individually From the Gel of Bladder Cancer; Detected Protein Spots Were Marked, Numbered, and Excised for Further Analysis. (C) Spots Were Excised Individually, Digested in Gel With Trypsin; the Spots Nos. 17 and 18 Were Cut as an Example From Normal Control (left) and Bladder Cancer (right); They Are Overexpressed in Bladder Carcinoma Compared With Normal Controls, Which Were Identified as Apolipoprotein A-I (apoA-1) by Matrix-assisted Laser Desorption Ionization Time-ofFlight Mass Spectrometry

A KD pH 3 100

10

Normal

10 KD pH 3 100

B

Patients

10

KD pH 3 100

Before excision

C

Normal 17

After excision

10

10

10

10

10 KD pH 3 100

Patients 18

apoA-I

of TBST overnight at 4°C, washed with TBST 3 times for 15 minutes each and incubated in horseradish peroxidase conjugated goat antimouse immunoglobulin G antibody, diluted 1:400 in 5% (weight per volume) nonfat milk of TBST, at room temperature for 1.5 hours, washed with TBST 3 times for 15 minutes each. After washing, immunobands were detected by enhanced horseradish peroxidase diaminobenzidine (HRP-DAB) detection reagent (Tiangen, China).

Results To search for potential biomarkers of bladder carcinoma, we systematically analyzed urinary proteins secreted from patients with bladder cancer and from normal controls. After concentration, the proteins were resolved by 2-dimensional electrophoresis and Coomassie brilliant blue–stained (Figure 1A). The mean numbers of protein spots from normal controls and from patients with bladder cancer were 445 ⫾ 11 and 385 ⫾ 14, respectively, by using the image analysis software. Finally, we chose 30 protein spots that expressed much more differentially for further identified (Figure 1B). The spots were excised individually (Figure 1C, apoA-I as an example), digested in gel with trypsin, and identified by MALDI-TOF MS. A total of 14 urinary proteins were identified. The detailed information from the database are presented in Table 1. ApoA-I, keratin cytoskeletal 1, keratin cytoskeletal 8, prostaglandin D2 synthase 21 kDa (brain) were upregulated; beta-2-microglobulin, fatty acid– binding protein adipocyte, gelsolin, isoform 1 of gelsolin (GSN), myoglobin

17

18

isoform 2 of fibrinogen alpha chain, transthyretin, protein alpha-1microglobulin/bikunin protein (AMBP) were downregulated; albumin (ALB) and mannan-binding protein-associated serine protease 2 (MASP2) were putative proteins. Information from Swiss-Prot database are presented in Table 1. To analyze further knowledge about the identified proteins, we used the gene ontology (GO) platform to obtain much more information with aspects of molecular function and biologic process (Table 2). ApoA-I was highly expressed in the patients with bladder cancer. To verify the mass spectrometry–assisted identification of apoA-I and correlate urinary apoA-I levels with clinical significance, we examined urine samples from 7 new patients with bladder cancer and 5 normal controls for apoA-I expression (Figure 2A and B). ApoA-I was abundant in the cancer groups but virtually not detected in the control groups. Our results indicated that the urine level of apoA-I was much higher in the patients with bladder cancer than the normal controls. The GO project provides a controlled vocabulary for describing a protein in terms of molecular function, biologic process, and subcellular localization.17 A custom GO annotation file for the reference data set was created with the instructions on the BiNGO Web page (http://www.psb.ugent.be/cbd/papers/BiNGO).18 To analyze further knowledge about the 14 identified proteins, we used the GO platform and GeneCodis 2.0 to obtain much more information with aspects of molecular function and biologic process (Table 2). We

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Potential Bladder Cancer Biomarkers Table 1 Differentially Expressed Urinary Proteins in Bladder Cancer Groups Identified by MALDI-TOF Mass Spectrometry Spot No.

Accession No.

Protein Name

Mr (KD)

PI

Total Ion Score C.I. %

Expression

2

IPI00215746

Fatty acid binding protein, adipocyte

14.7

6.59

99.079

Down

4

IPI00004656

Beta-2-microglobulin

13.7

6.06

99.919

Down

5

IPI00878517

Putative uncharacterized protein ALB

56.2

6.85

99.956

—

8

IPI00641047

Gelsolin

28.9

7.71

99.999

Down

9

IPI00878623

Myoglobin

16

7.14

100

Down

11

IPI00026314

Isoform 1 of gelsolin

85.6

5.90

99.953

Down

12, 23, 24, 28

IPI00029717

Isoform 2 of fibrinogen alpha chain

69.7

8.23

100

Down

14

IPI00871597

Putative uncharacterized protein MASP2 (fragment)

21.4

5.73

100

—

16, 20

IPI00022426

Protein AMBP

38.9

5.95

100

Down

17, 18

IPI00021841

Apolipoprotein A-I

30.7

5.56

100

Up

19

IPI00513767

Prostaglandin D2 synthase 21 kDa

22.9

9.92

98.617

Up

21

IPI00022432

Transthyretin

15.8

5.52

99.983

Down

15, 22, 25

IPI00220327

Type II cytoskeletal 1

65.9

8.16

85.97

Up

26

IPI00554648

Type II cytoskeletal 8

53.6

5.52

99.918

Up

Abbreviations: CI ⫽ confidence interval; MALDI-TOF MS ⫽ matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Mr ⫽ molecular weight; PI ⫽ isoelectric point.

mapped the GOSlim categories of these target genes for the GOSlim process and GOSlim function ontologies. The annotations at the deepest level of the ontology were used for each gene. These annotations were directly obtained from the database. P values were obtained by using the hypergeometric analysis corrected by the false discovery rate (FDR) method. The graph in Figure 3A and B shows the number of genes per singular annotation as aspects of function and process that were found by GeneCodis 2.0. Only the results with P ⬍ .05 are shown. By combining the data of Figure 3A and B, we could deduce Figure 3C, which included all the 14 proteins as the aspects of both molecular function and biologic process more visualizedly. However, these targets were all predictions and were not validated by the experiment.

Discussion At present, cystoscopy with cytology is the standard for diagnosing bladder cancer. Cytology is specific for diagnosing bladder carcinoma but less sensitive, particularly for detecting low-grade disease. However, cystoscopy is an invasive, relatively costly technique that may also be affected at times, particularly in cystitis cases. Thus, a simple, noninvasive marker to detect bladder cancer would be beneficial. The proteome is much more complex and dynamic than the genome. Thus, it could potentially overcome some limitations of other approaches to identify new marker molecules. Proteins secreted from tumor cells are potential biomarkers for disease diagnosis and/or prognosis. MALDI-TOF MS has been used to identify serum protein patterns of patients with bladder cancer with high sensitivity and specificity regardless of tumor stage.19 In this study, we used the

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sample of urine combined with 2-dimensional (2D) SDS-PAGE and MALDI-TOF MS to analyze the differences between the patients with bladder cancer and normal controls. Expression profiles of urinary proteins in the bladder cancer and normal control groups were similar but not identical. As a result, a set of 14 urinary proteins were systematically identified: beta-2-microglobulin, fatty acid binding protein adipocyte, gelsolin, isoform 1 of gelsolin, myoglobin, isoform 2 of fibrinogen alpha chain, apoA-I, prostaglandin D2 synthase 21 kDa (brain), protein AMBP, transthyretin, keratin type II cytoskeletal 1, type II cytoskeletal 8, putative uncharacterized protein ALB, and putative uncharacterized protein MASP2 (fragment). 8 out of the 14 identified urinary proteins have been reported to be related to bladder cancer. Fatty acid binding protein adipocyte,20 gelsolin, isoform 1 of gelsolin,21 and protein AMBP22 have low expression in the tissue, cell, or urine of bladder cancer compared with normal controls. Keratin type II cytoskeletal 8,23 apoA-I24 are overexpression in the urine of bladder carcinoma. The relationship among myoglobin,25,26 beta-2-microglobulin,27,28 and bladder tumor are sill disputed. ApoA-I identified in this study was differentially expressed in the urine of patients with bladder cancer compared with that in the normal controls. The results suggest that a high level of urine ApoA-I may be associated with bladder carcinoma. ApoA-I, which mediates the reverse transport of cholesterol from peripheral cells to the liver for excretion, is a major high-density lipoprotein component in plasma, which constitutes approximately 70% of the apolipoprotein content of high-density lipoprotein particles. In the lipid-bound state, ApoA-I governs lipid transport, receptor recognition, and other functions, including activation of lecithin-cholesterol acyltrans-

Ting Lei et al Table 2 Informations of Differentially Expressed Urinary Proteins in Bladder Cancer Groups Identified From GO Platform Spot No.

Protein Name

Molecular Function

Biologic Process

2

Fatty acid binding protein, adipocyte

Fatty acid binding; protein binding; transporter activity

Transport

4

Beta-2-microglobulin

Protein binding

Antigen processing and presentation of peptide antigen via MHC class I; immune response

5

Putative uncharacterized protein ALB

DNA binding; antioxidant activity; chaperone binding; drug binding; fatty acid binding; pyridoxal phosphate binding; toxin binding; zinc ion binding

Cellular response to starvation, hemolysis by symbiont of host erythrocytes; maintenance of mitochondrion location; negative regulation of apoptosis; transport

8

GSN gelsolin

Actin binding

—

9

MB myoglobin

Heme binding; oxygen binding; oxygen transporter activity

Oxygen transport

11

Isoform 1 of gelsolin

Actin binding; calcium ion binding

Actin filament polymerization; actin filament severing; barbed-end actin filament capping

Isoform 2 of fibrinogen alpha chain

Eukaryotic cell surface binding; protein binding, bridging; receptor binding

Platelet activation; protein polymerization; response to calcium ion; signal transduction

MASP2 putative uncharacterized protein MASP2 (fragment)

Calcium ion binding; calciumdependent protein binding; serinetype endopeptidase activity

Complement activation; classic pathway; complement activation; lectin pathway proteolysis

Protein AMBP

Immunoglobulin A binding; calcium channel inhibitor activity; calcium oxalate binding; heme binding; protein homodimerization activity; serine-type endopeptidase inhibitor activity transporter activity

Cell adhesion; pregnancy; heme catabolic process; interspecies interaction between organisms; negative regulation of JNK cascade; negative regulation of immune response; protein-chromophore linkage; transport

Apolipoprotein A-I

Apolipoprotein A-I receptor binding; beta-amyloid binding; cholesterol binding; cholesterol transporter activity; enzyme binding; highdensity lipoprotein receptor binding; identical protein binding; phosphatidylcholine sterol Oacyltransferase activator activity; phospholipid binding

Cdc42 protein signal transduction; G protein coupled receptor protein signaling pathway; cholesterol efflux; cholesterol homeostasis; cholesterol import; highdensity lipoprotein particle assembly; high-density lipoprotein particle clearance; high-density lipoprotein particle remodeling; negative regulation of cytokine; secretion during immune response; negative regulation of interleukin-1␤ secretion; negative regulation of verylow-density lipoprotein particle remodeling; phosphatidylcholine biosynthetic process; phospholipid efflux; positive regulation of cholesterol esterification; positive regulation of hydrolase activity; protein stabilization; reverse cholesterol transport

19

Prostaglandin D2 synthase 21 kDa

Binding; transporter activity

Lipid metabolic process; transport

21

Transthyretin

Hormone activity

Transport;

15, 22, 25

Type II cytoskeletal 1

Protein binding; receptor activity; structural constituent of cytoskeleton; sugar binding

Complement activation, lectin pathway; epidermis development; fibrinolysis regulation of angiogenesis; response to oxidative stress

26

Type II cytoskeletal 8

Protein binding; structural molecule activity

Cytoskeleton organization; interspecies interaction between organisms

12, 23, 24, 28

14

16, 20

17, 18

Abbreviations: ALB ⫽ albumin; AMBP ⫽ alpha-1-microglobulin/bikunin protein; GO ⫽ gene ontology; GSN ⫽ gelsolin; MASP2 ⫽ mannan-binding protein-associated serine protease 2; MB ⫽ myoglobin.

ferase, which converts cholesterol to cholesterylester.29 Several studies reported changes in serum lipids and lipoprotein in cancer patients. For example, apoA-1 is decreased in the serum of patients with pancreatic cancer30 and ovarian cancer;31,32 apoA-1 is downregulated in hepatocellular cancer tissue compared with surrounding nontumor tissue acquired by laser capture microdissection.33 In contrast, several forms of serum apoA-1, which may indirectly promote tumor survival through kinase activation, were found to be overexpressed in patients with recurrent head

and neck squamous cell carcinoma.34 Recent attention has focused on the level of lipoproteins in tumors because of their possible role in tumor angiogenesis. There also is a significant association between lipoprotein and the presence and stage of lung cancer.35 However, the association of changes in lipoprotein levels and lipoprotein metabolic pathways with cancer progression and differences in the lipoprotein-related proteome between the urine of patients with bladder cancer and normal controls remain unclear and warrants further in-depth investigation.

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Potential Bladder Cancer Biomarkers Figure 2 (A) Western Blot Analysis of Apolipoprotein A-I With 60 ␮g Urinary Protein Samples From 3 Patients of Bladder Cancer (P1-P3) and 3 Normal Controls (N1-N3). (B) Western Blot Analysis of Apolipoprotein A-I With 30 ␮g Urinary Protein Samples From 4 Patients of Bladder Cancer (P4-P7) and 3 Normal Controls (N4-N5); Samples Were Resolved on 12% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, Blotted onto Polyvinylidene Difluoride Membrane and Probed With Specific Antibodies Against Apolipoprotein A-I

A

M

P1

P2

P3

N1

N2

N3

B

M

P4

P5

P6

P7

N4

N5

36kd 25kd

25kd

Figure 3 Information of 14 Identified Proteins by Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry by GeneCodis 2.0 (A) and (B) Pie Graphs Showing the Number of Genes per Singular Annotation Determined From the Views of Function and Process, Respectively. (C) Transforming (A) and (B) to a Pie Graph to Directly Show the Intersection of 14 Proteins on the Aspects of Both MR and BP. The Statistical Significance Was Determined by Calculating the P Value Through Hypergeometric Analysis Correct by the FDR Method. Only Results With P < .05 are Shown. MF: molecular function BP:biology progress FDR:false discovery rate

Abbreviations: BP ⫽ biology progress; FDR ⫽ false discovery rate; MF ⫽ molecular function.

Conclusion Identification of the urinary proteomes developed in this study may serve as an ideal efficient method to establish a panel of potential biomarkers. Noninvasive detection of biomarkers in urine may be useful for clinical application in bladder cancer diagnosis and prognosis. Proteins secreted from bladder tumor or from normal cells and that are present in urine have potential diagnostic or prognostic

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value. The biomarkers identified in this study may be potential therapeutic targets for the future development of novel antitumor agents.

Clinical Practice Points ●

Bladder cancer is the most prevalent tumor of the urinary tract. In China, during the male urinary system diseases, both the incidence rate and the case fatality are the highest.

Ting Lei et al ●

●

●

At present, direct cystoscopic visualization and void urinary cytology are the traditional diagnosis and surveillance methods for patients with bladder carcinoma. The former is invasive and expensive, and requires high technology; the latter has low sensitivity. Recently, research has focused on urinary biomarkers of bladder cancer. Many urine markers, such as bladder cancer antigen, cytokeratins, survin, nuclear matrix protein 22, hyaluronic acid, CYFRA21.1, have been reported. However, none of them satisfied the clinical requirement both in sensitivity and specificity. In our study, we discovered a new biomarker, called apoA-I), which was highly expressed in the bladder cancer group compared with the normal control group. There were many reports about the relationship of apoA-I and cancers such as ovarian, breast, pancreatic, esophageal, colorectal, to name a few. However, the research on bladder cancer and apoA-I has been rare. Further, we confirmed in another 7 patients with bladder cancer and 5 normal controls by western blot analysis. The results were consistent with the MALDI-TOF MS. Although the clinical samples were limit, the tendency was obvious. We speculated that apoA-I would be a potential urinary boimarker for diagnosis and surveillance of bladder cancer.

Acknowledgment This work was funded by Beijing Natural Science Foundation, China (7122086) and 863 program, China (2011AA02A111).

Disclosure The authors have stated that they have no conflicts of interest.

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Clinical Genitourinary Cancer Month 2012

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