Serum and Tissue Profiling in Bladder Cancer Combining Protein and

Serum and Tissue Profiling in Bladder Cancer Combining Protein and Tissue Arrays Esteban Orenes-Pin ˜ ero,† Rodrigo Barderas,‡,§ Daniel Rico,| J. Ignacio Casal,‡,§ David Gonzalez-Pisano,| Jose Navajo,⊥ Ferran Algaba,# Josep Maria Piulats,∇ and Marta Sanchez-Carbayo*,† Tumor Markers Group, Spanish National Cancer Research Center, Madrid, Spain, Protein Unit, Spanish National Cancer Research Center, Madrid, Spain, Functional Proteomics Laboratory, Centro de Investigaciones Biologicas, Madrid, Spain, Bioinformatics Unit, Spanish National Cancer Research Center, Madrid, Spain, Biochemistry Department, Hospital Universitario de Salamanca, Salamanca, Spain, Pathology Department, Fundacio´ Puigvert, Barcelona, Spain, and Oncology Department, Instituto Catala´ d’Oncologia, Barcelona, Spain Received March 24, 2009

Aiming at identifying biomarkers for bladder cancer, the serum proteome was explored in a pilot study through a profiling approach using protein arrays. Supervised analyses identified a panel 171 immunogenic proteins differentially expressed between patients with bladder cancer (n ) 12) and controls without the disease (n ) 10). The microanatomical expression patterns of novel immunogenic proteins, especially dynamin and clusterin, were found significantly associated with histopathologic variables and overall survival, as confirmed by immunohistochemistry using an independent series of bladder tumors contained in tissue microarrays (n ) 289). Thus, the protein arrays approach has identified a panel of immunogenic candidates that may potentially play a role as diagnostic biomarkers, especially for muscle invasive disease. Moreover, the protein expression patterns of dynamin and clusterin in bladder tumors were shown to adjunct for histopathologic staging and clinical outcome prognosis. Keywords: Autoantibodies • protein array • bladder cancer • clusterin • dynamin

Introduction Bladder cancer is the fourth most frequent neoplasia in men, clinically characterized by high recurrent rates and poor prognosis once tumors invade the muscular uroepithelial layer.1 Bladder cancer can be classified based on the depth of invasion of tumor cells. Clinically, around 75% of uroepithelial transitional cell carcinomas (TCCs) are nonmuscle invasive (TIS, Ta, and T1), 20% are muscle infiltrating (T2-T4), and 5% are metastatic at the time of diagnosis.1,2 Bladder cancer diagnosis is based on cystoscopy, which is considered the gold standard. Urinary cytology remains a valuable adjunct, especially for detecting carcinoma in situ and high grade lesions.2 In serum, none of the 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 * To whom correspondence should be addressed. Marta Sanchez-Carbayo, Ph.D., Group Leader Tumor Markers Group, 310A 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, Spanish National Cancer Research Center. ‡ Protein Unit, Spanish National Cancer Research Center. § Functional Proteomics Laboratory, Centro de Investigaciones Biologicas. | Bioinformatics Unit, Spanish National Cancer Research Center. ⊥ Biochemistry Department, Hospital Universitario de Salamanca. # Pathology Department, Fundacio´ Puigvert. ∇ Oncology Department, Instituto Catala´ d’Oncologia.

164 Journal of Proteome Research 2010, 9, 164–173 Published on Web 11/02/2009

as well, and the use of such markers would ultimately distinguish indolent cancers from those that are potentially lethal so that therapeutic procedures can be tailored to each individual patient depending on the aggressiveness of each tumor.2,3 Cancer cells communicate with the blood directly or through extracellular fluids, and may secrete or release at least part of their contents into the bloodstream upon cell damage or death. Cancer proteins absent in non-neoplastic cell subpopulations might contain groups of autologous cellular antigens called tumor-associated antigens (TAAs), whose aberrant regulation or function could then elicit a host immune response.4-26 Emerging evidence of the association of autoimmunity with cancer suggests that each type of cancer might trigger unique autoantibody signatures that reflect the nature of the malignant process in the affected organ.4-26 The serum autoantibody repertoire from cancer patients might, therefore, be exploited for the identification of TAAs. The recognition that human tumors stimulate the production of autoantibodies against TAAs has opened the door to the possibility that autoantibodies could be exploited as serological tools for early diagnosis and/ or disease management. Since cancer-associated autoantibodies often target proteins that are mutated, modified, or aberrantly expressed in tumor cells, they could also be considered immunologic reporters that could uncover molecular events underlying tumorigenesis and/or tumor progression. The advent of novel high-throughput proteomic approaches including 10.1021/pr900273u

 2010 American Chemical Society

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Serum and Tissue Profiling in Bladder Cancer

Table 1. Demographic and Clinical Information of Bladder Cancer Patients Including Cases with Nonmuscle Invasive (NI) and Muscle Invasive (INV) Bladder Tumors and Controls Providing Serum Samples for Autoantibody Profiling Using Protein Arrays

Figure 1. Experimental design. (A) Serum samples were labeled and hybridized on protein arrays. Data analyses served to identify autoantibodies differentially expressed between patients with bladder cancer and controls. (B) Validation analyses were performed to evaluate whether the immunogenic proteins were differentially expressed in bladder tumors by means of IHC in custom-made tissue arrays.

designed antigen multiplexed panels and/or protein microarrays is accelerating the interest in the serum autoantibody repertoire in human cancers for the discovery of candidate TAAs. Protein arrays have been utilized for the identification of autoantibodies characteristic of several tumor types.4-26 To the best of our knowledge, the technology has not been applied to bladder cancer specimens to date. This report represents a pilot study in which we attempted to explore the serum proteome of patients with bladder cancer and control specimens using protein arrays. Validation analyses using wellcharacterized bladder tumors spotted in custom-made tissue arrays served to associate the microanatomical protein expression patterns of identified proteins with clinicohistopathologic variables of bladder tumors (Figure 1). The proof of concept analyses performed in this study provided critical information that can be utilized not only to identify candidate biomarkers for bladder cancer and potential immunologic therapeutic targets, but also to further characterize molecular events involved in bladder cancer progression.

Materials and Methods Serum Samples. Serum samples belonging to 12 patients with bladder cancer were collected immediately before surgery and frozen at -80 °C. Sera from 10 individuals without bladder cancer were used as controls, and were divided in two sets: a heterogeneous set (a) containing healthy individuals, patients with benign urologic conditions and cured of other malignancies; and a homogeneous set (b) including only healthy individuals (Table 1). These two sets served also to test the ability of the protein arrays to segregate bladder cancer patients from controls using different batches of protein arrays. Demographic and clinicopathologic variables of the cases and controls under study are shown in Table 1. Samples were handled anonymously following ethical and legal guidelines at the University Hospital of Salamanca. Protein Array Analyses. Serum samples were labeled and hybridized onto the Protoarray Human Protein Microarray (Invitrogen, Carlsbad, CA), consisting of over 8274 distinct, wellcharacterized proteins printed in duplicate, as described by the manufacturer. In brief, the protein arrays were blocked with

bladder cancer patients

age

sex

stage

grade

BT1 BT2 BT3 BT4 BT5 BT6 BT7 BT8 BT9 BT10 BT11 BT12

64 58 64 68 78 69 75 73 69 66 61 65

M F M M F M M M F F M M

INV_PT2_3 INV_PT3_3 INV_PT3_1 NI_PT1_2 INV_PT3_4 INV_PT2_2 INV_PT2_1 INV_PT4_2 NI_PT1_1 INV_PT4_1 NI_PTA_1 INV_PT3_2

high high high high high high high high high high low high

control individuals

(Set (Set (Set (Set (Set (Set (Set (Set (Set (Set

A) A) A) A) A) A) B) B) B) B)

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10

age

sex

clinical information

38 55 56 58 70 80 69 81 79 58

F M M M M F M M M M

healthy healthy benign prostatic hyperplasia benign prostatic hyperplasia prostate cancera breast cancera healthy healthy healthy healthy

a Control patients with malignancies other than the bladder were cured and did not display an active cancer disease when serum sampling was performed 2 years after surgery. Follow-up has been performed, and these two patients continue to be free of cancer disease 4 years after surgery.

1% BSA, 0.1% Tween-20 and phosphate buffered saline (PBS) (1×) with gentle agitation at 4 °C. Then, the serum samples (1/50 dilution) were added and incubated for 90 min at 4 °C without shaking. The unbound autoantibodies were washed away with the probe buffer (3 washes of 10 min). Following binding, a secondary antibody (anti-human antibody against IgG conjugated to AlexaFluor 647 dye) was added, incubated for 90 min at 4 °C, and washed away. Finally, the slides were dried and scanned with a fluorescent microarray scanner (Agilent, Santa Clara, CA). Data were acquired using the GenePix Pro 4.0 software (Molecular Devices Corporation, Sunnyvale, CA). The initial set of analyses dealt with the evaluation of the distribution of the negative (buffer spots in which bovine serum albumin (BSA) was printed) and positive controls (a gradient of human IgG) among the serum samples analyzed. The printed anti-human IgG antibody interacts with the human IgG present in the serum, an interaction which is detected by the Alexa Fluor 647 goat anti-human IgG antibody. These signals are used to verify assay procedure and proper dilution of the serum. The anti-human IgG is printed on the microarray at the following concentrations: IgG1 at 10.41 nM, IgG2 at 41.63 nM, and IgG3 at 166.5 nM. The next set of analyses were performed to search for differentially expressed autoantibodies between bladder cancer cases and the set (a) of controls using the ProtoArray Prospector software (version 4.0.0, Invitrogen), which estimated a detection test for each individual array calculating the Chebyshev’s Inequality (CI) p-value for each protein spotted onto the array. The significant autoantibodies differentially expressed between bladder cancer patients and controls taking a fold change higher than 2 at a Journal of Proteome Research • Vol. 9, No. 1, 2010 165

research articles significant p-value lower than 0.05 were displayed in clustered heatmaps using the Protoarray software, applying the Manhattan distance and average linkage metric. Two batches of proteins arrays (HA10758 and HA10768) were utilized to evaluate the autoantibody profiles of two independent sets of control individuals (a) and (b), respectively. An unsupervised hierarchical clustering was performed, using the autoantibodies identified to be differentially expressed between bladder cancer cases and the control set (a), to test the ability of this panel of autoantibodies to segregate between bladder cancer cases and the control set (b). The functional annotation of proteins differentially expressed between bladder cancer cases and controls was searched in the Swiss-Prot and NCBI protein databases. Cell Lines and Western Blotting Analysis. Nine bladder cancer cell lines were obtained from the American Type Culture Collection and cultured following standard procedures.28,29 These included transitional papillary RT4 and RT112 and lowgrade (5637), muscle invasive (T24, UM-UC-3, J82, EJ138), metastatic (TCCSUP); and squamous (ScaBER) cell lines. Total protein was extracted from 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, 0.005% bromophenol blue) and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gels. Proteins were detected immunologically following electrotransfer onto Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MC) after activation with methanol. The membranes were blocked with 5% nonfat 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-cytokeratin 20 (46 kDa) (dilution 1/750, mouse monoclonal, clone KS20.8, DAKO, Glostrup, Denmark), anti-dynamin (97 kDa) (dilution 1/500, mouse monoclonal, clone 41, BD Bioscience, San Jose, CA), and anti-clusterin (80 kDa) (dilution 1/200, mouse monoclonal, clone 7D1, Novocastra, Newcastle, U.K.). Blots were washed three times for 10 min in PBS and 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated (HRP) anti-mouse secondary antibodies for 1 h at room temperature at 1/1000 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 displaying a single predominant band at the expected molecular weights. R-Tubulin (50 kDa, dilution 1:4000, mouse monoclonal, clone DM1A, Sigma, St. Louis, MO) was utilized as the loading control. Tissue Microarrays. We constructed different tissue microarrays including triplicate cores of the paired bladder tumors belonging to the patients providing serum specimens and independent sets of primary TCCs cases (n ) 289), recruited from several collaborating clinical institutions under Institutional Review Board (IRB) approved protocols. One section was stained with hematoxylin and eosin to evaluate the presence of the tumor by light microscopy. After carefully choosing the morphologically representative region on the paraffin-embedded blocks (donor blocks), three core tissue biopsies of 1.0 mm were punched and transferred to the donor paraffin-waxembedded block (recipient block). The distribution of tumor stage among the bladder tumors spotted onto the tissue arrays was: non-muscle invasive (210), and muscle-invasive (79), while 166

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Orenes-Piñero et al. their tumor grade was low grade (LG) (65), and high grade (HG) (224), defined under standard criteria.2 Their clinicopathologic information and annotated follow-up allowed evaluation of associations of the identified protein expression patterns to be differentially immunogenic in serum specimens belonging to patients with bladder cancer and control individuals by the protein array approach, among them and with clinicopathologic variables. For overall survival analyses, only patients with available follow-up (either “dead as a result of disease” or “alive with no evidence of disease”) were included. Cases with unknown follow-up were excluded from clinical outcome analyses. Immunohistochemistry (IHC). Protein expression patterns of several differentially expressed immunogenic proteins were assessed at the microanatomical level by IHC analysis using both cytospins from cancer cell lines (data not shown), and the tissue arrays mentioned above (n ) 289). Standard avidin-biotin immunoperoxidase procedures were applied for IHC.27,28 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 were utilized at the following conditions for IHC: anti-clusterin (1/50 dilution) and anti-dynamin (1/500 dilution). Secondary antibodies (Vector Laboratories) were biotinylated horse anti-mouse antibodies (1:500 dilution). The staining for clusterin was scored based on the number of cancer cells presenting a nuclear protein sublocalization. For dynamin, it was found to be expressed in none or all of the cytoplasms of the cancer cells, with different intensities. Thus, cytoplasmic dynamin staining was scored based on its intensity varying from 0 (-: absence) to 3 (+/++/ +++: weak, moderate or strong intensity, respectively). Scores for both proteins were recorded using colon, and brain as positive controls, respectively. The absence of the primary antibody was used as negative control. Diaminobenzidine was the final chromogen and hematoxylin was the nuclear counterstain. The individual scores were reviewed, and the agreement between two independent observers was calculated. Whenever a discrepancy was noted between the first and the second interpretations, the pathologist decided on the final scoring.27,28 Statistical Methods. The consensus (mean) value of the three representative cores from each tumor sample arrayed was used for statistical analyses. The association between protein expression measured on tissue arrays by IHC and histopathologic stage and tumor grade was evaluated using the nonparametric Wilcoxon-Mann-Whitney test.29 There is no consensus on the cutoffs for the immunohistochemical expression for clusterin and dynamin. The number of cells expressing a nuclear sublocalization of clusterin was analyzed continuously. The cutoff value for weak and strong expressing cases for dynamin was specified at the median percentage score of positive cytoplasmic tumor cells resulting in a value of intensity of 2 (++). Dynamin intensity was then analyzed taking the cutoff of 2 (++) when considered as a categoric variable. The association between protein expression patterns with diseasespecific overall survival was estimated using the log-rank test in those cases for which follow-up information was available. Disease-specific 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 those lost to follow-up were censored. The association of protein expression patterns


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Figure 2. Quality control analyses. (A) Distribution of negative and positive (anti-human IgG gradient) controls in the hybridizations. The figure represents the boxplots of the signal intensities for all probes in each array (white boxes) compared with the distribution of positive and negative controls. IgG3 (166.5 nM) is shown in red, IgG2 (41.63 nM) in orange and IgG1 (10.41 nM) in yellow; and negative buffer controls in blue. (B and C) Histograms representing the within-array distributions of the densities of each control in two representative bladder cancer patients. The height of a rectangle is proportional to the number of spots falling into the same signal intensity range, so that the histogram has a total area of one for each specific IgG. While for example in a PT2 case (B), the signal distributions are very homogeneus for each IgG control and different among them, in other cases the distributions are more heterogeneous, as shown for a serum of a patient with a PT3 tumor (C). IgG3 is shown in red, IgG2 in orange and IgG1 in yellow; and negative buffer controls in blue.

with overall survival was analyzed using the log-rank test.29 The survival curves were plotted using the standard Kaplan-Meier methodology. Only p-values lower than 0.05 were considered statistically significant. Statistical analyses were performed using the SPSS statistical package v13.0 (SPSS, Inc., Chicago, IL).

Results Quality Control Assessment. The initial set of analyses dealt with the evaluation of the distribution of the negative and positive controls among the serum samples analyzed. This strategy served to assess the interindividual biological or potential variation in the labeling and hybridization of the serum specimens on the arrays. The duplicated spots supported the evaluation of the signals observed in the protein arrays. The intensities of the negative (buffer spots in which bovine albumin was printed) and positive controls (spots estimating the gradient of IgG) were evaluated among the total of spots analyzed in the arrays. Although the distributions of the intensities between negative and positive controls among arrays showed interindividual differences among specimens, such variation was not significantly associated with the presence of cancer. Moreover, there was no statistical difference among nonmuscle invasive and muscle invasive patients, as shown in Figure 2A. The intensities of IgG3 were found to be higher than the corresponding more diluted IgG2 and IgG1. However, the variability in the signal intensities revealed different degrees

of homogeneity among the density of the distribution of the intensity of each IgG control among bladder cancer patients, as shown for two representative cases with a PT2 (Figure 2B) or a PT3 (Figure 2C) tumor. Differential Autoantibody Expression among Serum Samples Belonging to Patients with Bladder Cancer and Controls. The next set of analyses dealt with the identification of immunogenic proteins differentially expressed between bladder cancer patients and control individuals without the disease. A series of 171 candidates were identified to be differentially expressed between bladder cancer cases and the heterogeneous set of controls (a) using the ProtoArray Prospector software (Figure 3A). The fold changes varied between 2.94 and 5.74, while the p-values ranged from 5.54 × 10-5 to 0.01. Such analyses revealed the presence of 136 immunogenic proteins overexpressed in bladder tumors and 35 overexpressed candidates in control individuals. The complete set of 171 immunogenic proteins including their functional annotation, p-value and fold changes are provided in Supplementary Table 1. Importantly, the panel of the 171 proteins identified using the heterogeneous control set (a) served to distinguish bladder cancer patients from the healthy controls of the homogeneous control set (b) measured with a different batch of protein arrays, as shown by an unsupervised hierarchical clustering (Supplementary Figure 1). The search for the functional annotations of the immunogenic proteins revealed that the most frequent biological processes in which these proteins were involved Journal of Proteome Research • Vol. 9, No. 1, 2010 167

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Figure 3. Autoantibodies differentially expressed in bladder cancer patients. (A) Supervised clustering analyses showing the proteins differentially expressed in serum samples from bladder cancer patients comparing with controls. (B) Summary of the functional annotations according to the GO biological processes classification of the proteins against which the autoantibodies are differentially expressed in serum samples from bladder cancer patients. (C) Summary of the localization of the immunogenic proteins according to the annotations provided by the GO cellular components classification.

belong to structural and cancer related cellular processes such as apoptosis, signal transduction, or transport, among others (Figure 3B). A summary of the top differentially overexpressed proteins in bladder cancer cases as compared to controls presenting the most frequent biological processes are summarized in Table 2. The cellular sublocalization revealed that the majority of these proteins were located in the membrane (Figure 3C). Selection of Identified Proteins for Validation Analyses. Several proteins along the list of identified immunogenic proteins were selected for further validation analyses. Keratin 20, clusterin and dynamin were chosen among the 171 differentially expressed proteins among the bladder cancer cases and control sets of specimens (a) in the protein arrays based on the availability of antibodies and the functional annotation and the known involvement of such proteins in cancer biology. The differential expression of these autoantibodies in all the serum samples under analyses, including bladder cancer cases and both sets (a and b) of control specimens is shown in Figure 4 A. Representative spots targeting these proteins in the array are shown in Figure 4B for different patients. The specificity of the available antibodies against Keratin 20, clusterin and dynamin was then tested by immunoblotting to verify the presence of single bands at the specific molecular weights of the target proteins. Moreover, they served to confirm that these proteins were differentially expressed in a series of nine bladder cancer cells derived from bladder tumors (Figure 4C), before such antibodies were optimized for immunohistochemistry analyses and evaluated in a large number of bladder tumor in 168


tissue arrays. Immunoblotting of clusterin and dynamin supported further analyses to test whether identification of these immunogenic proteins in serum could be due to an increased expression of these proteins in the paired bladder tumors of the patients that provided serum specimens, and to assess the clinical relevance of these proteins in independent sets of human clinical material. The optimization and characterization of the protein expression patterns of the identified proteins by means of IHC revealed an increased expression for clusterin and dynamin in the matching tumors of cases showing the presence of the autoantibody in the serum (data not shown), with staining patterns similar to those shown below (Figures 5A, and 5C). Clusterin and Dynamin Are Associated with Tumor Progression and Clinical Outcome in Patients with Bladder Tumors. The next set of analyses searched for associations of identified proteins with clinicopathologic variables of patients with bladder cancer. Protein expression patterns of identified proteins were characterized by means of IHC on independent series of bladder tumors contained in several custom-made tissue arrays. For clusterin, we evaluated the number of cancer cells showing expression in their nuclei. Although nuclear clusterin was not differentially expressed between tumors regarding their tumor grade, it was found to be decreased in patients with muscle invasive bladder tumors as compared to nonmuscle invasive tumors (p ) 0.018) (Figure 5A,B). For dynamin analyses (Figure 5C,D), the cytoplasmic protein staining intensity was categorized from 0 to 3. Interestingly, a low protein expression of dynamin was significantly

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Table 2. Summary of Immunogenic Proteins Differentially Expressed among Serum Specimens of Patients with Bladder Cancer and Controls in a Significant Manner Involved in the Most Frequent Biological Processes, Apoptosis and Signal Transduction protein ID

protein name

adjusted P-value

NM_019010 BC024590 NM_014410

Homo sapiens keratin 20 (KRT20) Homo sapiens dynamin 1-like (DNM1L) Homo sapiens clusterin-like 1 (retinal) (CLUL1), transcript variant 1 Homo sapiens nerve growth factor receptor (TNFRSF16) associated protein 1 (NGFRAP1) Homo sapiens NLR family, pyrin domain containing 3 (NLRP3) Homo sapiens lectin, galactoside-binding, soluble, 1 (galectin 1) (LGALS1) Homo sapiens interleukin 11 receptor, alpha (IL11RA) Homo sapiens syntaxin binding protein 4 (STXBP4) Homo sapiens sclerostin domain containing 1 (SOSTDC1) (Ectodin) Homo sapiens olfactory receptor, family 5, subfamily P, member 3 (OR5P3) Homo sapiens glycoprotein A33 (transmembrane) (GPA33) Homo sapiens formyl peptide receptor 1 (FPR1) Homo sapiens histidine triad nucleotide binding protein 1 (HINT1) Homo sapiens ATPase, H+ transporting, lysosomal accessory protein 2 (ATP6AP2) (Renin receptor) Homo sapiens chemokine (C-X-C motif) ligand 14 (CXCL14) Homo sapiens interleukin 10 receptor, beta (IL10RB) Homo sapiens ADP-ribosylation factor-like 5A (ARL5A) Homo sapiens glutamate receptor, metabotropic 2 (GRM2) Homo sapiens chromosome 9 open reading frame 86 (C9orf86) Homo sapiens protein kinase, cAMP-dependent, catalytic, gamma (PRKACG) Homo sapiens reticulon 1 (RTN1), transcript variant 3 Homo sapiens poly(rC) binding protein 4 (PCBP4) Homo sapiens linker for activation of T cells transcript variant 1 (LAT1) Homo sapiens interleukin 8 receptor, alpha (IL8RA) Homo sapiens CD79b molecule, immunoglobulin-associated beta (CD79B) Homo sapiens lymphocyte antigen 9 (LY9)

0.0001 0.0054 0.0071

+5.694 +3.334 +3.147

0.0068

+3.139

0.0082 0.0010

+3.043 +4.066

0.0011 0.0014 0.0020

+4.028 -3.922 +3.789

0.0021

+3.760

0.0051 0.0023 0.0026

+3.677 +3.658 +3.603

0.0027

+3.593

0.0030 0.0040 0.0060 0.0062 0.0100 0.0051

-3.539 +3.484 -3.468 +3.257 +2.947 -3.415

0.0051 0.0049 0.0001

-3.318 +3.434 +5.296

0.0006 0.0061

+4.319 +3.270

0.0062

-3.184

NM_014380 NM_004895 NM_002305 NM_147162 NM_178509 NM_015464 NM_153445 NM_005814 NM_002029 NM_005340 NM_005765 NM_004887 NM_000628 NM_012097 NM_000839 BC002945 NM_002732 NM_206852 BC017098 BC011563 NM_000634 BC030210 BC064485

associated with an increased tumor stage (p e 0.0005). Tumor grade was also associated with low intensity in dynamin expression (p e 0.0005). Interestingly, taking a subseries of T1G3 tumors (n ) 92) with available follow-up revealed that a low dynamin expression was significantly associated with higher recurrence rate within the 3 months after transurethral resection (p ) 0.042). Furthermore, patients with low intensity in the cytoplasmic dynamin expression had shorter diseasespecific survival than those with high intensity (log rank, p ) 0.014, Figure 5E). Overall, IHC validation analyses on tissue arrays containing an independent large set of bladder tumors served to associate clusterin and dynamin expression with histopathologic variables of tumor progression and clinical outcome of bladder cancer patients.

Discussion The novelty of this report deals with the application of a targeted proteomic approach using protein arrays to serum specimens to identify immunogenic signatures associated with bladder cancer. Our pilot study aimed to test whether it could be possible to detect any immunogenic protein and/or pattern associated with bladder cancer that could be further developed into biomarkers or targets. It was not designed for classification purposes of samples by the immunogenic patterns themselves, an objective that would have required a high number of

fold change

samples and an additional independent validation set of protein arrays. The strategy follows complementary serological analyses performed using antibody arrays to identify bladder cancer related tumor marker candidates.30 One of the advantages of the study of serum autoantibodies resides on their higher stability and half-life in the serum of cancer patients as compared to the antigens that elicit the immune response, which might be released by tumors but rapidly degraded or cleared after circulating in the serum for a limited time.12 However, proteins reaching the bloodstream that may not generate an immunogenic response could not be estimated using this approach. Interestingly, a panel of autoantibodies was found to be differentially expressed in a significant manner in serum specimens of patients with bladder tumors. The control group serving to identify specific signatures for bladder cancer included not only healthy individuals, but also benign urological conditions and cases with previous history of malignancies of other origin than the bladder, and did not display an active disease when serum sampling was performed 2 years after surgery. Follow-up has been performed, and these two patients continue to be free of disease 4 years after surgery. This is an important and critical step in the experimental design because for biomarker research it is very important to contrast benign conditions that could be simultaneously present in the cancer patients under study, and other frequent malignancies Journal of Proteome Research • Vol. 9, No. 1, 2010 169

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Figure 4. Selection of proteins for validation analyses. (A) Heatmap of the differential expression of clusterin, dynamin and CK20 in the bladder cancer cases and control specimens. (B) Differential fluorescent signals observed in the protein arrays for the selected proteins for validation analyses in representative bladder cancer cases and controls. (C) Immunoblotting validation of proteins using extracts from nine different bladder cancer cell lines. Antibodies were accepted as displaying a single predominant band at the expected molecular weights. CK 20, 46 kDa; clusterin, 80 kDa; dynamin, 97 kDa; R-tubulin, 50 kDa, was used as loading control.

such as prostate for males or breast for women, which could be confounding factors for biomarker interpretation.2,30 Moreover, the bladder cancer specificity of the expression of the immunogenic proteins was validated by means of IHC. Confirmatory studies showed an increased protein expression of the identified TAAs in bladder tumors as compared to normal urothelium counterparts from patients that provided serum samples for the protein array analysis. This observation served to support phenotypically the tumor specificity of the autoantibodies detected in the serum by the protein array. The increased expression of TAAs in matching tumors supports the working hypothesis by which profiling serum specimens would serve to characterize the tumor from which the cancer proteins are originated, released into the bloodstream and then become immunogenic. Indeed, these TAAs included proteins participating in signaling networks known to be involved in tumor progression such as apoptosis, signal transduction, or transport, among others. Immunoblotting analyses of cell lines derived from early and late tumors also supported a differential expression of the identified proteins in association with bladder cancer progression. Further research will characterize the biological relevance of the identified proteins in tumorigenesis using appropriate in vitro and in vivo models. To evaluate the clinical relevance of the identified proteins along bladder cancer progression, IHC was performed on several tissue arrays containing independent series of tumors comprising early and advanced stages of bladder cancer. These analyses served to assess the associations of the identified proteins with histopathologic progression variables and clinical outcome. One of the immunogenic proteins most differentially expressed was keratin 20. This is an interesting finding since its overexpression was previously found in peripheral blood,31-33 170


and in bladder tumors in association with increased tumor stage and grade.34 Furthermore, it was taken into consideration by the WHO/ISUP system for the histologic grading of papillary tumors.35 Interestingly, the other two selected immunogenic proteins for IHC validation, clusterin and dynamin, were revealed to be associated with tumor progression and clinical outcome in bladder cancer. Clusterin, is involved in several physiologic processes important for carcinogenesis and tumor growth, including tissue remodelling, cell cycle, DNA repair, and apoptosis.36-40 Three isoforms with distinct subcellular locations and contradictory functions have been described: a prosurvival secretory, a prosurvival cytoplasmic and a proapoptotic nuclear form.40,41 Although the cytoplasmic form was overexpressed in several tumors,37-41 the microanatomical expression patterns we observed for clusterin are consistent with the lower levels reported for its nuclear counterpart in several tumor types in cancer progression.40,41 Therapeutically, an antisense oligodeoxynucleotide against the clusterin gene has been combined in a clinical trial with cisplatin, which is the mainstay of treatment for patients with advanced bladder cancer.38 The identification of autoantibodies against clusterin observed in our report suggests that clusterin could also be immunotherapeutically targeted in bladder cancer. Dynamin is involved in several physiologic processes such as the endocytic membrane fission,42-45 caveolae internalization, protein trafficking at the Golgi apparatus and maintenance of actin cytoskeleton.45,46 Dynamin has been shown to be related to key critical cancer processes such as regulation of apoptosis,44,47,49 and invasiveness.45,48 These functional aspects of dynamin are consistent with the lower expression of dynamin observed with an increased aggressiveness in bladder tumors. Our findings are consistent with the decreased pattern of dynamin immu-


research articles A following step of our work would also be to test the ability of this approach to subclassify bladder tumors and to predict clinical outcome or response to current immunotherapy and chemotherapy strategies. Moreover, the novel application of protein arrays for serum autoantibody profiling could be exploited to identify potential biomarkers and to develop novel therapies targeting the immune system and/or the tumor.

Figure 5. Validation of the differential protein expression patterns by IHC on tissue arrays. (A and B) clusterin; (C and D) dynamin; (E) Kaplan-Mayer curve survival analysis indicating that a decreased protein expression of dynamin measured by IHC on tissue arrays was associated with poor survival (log rank, p ) 0.014).

noreactivity in pancreatic tumors in correlation with tumor progression.43 Several hypotheses have been postulated to explain how intracellular proteins become targets of autoantibodies. Posttranslational modifications (e.g., proteolytic cleavage, phosphorylation, and oxidation) associated with aberrant cell death may enhance their immunogenicity under a proinflammatory environment.49,50 Fetal proteins aberrantly expressed in tumor cells due to mutations or truncations,51 or expressed in abnormally high amounts in neoplastic diseases may contribute to a loss of immune tolerance to such antigens.52 It is likely that, for clusterin, the immunogenic mechanism could be related to altered levels of expression in any of the 3 isoforms,40,41 while for dynamin, it might be due to the presence of mutations.47 This pilot study aiming to test the potential utility of protein arrays to uncover cancer related biomarkers has shown the variability of the humoral response even among bladder cancer cases. The lack of explanation why only a subset of patients with a particular tumor type develops a humoral response to a particular antigen could be related to the variability among tumors and individuals in MHC molecules and in antigen presentation. The interindividual variability of the immune response pattern suggests that the use of TAA panels for early detection of tumors will require evaluating large training and validation series of high-risk individuals, objectives out of the scope of this pilot proof of principle analysis in bladder cancer.

In this pilot study, we utilized one of the early batches versions of the protein array comparing the set (a) of controls and bladder cancer patients. Statistics were performed using the Prospector software from which our validation analyses were decided. This software provides an adjusted p-value obtained as claimed by the manufacturer after iterative analyses similar to the FDR concept. It is very well-known in array field that different statistic approaches applied would render noncoincident results, an observation emphasizing the importance of verification strategies. Two important pieces of data support the confidence in our results. First, a nonsupervised hierarchical clustering served to classify two independent sets of control individuals performed using different batches of protein arrays using the panel of 171 proteins identified with the most heterogeneous set of control specimens. Moreover, an important validation part of the manuscript performed on an independent large set of bladder tumors spotted on tissue arrays served to confirm the increased expression of clusterin and dynamin as immunogenic proteins associated with clinicopathologic correlates of bladder cancer, with potential biomarker utility for tumor stratification and clinical outcome prediction. Thus, these two main claims of the study are based on validation analyses, and represent novel and clinically relevant pieces of information to support the contribution of combined serological and protein profiling for further translational research interventions in bladder cancer. In summary, this pilot study suggested that protein arrays can serve to identify serum immunogenic proteins indicative of the presence of bladder cancer, especially for muscle invasive disease. Several autoantibodies were found to be differentially expressed in bladder cancer patients as compared to controls in a significant manner. These immunogenic proteins were shown to be highly expressed in matching and independent bladder tumors. Clusterin and dynamin were identified to play a potential role as bladder cancer progression biomarkers. In conclusion, the use of this high-throughput protein arrays served to identify a panel of immunogenic proteins that may play a role as biomarker candidates for bladder cancer diagnosis in serum samples, and novel protein estimates that may adjunct for tumor staging and clinical outcome prognosis in tissue specimens. Abbreviations: BSA, bovine serum albumin; HG, high grade; HRP, horseradish peroxidase-conjugated; IHC, immunohistochemistry; IRB, Institutional Review Board; LG, low grade; MHC, major histocompatibility complex; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAA, Tumor-Associated Antigen; TCC, Transitional Cell Carcinoma; RIPA, Radio-Immuno Precipitation Assay.

Acknowledgment. The authors would like to thank all members of Dr. Sańchez-Carbayo’s laboratory for their technical support and constructive suggestions in the preparation of this manuscript. We would like also to thank all the members of our clinical collaborators for their support in facilitating specimens and clinical follow-up of Journal of Proteome Research • Vol. 9, No. 1, 2010 171

research articles the bladder cancer cases analyzed in this study. This work was supported by a grant to Dr. Sańchez-Carbayo from the Spanish Ministry of Education and Science (SAF2006-08519). Esteban Orenes and Rodrigo Barderas are recipient of Postdoctoral Contracts of the FIS supported by the Spanish Ministry of Health.

Supporting Information Available: Supplementary Table 1, complete set of 171 immunogenic proteins including their functional annotation, p-value and fold changes. Supplementary Figure 1, (A) nonsupervised hierarchical clustering using the Manhattan metrics displaying how the panel of the 171 proteins identified using the heterogeneous (a) set of controls served to distinguish bladder cancer patients from the healthy controls of the homogeneous (b) control set measured with a different batch of protein arrays. (B) Classification of bladder cancer cases and control set (b) samples is magnified. This material is available free of charge via the Internet at http://pubs.acs.org.

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Serum and Tissue Profiling in Bladder Cancer Combining Protein and

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