Searching Urinary Tumor Markers for Bladder Cancer Using a Two

Searching Urinary Tumor Markers for Bladder Cancer Using a Two-Dimensional Differential Gel Electrophoresis (2D-DIGE) Approach Esteban Orenes-Pin ˜ ero,†,# Marta Corto´ n,†,# Pilar Gonza´ lez-Peramato,‡ Ferra´ n Algaba,§ Ignacio Casal,| Alvaro Serrano,O and Marta Sa´ nchez-Carbayo*,† Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Research Center, Madrid, Spain, Pathology Department, Hospital de Guadalajara, Guadalajara, Spain, Pathology Department, Fundacio´ Puigvert, Barcelona, Spain, Protein Unit, Biotechnology Program, Spanish National Cancer Center, Madrid, Spain, and Urology Department, Hospital de Guadalajara, Guadalajara, Spain Received June 13, 2007

Aiming at identifying biomarkers for bladder cancer, the urinary proteome was explored through a two-dimensional gel-based proteomic approach (2D-DIGE) coupled with mass spectrometry and database interrogation. The increased expression of proteins differentially expressed between patients with bladder tumors and controls such as Reg-1 and keratin 10 was confirmed to be associated with bladder cancer progression on bladder cancer cell lines by immunoblotting, and bladder tumors by immunohistochemistry. Moreover, the association of these proteins, especially Reg-1, with tumor staging and clinical outcome was confirmed by immunohistochemistry using an independent series of bladder tumors contained in tissue microarrays (n ) 292). Furthermore, Reg-1 was quantified using an independent series of urinary specimens (n ) 80) and provided diagnostic utility to discriminate patients with bladder cancer and controls (area under the curve (AUC ) 0.88)). Thus, the 2D-DIGE approach has identified Reg-1 as a biomarker for bladder cancer diagnostics, staging, and outcome prognosis. Keywords: 2D-DIGE • bladder cancer • MALDI-TOF-TOF • Reg-1

Introduction Bladder cancer is one of the tumors associated with the highest morbidities and follow-up expenses. It is the fourth most frequent neoplasia in men, clinically characterized by high recurrent rates and poor prognosis once tumors invade the lamina propia.1 Bladder cancer can be classified based on the depth of invasion. Clinically, 75% of urothelial cell carcinomas are non-muscle invasive (TIS, Ta, and T1), 20% are muscle infiltrating (T2-T4), and 5% are metastatic at the time of diagnosis.1,2 Cystoscopy is considered the gold standard diagnostic method of bladder cancer. Urinary cytology remains a valuable adjunct, especially for detecting carcinoma in situ and high grade lesions.2,3 Availability of urinary tumor biomarkers represents a convenient alternative for early detection and disease surveillance because of its direct contact with the tumor and sample accessibility.3 None of the serum or urinary * To whom correspondence should be addressed: Marta SanchezCarbayo, Ph.D., Group Leader, Tumor Markers Group, 208A, Spanish National Cancer Research Center, Melchor Fernandez Almagro 3, E-28029 Madrid, Spain. Phone: + 34 91 732 8053. Fax: + 34 91 224 6972. E-mail: [email protected]. † Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Research Center. # Both authors contributed equally to this work. ‡ Pathology Department, Hospital de Guadalajara. § Pathology Department, Fundacio´ Puigvert. | Protein Unit, Biotechnology Program, Spanish National Cancer Center. O Urology Department, Hospital de Guadalajara.

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Journal of Proteome Research 2007, 6, 4440-4448

Published on Web 09/29/2007

diagnostic tumor biomarkers evaluated to date have provided sufficient sensitivity and specificity to be used for the detection or follow-up of patients with bladder cancer in clinical routine practice. Improved specific prognostic biomarkers are needed as well, and the use of such markers would ultimately distinguish indolent cancers from those that are potentially lethal so that therapeutic procedures could be tailored to each individual patient.2,3 Similarly to genomic technologies, proteomic approaches allow the discovery of disease-specific targets and biomarkers, providing comprehensive diagnostic and prognostic information. The challenge of proteomics versus genomics resides on the complexity of protein chemistry and multiple potential post-translational functional modifications contrasting with the unique binding nucleotide complementarity on which genomics relies. Proteomic techniques are usually classified depending on whether the protein measured is known or unknown when defining the experimental design.4-9 Bidimensional gels (2D) and mass/weight spectrometry techniques are ideal approaches for identification purposes. Protein and antibody arrays allow differential quantification of known proteins.5,9 In bladder cancer, 2D electrophoresis6,7 and mass spectrometry techniques8 have previously been utilized to identify proteins differentially expressed between bladder tumors and urinary protein extracts using Coomassie, silver, or radiolabeled staining.6-8 However, to the best of our knowledge, fluorescent 10.1021/pr070368w CCC: $37.00

 2007 American Chemical Society

research articles

Searching Urinary Tumor Markers for Bladder Cancer Using 2D-DIGE

Figure 1. Experimental design. (A) Urine samples handling was optimized by removing salts and other potential interfering substances. (B) DIGE analyses revealed proteins differentially expressed in a significative manner between urinary specimens from bladder cancer and control patients. (C) Preparative new gels were visualized using SYPRO Ruby staining for picking up selected spots. (D) Proteins of interest were identified using MALDI-TOF-TOF. (E) Immunohistochemistry on tissue arrays containing bladder tumors served to validate associations of identified proteins with clinicopathological variables. (F) Enzymeimmunoanalysis served to validate the bladder cancer diagnostic properties of identified proteins on independent series of urinary specimens.

labeling of urinary protein extracts using a two-dimensional differential gel electrophoresis (2D-DIGE) strategy has not been reported to date. The advantage of 2D-DIGE relies on defining statistical significance on proteins identified differentially expressed between disease and control specimens. As part of the experimental design (Figure 1), a combination of proteomic technologies (2D-DIGE and MALDI-TOF-TOF) was explored as a means to identify differentially expressed proteins in urinary protein extracts of patients with bladder cancer. Identified proteins were tested to be overexpressed in bladder tumors in association with cancer progression. Moreover, their presence in the urine was detected using independent analytical methods.

Table 1. Clinical Information of Patients Providing Urinary Samples Included for 2D-DIGE Analysisa gel

ID

age

cytology

stage

grade

1

16 55 23 73 14 62 21 4 26 9 36 60 29 2

84 79 77 62 63 61 78 65 71 57 70 69 67 34

Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative

pTa pTa pT2 pT2 pTa CIS pT1 -

G3 G2 G3 G3 G1 G3 G3 -

2 3 4 5 6 7

Materials and Methods Urinary Samples and Patients. Urine specimens belonging to individuals presenting microscopic hematuria under suspicion of bladder cancer were collected immediately before urinary cytology and frozen at -80 °C. Samples were handled anonymously following ethical and legal guidelines at the University Hospital of Guadalajara. The presence of bladder cancer was confirmed by cystoscopy, together with histopathological information after subsequent surgical interventions. Urinary samples of 14 individuals (Table 1) were lyophilized, and protein pellets were resuspended in 1 mL of distilled water for 2D-DIGE analyses. Protein concentration was determined using the 2D Quant Kit (GE Healtcare, Uppsala, Sweden). Inorganic salts from urine samples were removed using PD 10 Columns (GE Healthcare). Following extraction, any other interfering components were removed using 2D Clean-Up Kit (GE Healthcare). An independent set of 80 urinary specimens

a Urinary specimens from patients with a positive cytology were labeled with Cy5, while controls with a negative cytology were labeled with Cy3. All the cases and controls analyzed were males.

of patients under suspicion for bladder cancer collected following the same protocol was centrifuged for 5 min at 1500 rpm, and supernatants utilized for enzymeimmunoanalysis. From these 80 urinary samples, 32 were found to be positive for bladder cancer by urinary cytology. Surgical biopsy revealed that the distribution of tumor stage among these patients was pTa (11), pTis (5), pT1 (6), and pT2+ (10). The distribution of tumor grade was grade 1 (5), grade 2 (6), and grade 3 (21). 2D-DIGE. Protein extracts obtained from urine samples were labeled with cyanine dyes according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ). Briefly, 75 µg of urinary protein extract belonging to non-neoplastic patients were minimally labeled with 400 pmol of Cy3 dye, and Journal of Proteome Research • Vol. 6, No. 11, 2007 4441

research articles protein extracts from bladder cancer patients were labeled with Cy5 dye on ice for 30 min, in the dark. An internal pool was generated by combining equal amounts of extracts from all neoplastic and non-neoplastic urinary samples included in the study. This pool labeled with Cy2 was included in all gel runs, serving to assess reproducibility and statistical inferences (Table 1). The labeling reaction was quenched with 0.2 mM lysine (Sigma, St. Louis, MO). Following the labeling reaction, the neoplastic and non-neoplastic urinary protein extracts, together with a pool aliquot, were mixed and run in a single gel. The samples were focused using immobilized pH gradient (IPG) strips (3-10 pH range, NL, 14 × 16 cm) on an IPGphor apparatus (GE Healthcare). The IPG strips were equilibrated for 15 min with gentle shaking in 50 mM Tris-HCl, pH 8.8, containing 6 M urea, 4% (w/v) SDS, 65 mM DTT, 30% glycerol, and a trace of bromophenol blue. Iodoacetamide (53 mM) (Wako Pure Chemical Industries, Osaka, Japan) was added to the second equilibration solution instead of DTT, and the strips were then incubated for 15 min in this solution.10 Standard continuous SDS-PAGE electrophoresis for the second dimension (12%) was carried out at 15 mA per gel for 16 h. Analysis of Gel Images. Proteins were visualized using a fluorescence scanner (Typhoon 9400; GE Healthcare). The images were processed using the DeCyder 5.01 software (GE Healthcare). Its differential in-gel analysis (DIA) module was used for pairwise comparisons of each neoplastic and nonneoplastic urinary protein sample pair to the mixed standard present in each gel, and for the calculation of normalized spot volumes/protein abundance. The spot maps corresponding to seven DIGE gels were used to calculate average abundance changes and paired Student’s t-test p-values for each spot across the different gels. This analysis was performed using the DeCyder biological variation analysis (BVA) module and the Cy3/Cy2 and the Cy5/Cy2 ratios for each individual protein.10 Protein spots that showed a significant change (p < 0.05) in abundance between the protein extracts of neoplastic and nonneoplastic urine samples were selected for further characterization using mass spectrometry. Identification of Proteins by MALDI-TOF-TOF Peptide Mass Fingerprinting (PMF). For preparative purposes, new 2DPAGE analyses were performed, and the resulting gels were stained with SYPRO Ruby for protein visualization. Changes detected by the 2D-DIGE analysis matching with SYPRO Ruby protein patterns were selected for spot picking according to the post-stained image. Spots of interest were excised from the gel automatically using an Ettan-Picker robot (GE Healthcare) and subjected to tryptic digestion according to a previous protocol10 with minor variations.11 Proteins were first reduced (10 mM DTT) and then alkylated (50 mM iodoacetic acid). Following vacuum-drying, the gel pieces were incubated with modified porcine 10 ng/µL trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate for 16 h at 37 °C. Supernatants were collected, vacuum-dried, redissolved in 0.5 µL of 0.1% trifluoroacetic acid (TFA), and added onto a matrix consisting of 0.5 µL of 5 mg/mL 2-5-dihydroxybenzoic acid in water/ACN (2:1) with 0.1% TFA. MALDI-TOF-TOF MS analysis of the samples was carried on a mass spectrometer 4800 (Applied Biosystems, Framingham, MA) in a positive ion reflector mode. Following MALDI-TOF-TOF, the instrument was switched to MS/MS mode, and the five strongest peptides from the MS scan were isolated and fragmented by collision-induced dissociation with air. The ion acceleration voltage was 20 kV. The obtained MALDI-MS data were further processed using the GMS Explorer 4442

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Orenes-Pin ˜ ero et al.

software (Applied Biosystems) that acts as an interphase between the Oracle database containing the raw spectra and a local copy of the MASCOT search engine. The Homo sapiens subsets of the sequences in the Swiss-Prot (12 867 sequences in the Sprot_47.8 fasta file) and NCBI (131 447 sequences in the NCBInr_20050422 fasta file) nonredundant protein sequence databases were utilized for MASCOT searches. Carbamidomethylation (C) and oxidation (M) as fixed and variable modifications, respectively, were taken into account for database searching. The following search parameters were used in all MASCOT searches: tolerance of two missed trypsin cleavages, variable modification on the methionine residue (oxidation +16 Da), and a maximum error tolerance of (2.0 Da in the MS data and (1.0 Da in the MS/MS data. Protein hits with more than two significant matched peptides with the distinct sequences, a mass accuracy between 35 and 100 ppm (SwissProt Database) and between 42 and 100 (NCBI Database) were statistically considered to estimate the confidence of protein identifications. In addition, the MS/MS spectra of the identified peptides were manually inspected. Bladder Cancer Cell Lines. Eight bladder cancer cell lines (RT4, 5637, UM-UC-3, T24, SW780, EJ138, TCCSUP, and ScaBER) were obtained from the American Type Culture Collection and cultured following standard procedures, as previously described.12 Immunoblotting. Total protein was extracted from eight different bladder cancer cell lines using RIPA lysis buffer and quantified with the Bradford assay using BSA as standard (Protein Assay Kit, Bio-Rad, Hercules, CA). Total protein extracts (75 µg) were mixed with 5× SDS sample buffer (62.5 mM TrisHCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.005% bromophenol blue) and resolved by SDS-PAGE on 10% acrylamide gels. Proteins were detected immunologically following electrotransfer onto PVDF membranes (Millipore, Bedford, MA) after activation with methanol. The membranes were blocked with 5% non-fat dry milk in PBS and 0.1% Tween-20 for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-Reg-1 (rabbit polyclonal, 1:1000 dilution, kindly supplied by Dr. Iovanna), anti-prefoldin (goat polyclonal, 1:200 dilution, IMGENEX, San Diego, CA), anti-cytokeratin 2 (mouse monoclonal, 1:200 dilution, PROGEN Biotechnik, Heidelberg, Germany), and anticytokeratin 10 (mouse monoclonal, 1:200 dilution, Neomarkers, Fremont, CA). Blots were washed three times for 10 min in PBS and 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated (HRP) secondary antibodies for 1 h at room temperature: HPR-conjugated anti-mouse (1/1000 dilution), anti-rabbit (1/2000 dilution), and anti-goat IgG (1/2000 dilution) (Dako, Glostrup, Denmark). Blots were developed using a peroxidase reaction with the enhanced chemiluminescent immunoblotting detection system (ECL, GE Healthcare). Antibodies were accepted when they displayed a single predominant band at the expected molecular weights. R-Tubulin (mouse monoclonal, 1:4000 dilution, Sigma) was utilized as the loading control. Tissue Microarrays. Five different bladder cancer tissue microarrays were constructed at the Spanish National Cancer Center and Fundacio´ Puigvert and used in this study. These arrays included a total of 292 primary urothelial cell carcinomas of the bladder, belonging to patients recruited under Institutional Review Board (IRB)-approved protocols. The distribution of tumor stage among the 292 bladder tumor samples spotted on the tissue arrays was pTa (93), pT1 (95), and pT2+ (104).


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Figure 2. Representative 2D-DIGE staining of proteomic profiling of urine samples. Protein extract from non-neoplastic and neoplastic urine samples was differentially labeled with Cy3 and Cy5, respectively. A mixed internal standard combining all the proteins from tumor and normal urinary specimens, labeled with Cy2, was included in all gels run. Spots marked with a number indicate identified proteins whose abundance changes between neoplastic and control specimens reached statistical significance.

The distribution of tumor grade was grade 1 (29), grade 2 (64), and grade 3 (199). Clinicopathological and annotated followup information of the tumors spotted onto the tissue microarrays allowed the evaluation of the staging properties and outcome assessment of the proteins identified by the proteomic DIGE approach. Immunohistochemistry. Protein expression patterns of the differentially expressed proteins were assessed at the microanatomical level on these tissue microarrays by immunohistochemistry using standard avidin-biotin immunoperoxidase procedures. Antigen retrieval methods (0.01% citric acid for 15 min under microwave treatment) were utilized prior to incubation with primary antibodies overnight at 4 °C. The same primary antibodies used in Western blotting worked for immunohistochemistry at the following conditions: Reg-1 (1/1000 dilution), anti-cytokeratin 2 (1/100 dilution), anti-cytokeratin 10 (1:100 dilution), and anti-CD5 (mouse monoclonal, homemade at CNIO, clone 36/G2, 1/100 dilution). Secondary antibodies (Vector Laboratories) were biotinylated goat anti-rabbit antibodies (1:1000 dilution) and biotinylated goat anti-mouse antibodies (1:500 dilution). The absence of primary antibody was used as negative control. Diaminobenzidine was utilized as the final chromogen and hematoxylin as the nuclear counterstain.12 Enzymeimmunoassays. Soluble urinary Reg-1 concentrations were measured using an enzymeimmunoassay (Dynabio, Marseille, France). Urinary samples tested for soluble Reg-1 were centrifuged for 5 min at 1500 rpm, and supernatants utilized for enzymeimmunoanalysis. Statistical Analysis. The association of the expression of these proteins measured by immunohistochemistry on tissue arrays with histopathologic stage and tumor grade was evalu-

ated using the nonparametric Wilcoxon-Mann-Whitney and Kruskall-Wallis tests.13 The associations of these proteins with overall survival were also evaluated using the log-rank test in those cases for which follow-up information were available. Overall survival time was defined as the years elapsed between transurethral resection or cystectomy and death as a result of disease (or the last follow-up date). Patients who were alive at the last follow-up or lost to follow-up were censored. Survival curves were plotted using the standard Kaplan-Meier methodology.13 Urinary specimens (n ) 80) were utilized to analyze the clinical utility of Reg-1 at discriminating patients with bladder cancer from healthy individuals and patients with benign urological diseases. Urinary samples categorization was based on cytology and cystoscopic observations, the latter being utilized as the gold standard. Receiving operating curve (ROC) analyses were used to define the most optimal diagnostic cutoff as well as the diagnostic performance given by the area under the curve (AUC), estimating its 95% confidence interval at optimal cutoffs.13 Statistical analyses were performed using the SPSS statistical package (version 11.0).

Results Differential Protein Expression among Urinary Specimens belonging to Patients with Bladder Cancer and Controls. Seven independent 2D-DIGE gels were performed comparing the urinary proteomes of neoplastic (labeled with Cy5) and non-neoplastic (labeled with Cy3) protein extracts. An internal pool containing protein extracts belonging to all the patients and controls included in the study was labeled with Cy2 (Table 1). Analysis of the Cy2, Cy3, and Cy5 gel images with the BVA module of the DeCyder software revealed the presence of 12 spots changing in a significant manner (Figure 2), with statistiJournal of Proteome Research • Vol. 6, No. 11, 2007 4443

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Figure 3. 3D view of differentially expressed proteins in urinary samples from bladder cancer patients comparing with control patients. Changes in protein expression from control and tumor specimen of each gel were shown by each line. The average ratio of expression for each selected protein grouping non-neoplastic and neoplastic specimens as obtained by computational analysis with DeCyder Software was represented by lines marked with a cross. Statistical analysis allowed detection of significant abundance changes (95th confidence level) based on the variance of the mean change within the cohort: (A) Reg-1, (B) CK-10, (C) CK-2, (D) CK-1, (E) T-cell surface protein CD5, (F) Prefoldin. Table 2. Differentially Expressed Proteins Identified by MALDI-TOF-TOF after 2D-DIGE Analysis of Neoplastic versus Non-Neoplastic Urine Samples in a Significant Manner. master number

protein AC (Swiss-Prot)

1637

P05451

1002

t test

MASCOT score

coverage (%)

peptides matched/ unmatched

5.65

0.013

88

17

2/29

2.43

59519

5.13

0.048

128

14

8/21

1.61

CK2 CK1 CD5

65865 66018 54625

8.07 8.16 8.66

0.048 0.05 0.041

165 82 91

17 11 13

8/20 7/19 6/20

2.36 -2.7 2.38

Prefoldin

13284

4.70

0.045

212

35

4/26

1.63

size (Da)

pI

Reg-1

18731

P13645

CK10

1336 1538 577

P35908 P04264 P06127

1242

P52553

name

cal variance of the tumor versus normal spot volume ratios within the 95th confidence level (Student’s t test; p < 0.05). Figure 3 illustrates the 3-D view corresponding to the pixel volume distribution for the Cy5/Cy3 ratio of the proteins differentially expressed between urinary specimens belonging to patients with bladder cancer and controls without the disease. Certain spots showed increased expression in urinary samples of patients with bladder cancer, while other spots showed lower expression levels. Identification of Urinary Proteins after SYPRO Ruby Staining. Proteins identified by the 2D-DIGE approach to be significant among neoplastic versus non-neoplastic urinary specimens were excised from SYPRO Ruby stained gels. Description of the spots and the identified proteins differentially 4444


av ratio

function (Swiss-Prot)

positive regulation of cell proliferation constituent and development of epidermis terminal cornification epidermis development cell proliferation, cell recognition protein and tubulin binding, tubulin folding

expressed between urinary specimens belonging to patients with bladder cancer and controls in a significant manner is shown in Table 2. Identification numbers in Table 2 matched with those associated to the 2D-DIGE spots shown in Figure 2. In our study, 12 spots were found to be differentially expressed between urinary specimens from patients with bladder cancer and controls in a statistically significant manner. Mass spectrometry identified 6 proteins, because more than one spot revealed the presence of the same protein. Because of the intrinsic variability associated with patient and sample heterogeneity, stringent criteria were chosen to select proteins for further analyses. Only proteins differentially expressed in at least four of the seven gels and for which antibodies were available were considered for validation analysis. Five proteins


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Figure 4. Immunoblotting validation of proteins identified by DIGE analysis using protein extracts from eight different bladder cancer cell lines. Antibodies were accepted as displaying a single predominant band at the expected molecular weights. (A) Reg-1, 16 kDa; (B) CK10, 55 kDa; (C) CK2, 65 kDa; (D) Prefoldin, 13 kDa; (E) R-tubulin, 50 kDa, was used as loading control.

from these 12 spots followed these criteria and were submitted to further verification analyses. The following proteins were identified to be overexpressed in the urinary specimens belonging to patients with bladder cancer: Regenerating protein (Reg-1), cytokeratins 2 and 10, prefoldin, and T-cell surface glycoprotein CD5 precursor. Cytokeratin 1 was an underexpressed protein. Triptic digestion and protein identification was additionally performed on other detectable spots not statistically significant through 2D-DIGE analyses (data provided in Supplementary Table 1 and Supplementary Figure 1 of Supporting Information which provides the gel stained with SYPRO Ruby, highlighting the spots statistically significant as well). Identified Proteins Are Differentially Expressed in Bladder Cancer Cell Lines. As part of validation analyses of the potential relevance of the identified proteins in bladder cancer progression, immunoblotting analyses were performed on a series of eight bladder cancer cell lines derived from bladder tumors representing several steps along bladder cancer progression (Figure 4). Immunoblotting analyses of these bladder cancer cell lines showed differential expression of these proteins in cells from the papillary RT4 and low-grade 5637 cell lines, to invasive (T24, UM-UC-3, SW780, EJ138), metastatic (TCCSUP), and squamous (ScaBER) cell lines. These validation analyses revealed a differential protein expression of the identified proteins using the 2D-DIGE approach in bladder cancer cell lines, in high concordance with the disease progression. Further analyses were then conducted to assess the clinical relevance of these proteins using independent sets of bladder tumors and urinary specimens of patients with bladder cancer. Identified Proteins Are Differentially Expressed in Bladder Tumors. The next set of analyses dealt with the optimization and characterization of protein expression patterns of the identified proteins by means of immunohistochemistry. These analyses were performed aiming to test whether identification of these proteins in the urine could be due to an increased expression of these proteins in the bladder tumors of the patients that provided urinary specimens before their scheduled urinary cytology. In this regard, differential and increased expression was confirmed for Reg-1, cytokeratin 10, and CD5 (data not shown, representative immunohistochemical analyses are shown in Figure 5). Reg-1 Is Associated with Tumor Progression and Clinical Outcome of Patients with Bladder Tumors. The next set of analyses searched for associations of identified proteins with clinicopathological variables of patients with bladder cancer.

Figure 5. Validation of the differential protein expression patterns at the tissue level by immunohistochemistry on tissue arrays with bladder tumors. (A and B) Reg-1, (C and D) CK10; (E and F) CD5.

Protein expression patterns of identified proteins were characterized by means of immunohistochemistry on independent series of bladder tumors contained in several tissue arrays (Figure 5). These analyses revealed statistical associations between Reg-1 expression and tumor stage (p ) 0.001, n ) 292). Interestingly, in a series of T1G3 tumors (n ) 92), an increased protein expression of Reg-1 was significantly associated with T1 substaging (T1a vs T1b and T1c) (p ) 0.024, n ) 92), tumor size (p ) 0.007, n ) 92), and T1 progression into muscle invasive T2-4 disease (p ) 0.009, n ) 92). Clinical follow-up availability of this T1G3 subset of cases allowed further prognostic analyses at the protein level. Increased expression of Reg-1 was observed in cases with poor survival while low levels were detected for those patients with longer survival (log rank, p ) 0.005). Kaplan-Mayer analyses displayed the statistical association found between Reg-1 overexpression and poorer survival (Figure 6A). Overall, expression patterns of Reg-1 on these independent series of patients with bladder tumors further confirmed the clinical associations of overexpression Journal of Proteome Research • Vol. 6, No. 11, 2007 4445

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Figure 6. (A) Kaplan-Mayer curve survival analysis indicating that an increased protein expression of Reg-1 measured by immunohistochemistry on tissue arrays (n ) 92) was associated with poor survival. (B) Urinary Reg-1 discriminates bladder cancer on independent series of urinary specimens of patients with bladder cancer and controls. ROC curve of Reg-1 urinary tumor marker as a detection marker for bladder cancer on a series of 80 urinary specimens collected immediately before urinary cytology. Among these, 32 had positive cystoscopy. The optimal cutoff was 0.0038 ng/mL, and the AUC obtained was 0.881 (95% CI: 0.801-0.961). Sensitivity and specificity were 81.3% and 81.2%, respectively.

of Reg-1 with tumor progression and clinical outcome at the protein level. In the series of T1G3 tumors, CK10 protein expression patterns were associated with T1 progression into muscle invasive T2-4 disease (p ) 0.025, n ) 92). Increased number of CD5 positive cells were observed in multifocal bladder tumors as compared to those with unifocal tumors (p ) 0.037, n ) 92). Reg-1 Is Detected in Urinary Specimens and Segregated Bladder Cancer Patients from Controls. Once the protein identified in the urinary specimens through the DIGE approach was observed overexpressed in bladder cancer cell lines and tumors in association with clinicopathological variables, it was tested whether quantitative measurement of Reg-1 could be utilized as a diagnostic tool to differentiate patients with bladder cancer and controls on an independent set of urinary specimens using an independent method. Once calibration curves were proven to be analytically optimal, Reg-1 was measured with the same antibody utilized for immunohistochemistry and Western blotting on 80 urinary specimens by an ELISA. Considering sample categorization given by urinary cytology, ROC analyses rendered a diagnostic accuracy of 0.714, given by this AUC, whose significance was p ) 0.002, and IC 95%: 0.590-0.837, at a 0.0086 ng/mL cutoff. Using the cystoscopy as the gold standard for urinary sample categorization, the diagnostic accuracy increased to 88.1%, with a sensitivity and specificity of 81.3% and 81.2%, respectively (Figure 6B). The majority of the cases not detected by Reg-1 had a pTaG1 tumor. Overall, these results indicated that Reg-1 was detectable in the urine and supported the role of Reg-1 to discriminate between bladder cancer patients and controls with high sensitivity and specificity. Remarkably, independent sets of specimens and methodology suggested the potential diagnostic utility of urinary Reg-1.

Discussion The novelty of this report deals with the application of 2DDIGE coupled with mass spectrometry to urinary specimens. This represents a promising approach for the identification of 4446


tumor biomarkers aiming at the detection of the presence of bladder cancer. Several proteins were found to be differentially expressed in a significative manner in urinary specimens of patients with bladder cancer as compared to controls as confirmed by gold standard diagnostic methods. Bladder cancer specificity of proteins such as Reg-1 or keratin 10 was observed by means of immunohistochemistry on tissue paraffin sections of tumors specimens from the same patients with bladder cancer that provided urinary specimens for 2D-DIGE analyses. Immunostainings on tissue arrays containing independent larger series of patients with bladder cancer served to assess the associations of novel proteins with clinicopathological variables. Thus, the combination of proteomic approaches has served to the identification of novel proteins differentially expressed along bladder cancer progression and in association with clinical outcome. Moreover, urinary measurement of Reg1 on independent series of urine specimens and independent methodologies served to discriminate patients with bladder cancer from controls with high diagnostic accuracy. The main advantage of 2D-DIGE fluorescent labeling relies on the availability of estimating statistically the extent of the differential protein expression among disease and control specimens. The labeling strategy was critical in the discovery process for urinary biomarkers using this 2D-DIGE approach. The internal pool generated by combining equal amounts of extracts from all the neoplastic and non-neoplastic urinary specimens was labeled with Cy2 dye, included in all gel runs, and served to assess inter-assay reproducibility. Even when applied to a small sample size (comparing urinary specimens belonging to seven bladder cancer patients versus controls), the use of the mixed internal pool with all the specimens under comparison in this experimental design allowed evaluating significant abundance changes among urinary protein extract within the cohort. These changes might not have been detectable through individual comparisons due in part to the large variation between samples. Such interindividual differences might even be more evident among urinary specimens. Nevertheless, it should be assumed that other potentially interesting


proteins might not be identified in a significant manner as the abundance changes may be underexpressed or not detected in certain patients and overexpressed in others. This variability justifies the limited number of statistically significant changes found in our series. To cover this variability, the majority of the detectable spots observed in each SYPRO image were also selected for tryptic digestion on protein identification (Suplementary Table 1 in Supporting Information). Cancer cell lines represent an ideal model for exploratory evaluations of potential biological relevance of targets or biomarker candidates.13 A common strategy of biological studies utilizes clinical samples to validate findings discovered in cancer cell lines. Since cancer cell lines are derived from human individual tumors, they might serve to estimate potential differences in expression of the proteins under study along cancer progression.13 In this study, both strategies were undertaken. Once the quality of our antibodies and the differential expression of the identified proteins were tested on the cell lines, independent sets of bladder tumors and urinary samples were utilized to confirm the relevance of these proteins in bladder cancer. Immunoblotting analyses of eight bladder cell lines derived from early and advanced TCC and squamous tumors revealed differential expression of the identified proteins in association with bladder cancer progression. The next set of validation analyses dealt with confirmatory studies showing increased protein expression of the identified proteins in bladder tumors as compared to normal urothelium counterparts of those patients that provided urinary specimens for 2D-DIGE analysis. This observation served to support phenotypically the cancer specificity of the proteins detected in the urine by the 2D-DIGE. Furthermore, it supported our experimental design searching for specific bladder tumor markers on urinary specimens. To evaluate the relevance of these identified novel proteins along bladder cancer progression, immunohistochemical stainings were performed on several tissue arrays containing independent larger series of bladder tumors comprising early and advanced stages of bladder cancer progression, with available follow-up. Interestingly, Reg-1 was associated with tumor progression and clinical outcome in bladder cancer. Other proteins such as keratin 10 and CD5 showed clinical associations as well. The proteins of the Pap/Reg family have been found to be expressed in the pancreas and other gastrointestinal tract organs.14-18 Significant overexpression of mRNA and protein Reg-1 has been associated with tumor progression, and poor prognosis in patients with colon,15 liver,16 and gastric cancer.17 It has been suggested that the regenerative response resulting from Reg-1 expression may be responsible for inhibition of apoptosis. This switch-off phenomenon of apoptosis may be caused by Reg-1 activation of proliferative activity.14 Suggested as a potential biomarker of gastrointestinal cancer activity, Reg-1 relevance in uroepithelial neoplasias had not been described yet. In this report, Reg-1 overexpression was also associated with several clinical variables related to bladder cancer progression and poor survival. Furthermore, it was identified in the urine of patients with bladder tumors and revealed to be a potential noninvasive adjunct for bladder cancer diagnosis. Increased secreted Reg-1 in body fluids had been reported in serum of gastrointestinal and pancreatic cancer,19 and even in the urine of patients with intestinal segments transposed into the urinary tract,20 but not in the context of bladder cancer detection or progression.

research articles

Keratins are expressed in a tissue-specific manner in human epithelia.4,6,13,21,22 In bladder cancer, several reports have evaluated the different expression of cytokeratins in normal and neoplastic uroepithelium.4,6,13,21,22 CK10 is a suprabasal differentiation related keratin associated with squamous differentiation.4,13 The novelty in this regard was that CK10 was identified in the urinary specimens. Immunoblotting served to confirm antibody specificity and the differential expression of CK10 in bladder cancer cell lines, with increased expression in ScaBER, the only squamous cell carcinoma cell line. Immunostainings on a series of T1G3 bladder tumors contained on a tissue array revealed a statistical association between CK10 overexpression and T1 progression into muscle invasive T2-4 disease. Supporting the relevance of CK10 in tumor staging are significant associations previously reported by our group using an independent series of bladder tumors (n ) 173) spotted on tissue arrays between CK10 and tumor stage (p ) 0.019), tumor grade (p ) 0.018), and squamous differentiation (p < 0.001).13 Consistent with such previous results, significant associations of CK10 with tumor progression were found in the series analyzed in this report. However, the number of muscleinvasive bladder tumors with clearly defined squamous differentiation was limited to allow addressing statistical associations between CK10 and squamous differentiation. Urinary detection of differential expression of CK1 and CK2 is also novel in bladder cancer. Although CK2 has been measured in parallel with CK6 on tissue stainings,21 it was not identified to be differentially expressed along bladder cancer progression in either bladder cancer cell lines resembling bladder tumors biology or human clinical material. CD5 is a T cell surface protein with a large cytoplasmic domain involved in modulating T cell responses.23,24 Leukocyte infiltration is a frequent event in bladder tumors, prevalently T “activated” cells (CD5+), as part of the immune response after BCG treatment.23,24 Thus, the presence of CD5 in the urine could be due either to an inflammatory response associated to cystitis or urinary tract infection, or to lymphocytes leaking from the tumor into the urine. Bladder immunostainings on tissue arrays revealed that CD5 was expressed not only in surrounding leukocytes, but also in bladder tumor cells. Increased number of CD5 positive T cells was observed in multicentric bladder tumors as compared to unifocal lesions. The statistical correlation with multifocality suggests the relevance of the interaction of the tumor and the immune system of the host in bladder cancer or increased tumor angiogenesis.23,24 The presence of CD5 staining in tumor cells is supported with previous reports indicating other T membrane antigens such as CD40 in endothelial cells.25 These observations indicate that urinary CD5 detection could be originated from T cells and bladder tumor cells, and suggest the possibility of shared antigens between human bladder cancer cells and T cells as has been suggested for normal host macrophages.23 Our concluding remark points out the utility of the 2D-DIGE proteomic strategy for the identification of urinary proteins indicative of the presence of bladder cancer. As a result of this approach, Reg-1 was associated with bladder cancer progression and clinical outcome. Interestingly, urinary Reg-1 protein levels served to identify patients with bladder cancer. These observations suggest a role for Reg-1 as a biomarker for bladder cancer diagnostics, staging, and outcome prognosis. Abbreviations: AUC, area under curve; BCG, Bacillus Calmette-Gue´rin; BVA, biological variation analysis; CI, confiJournal of Proteome Research • Vol. 6, No. 11, 2007 4447

research articles dence interval; CK, cytokeratin; DIA, differential in-gel analysis; ELISA, enzymeimmunoassay; Reg-1, Regenerating protein; RIPA, rapid immunoprecipitation assay; ROC, receiving operating curve; SCC, squamous cell carcinoma; TCC, transitional cell carcinoma; TFA, trifluoroacetic acid; TMA, tissue microarray.

Acknowledgment. The authors thank all members of Dr. Sa´nchez-Carbayo’s laboratory for their technical support and constructive suggestions in the preparation of this manuscript. We thank Dr. Iovanna for sharing Reg-1 reagents with us, as well as all the members of our clinical collaborators at the University Hospital of Guadalajara and Fundacio´ Puigvert, especially Joan Palou, for their support in facilitating specimens and clinical follow-up of the bladder cancer cases analyzed in this study. Supporting Information Available: Figure and table showing the identification of detectable spots not statisticaly significant through 2d-DIGE analysis. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Jemal, A.; Siegel, R.; Ward, E.; Murray, T.; Xu, J.; Thun, M. J. Cancer statistics, 2007. CA Cancer J. Clin. 2007, 57 (1), 43-66. (2) Kirkali, Z.; Chan, T.; Manoharan, M.; Algaba, F.; Busch, C.; Cheng, L.; Kiemeney, L.; Kriegmair, M.; Montironi, R.; Murphy, W. M.; Sesterhenn, I. A.; Tachibana, M.; Weider, J. Bladder cancer: epidemiology, staging and grading, and diagnosis. Urology 2005, 66 (6 Suppl. 1), 4-34. (3) Sanchez-Carbayo, M.; Cordon-Cardo, C. Molecular alterations associated with bladder cancer progression. Semin. Oncol. 2007, 34 (2), 75-84. (4) Celis, J. E.; Celis, P.; Østergaard, M.; Basse, B.; Lauridsen, J. B.; Ratz, G.; Rasmussen, H. H.; Orntoft, T. F.; Hein, B.; Wolf, H.; Celis, A. Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions. Cancer Res. 1999, 59 (12), 3003-3009. (5) Sanchez-Carbayo, M. Antibody arrays: technical considerations and clinical applications in cancer. Clin. Chem. 2006, 52 (9), 1651-1659. (6) Celis, J. E.; Wolf, H.; Østergaard, M. Bladder squamous cell carcinoma biomarkers derived from proteomics. Electrophoresis 2000, 21 (11), 2115-2121. (7) Sheng, K. H.; Yao, Y. C.; Chuang, S. S.; Wu, H.; Wu, T. F. Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis. Proteomics 2006, 6 (3), 1058-1065. (8) Kreunin, P.; Zhao, J.; Rosser, C.; Urquidi, V.; Lubman, D. M.; Goodison, S. Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling. J. Proteome Res. 2007, 6, 2631-2639. (9) Sanchez-Carbayo, M.; Socci, N. D.; Lozano, J. J.; Haab, B. B.; Cordon-Cardo, C. Profiling bladder cancer using targeted antibody arrays. Am. J. Pathol. 2006, 168 (1), 93-103. (10) Alfonso, P.; Nu ´n ˜ez, A.; Madoz-Gurpide, J.; Lombardia, L.; Sanchez, L.; Casal, J. I. Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis. Proteomics 2005, 5 (10), 2602-2611.

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Orenes-Pin ˜ ero et al. (11) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 850-858. (12) Sanchez-Carbayo, M.; Socci, N. D.; Charytonowicz, E.; Lu, M.; Prystowsky, M.; Childs, G.; Cordon-Cardo, C. Molecular profiling of bladder cancer using cDNA microarrays: defining histogenesis and biological phenotypes. Cancer Res. 2002, 62 (23), 69736980. (13) Dawson-Saunders, B.; Trapp, R. G. Basic & Clinical Biostatistics, 2nd ed.; Appleton & Lange: Norwalk, CT, 1994. (14) De Reggi, M.; Capon, C.; Gharib, B.; Wieruszeski, J. M.; Michel, R.; Fournet, B. The glycan moiety of human pancreatic lithostathine. Structure characterization and possible pathophysiological implications. Eur. J. Biochem. 1995, 230 (2), 503-510. (15) Macadam, R. C.; Sarela, A. I.; Farmery, S. M.; Robinson, P. A.; Markham, A. F.; Guillou, P. J. Death from early colorectal cancer is predicted by the presence of transcripts of the REG gene family. Br. J. Cancer. 2000, 83 (2), 188-195. (16) Harada, K.; Zen, Y.; Kanemori, Y.; Chen, T. C.; Chen, M. F.; Yeh, T. S.; Jan, Y. Y.; Masuda, S.; Nimura, Y.; Takasawa, S.; Okamoto, H.; Nakanuma, Y. Human REG I gene is up-regulated in intrahepatic cholangiocarcinoma and its precursor lesions. Hepatology 2001, 33, 1036-1042. (17) Yonemura, Y.; Sakurai, S.; Yamamoto, H.; Endou, Y.; Kawamura, T.; Bandou, E.; Elnemr, A.; Sugiyama, K.; Sasaki, T.; Akiyama, T.; Takasawa, S.; Okamoto, H. REG gene expression is associated with the infiltrating growth of gastric carcinoma. Cancer 2003, 98 (7), 1394-1400. (18) Rechreche, H.; Montalto, G.; Mallo, G. V.; Vasseur, S.; Marasa, L.; Soubeyran, P.; Dagorn, J. C.; Iovanna, J. L. pap, reg Ialpha and reg Ibeta mRNAs are concomitantly up-regulated during human colorectal carcinogenesis. Int. J. Cancer 1999, 81 (5), 688694. (19) Satomura, Y.; Sawabu, N.; Mouri, I.; Yamakawa, O.; Watanabe, H.; Motoo, Y.; Okai, T.; Ito, T.; Kaneda, K.; Okamoto, H. Measurement of serum PSP/reg-protein concentration in various diseases with a newly developed enzyme-linked immunosorbent assay. J. Gastroenterol. 1995, 30 (5), 643-650. (20) Nabi, G.; N′Dow, J.; Hasan, T. S.; Booth, I. R.; Cash, P. Proteomic analysis of urine in patients with intestinal segments transposed into the urinary tract. Proteomics 2005, 5 (6), 1729-1733. (21) Lopez, J. I.; Angulo, J. C.; Flores, N.; Toledo, J. D. Small cell carcinoma of the urinary bladder. A clinicopathological study of six cases. Br. J. Urol. 1994, 73, 43-49. (22) Ostergaard, M.; Rasmussen, H. H.; Nielsen, H. V.; Vorum, H.; Orntoft, T. F.; Wolf, H.; Celis, J. E. Proteome profiling of bladder squamous cell carcinomas: identification of markers that define their degree of differentiation. Cancer Res. 1997, 57, 4111-4117. (23) Devonec, M.; Hijazi, A.; Bringuier, P. P.; Dutrieux-Berger, N.; Perrin, P.; Revillard, J. P. Flow cytometric demonstration of shared antigens between human bladder cancer cells and normal host macrophages. Eur. Urol. 1989, 16 (1), 57-62. (24) Stefanini, G. F.; Bercovich, E.; Mazzeo, V.; Grigioni, W. F.; Emili, E.; D’Errico, A.; Lo, Cigno, M.; Tamagnini, N.; Mazzetti, M. Class I and class II HLA antigen expression by transitional cell carcinoma of the bladder: correlation with T-cell infiltration and BCG treatment. J. Urol. 1989, 141 (6), 1449-1453. (25) Bergmann, S.; Pandolfi, P. P. Giving blood: a new role for CD40 in tumorigenesis. J. Exp. Med. 2006, 203 (11), 2409-2412.

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Searching Urinary Tumor Markers for Bladder Cancer Using a Two

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