Modern Tumor Marker Discovery in Urology: Surface Enhanced Laser Desorption and Ionization (SELDI)

Technique Update

TECHNIQUE UPDATE Modern Tumor Marker Discovery in Urology: Surface Enhanced Laser Desorption and Ionization (SELDI) Matthew B. Gretzer, MD,* Alan W. Partin, MD, PhD,* Daniel W. Chan, PhD,† Robert W. Veltri, PhD* *The James Buchanan Brady Urological Institute, †Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD During the last two decades, biomarker research has benefited from the introduction of new proteomic analytical techniques. In this article, we review the application of surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy in urologic cancer research. After reviewing the literature from MEDLINE on proteomics and urologic oncology, we found that SELDI-TOF is an emerging proteomic technology in biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures. SELDI-TOF is a novel proteomic technology that has the potential to contribute further to the understanding and clinical exploitation of new, clinically relevant biomarkers. [Rev Urol. 2003;5(2):81–89] © 2003 MedReviews, LLC Key words: Proteomics • Markers • Prostate cancer • Renal cancer • Bladder cancer umor markers have positively impacted the detection, diagnosis, and prognosis of many genito-urinary malignancies. Unfortunately, many of these markers have inherent diagnostic performance issues, usually in the area of specificity, that limit their clinical utility. Optimal treatment and cure depend not only on an accurate early diagnosis and determination of the extent of disease, but also on reliable follow-up for early diagnosis of clinical recurrence. A review of our current U.S. Food and Drug Administration (FDA)-approved armamentarium reveals a paucity of new and cost-effective markers that will ultimately improve disease outcomes and the patient’s quality of life. T VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY 81 Surface Enhanced Laser Desorption and Ionization (SELDI) continued Among urologic malignancies, diagnosis and treatment for testicular, prostatic, and, more recently, bladder cancers have benefited from the discovery and application of tumor markers. Since the introduction of prostate-specific antigen (PSA) screening, more prostate cancers are being identified earlier, mortality has decreased, and a cure is likely with definitive therapy when the tumor remains confined to the prostate.1,2 Unfortunately, despite the routine of the noninvasive approaches, the high rate of false negatives, especially for low-grade tumors, illustrates the limitations of cytopathology as an adjunct to cystoscopy.6,7 Several attempts at applying monoclonal antibodies directed at tumor-related antigens have met with only moderate success because of problems in technical performance, interpretation, and specificity.6,7 One FDA-approved bladder cancer test available today is the NMP22 soluble antigen urine Identification of more accurate “interventional-based" markers would serve to improve cancer detection and reduce morbidity, and should continue to reduce cancer mortality. application of PSA assays, as many as 30% of potentially significant prostate cancers are missed by the current early detection protocols.1-3 In addition to PSA, the introduction of other derivations of PSA such as percent free PSA, PSA velocity, and PSA density have also contributed to earlier diagnosis, but patients’ diagnoses currently continue to suffer from compromised specificity.4,5 Although PSA-detected tumors appear clinically homogeneous, with most men having moderate grade disease (Gleason scores of 5, 6, and 7), not all of these men have the same outcome. Differences in biochemical and clinical recurrence rates among these men with different tumor grades illustrate another limitation of this marker. The discovery of new serum markers is greatly needed. Identification of more accurate “interventional-based" markers would serve to improve cancer detection and reduce morbidity, and should continue to reduce cancer mortality. Currently, the most reliable method for diagnosis and surveillance of bladder cancer is cystoscopic examination and biopsy.6,7 Although urine cytology remains the gold standard 82 VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY test.6,7 However, there is at least one report of specificity problems with this test.8 Another FDA-approved cytology, supplementary immunocytology assay, combines three monoclonal antibodies,9 but it also recently failed validation in a clinical trial.10 At least one new innovative technology, fluorescence in situ hybridization (FISH), uses multiple chromosomal microsatellite markers and has recently been approved by the FDA for monitoring the recurrence of bladder cancer.11,12 However, the test, though accurate, does require customized fluorescent microscope equipment and is technically challenging to perform in routine voided urine samples.12 An evaluation of the renal cancer biomarker field demonstrates a complete lack of FDA-approved, noninvasive molecular diagnostic tests for these malignancies, even though considerable knowledge has accumulated on the molecular alterations associated with renal cancer.13 Improved knowledge of the molecular pathogenesis of this disease14 has resulted in only very limited success of chemotherapy, immunotherapy, and radiotherapy for this cancer; hence, the importance of an accurate early diagnosis, which would provide the best chance for a definitive surgical or medical cure of renal cell carcinoma. Reliable, cost-effective, and accurate diagnostic and prognostic noninvasive biomarker tests for urologic cancers are needed to aid in the early primary diagnosis. In the case of prostate cancer, we are in the midst of a PSA biomarker crisis in which the specificity of PSA and its derivatives requires supplementation in order to further improve specificity and to differentiate cancer from benign diseases of the prostate, as well as predicting those cancers that are aggressive. One source for the discovery of new biomarkers may be found among the cancer-specific cellular proteome.15 The proteome encompasses the entire protein complement of a cell, including proteins that are modified after initial translation from the genetic blueprint. The profiling of differentially expressed proteins in healthy and diseased conditions represents the study termed “proteomics."15 Proteins that are expressed at higher levels in various cancer disease states may be potentially disease-state specific. Identification of these protein changes from various disease states will not only present new clues to the understanding of the molecular basis of cancer progression, but will also aid in the development of novel methods for improved disease management.16-18 During the last two decades, advances biomarker research have benefited from the introduction of new proteomic analytical techniques.19,20 Previously, the use of two-dimensional electrophoresis dominated this field and provided numerous urologic candidate biomarker discoveries. A novel proteomic technology that has the potential to contribute further to the understanding Surface Enhanced Laser Desorption and Ionization (SELDI) and clinical exploitation of new, clinically relevant biomarkers is surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy.21 SELDI-TOF represents an emerging proteomic technology in biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures.21-25 Following is an overview of proteomics in urologic cancer research that is specifically focused on SELDI-TOF. Proteomics and Cancer The functional products of a cell’s genetic-coding complement are proteins. These proteins—the proteome— are responsible for many structural components (eg, actin, tubulin, and desmin) and cell-cell communicative functions, as well as the regulation of several key biochemical pathways affecting cell division, programmed cell death or apoptosis, and cellular differentiation. The disruption of normal physiologic cell functions, as a result of various disease processes, will alter the protein structure and expression profiles. Such changes may or may not reveal a correlation between the gene that is turned on and the protein that is produced.17 This is particularly important in cancer research where modifications to proteins (eg, over-expression, mutations, polymorphisms, and posttranslational alterations) can specifically contribute to the pathogenesis of the disease and also provide new targets for diagnosis and therapy.16-18 Recent work by Li and colleagues26 illustrates how proteomic approaches, such as SELDI mass spectroscopy, in conjunction with bioinformatic tools, may facilitate the discovery of new and more accurate tumor markers for breast cancer. Using this new technology to screen banked serum from patients with and without breast cancer, these authors identified a panel of potential biomarkers with high sensitivity and specificity for breast cancer. Proteomic studies of cancer may identify this differential expression of proteins at varying stages of disease, from healthy to dysplasia and from preinvasive to invasive neoplasia. Identifying the array of different proteins that are expressed during missed.28,29 Furthermore, the lack of reproducibility of this technique creates difficulty in detecting differences in protein expression reflected in two different gels from two different runs. Attempts to overcome the technical limitations of gel electrophoresis are improving the reproducibility and application of this technique. SELDI-TOF represents one such tool that is able to comprehensively investigate the complex changes at the protein level in tissue that may be associated with tumor development and progression. this genetically controlled, stepwise progression represents a key challenge in the development of novel approaches to the diagnosis and treatment of disease. SELDI-TOF represents one such tool that is able to comprehensively investigate the complex changes at the protein level in tissue that may be associated with tumor development and progression, and will contribute to the discovery of more accurate biomarkers and therapeutic targets. Techniques in Proteomic Analysis To date, the combination of twodimensional (2-D) gel electrophoresis and mass spectroscopy (MS) has been the effective traditional technique for analyzing the protein complement of cells and tissues.27-32 Two-dimensional gel electrophoresis combines the separation of proteins in one dimension on the basis of charge (isoelectric points), and in a second dimension on the basis of molecular size. Once the proteins are separated, it is possible to identify the protein(s) of interest. Unfortunately, the resolving power of the 2-D gel electrophoresis has certain technical limitations regarding this separation and identification process in extremely complex protein solutions, and certain categories of differentially expressed proteins may be Advances such as the preprocessing of molecular species of interest in order to enrich for certain classes of proteins, the use of new immobilized pH gradients, and the availability of narrower-range pH gradients to help further separate the proteins within the gel, as well as more sophisticated imaging software to resolve complex data, are a few examples. These enhancements have improved the reproducibility of 2-D gel electrophoresis in resolving the spectrum of basic to acidic proteins.27-29 Although this technique continues to aid in biomarker discovery, it remains a cumbersome method, and newer techniques with global surveillance capability in a population of proteins are needed. To this end, the development of powerful and sensitive new methods, such as matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry, have facilitated the characterization of proteins by mass spectrometry.33,34 The application of this procedure in a routine practice is limited, however, because complex biological materials, such as blood, sera, plasma, whole cells, and cell lysates typically contain hundreds of biologically and clinically important molecules that require tedious sample preparation and purifi- VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY 83 Surface Enhanced Laser Desorption and Ionization (SELDI) continued A 1) Sample applied to ProteinChip. 2) Nonspecifically bound proteins washed away. 4) Ions are detected and mass of each protein calculated by time-offlight mass spectroscopy. Intensity Intensity 3) EAM is applied to each sample. A laser is fired that desorbs and ionizes the proteins in the EAM. Mass/Charge Mass/Charge B Lens Laser beam source Mirror Detector + + + ProteinChip® Array with protein samples coated with energy absorbing molecule. + - Accelerating Potential The smallest ionized proteins fly fastest and are detected first Figure 1. (A) The SELDI process: The sample is added to the ProteinChip; after washing, only the retained proteins are subjected to laser desorption and ionization as depicted in panel B. (B) Schematic of SELDI ProteinChip Reader: The ProteinChip is inserted into the reader; a laser beam is focused on the sample, which causes the proteins embedded in the sample to desorb and ionize. The resulting ions are then accelerated through a vacuum tube to an ion detector. The ionized proteins are detected and the mass is determined based on their time-of-flight. EAM, energy absorbing molecule. Data from Ciphergen Biosystems (http://www.ciphergen.com). cation steps prior to MS evaluation. Methods for sample purification, such as molecular sieve and ion chromatography, high performance liquid chromatography, and electrophoresis, have been introduced to minimize this limitation. However, these techniques are costly, labor 84 VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY intensive, and may require a significant sample size to process. They also suffer from sample loss during the entire procedure. In an effort to develop a direct, facile, mass spectrometric technology for the detection of numerous proteins in heterogeneous samples, that over- comes many of the problems associated with sample preparation inherent with MALDI-TOF, Hutchins and Yip21 introduced the concept of SELDI. This technology is a type of affinity-based mass spectrometry21-23,35,36 in which the protein sample is directly applied to a pretreated surface termed the “ProteinChip" (Ciphergen Biosystems ProteinChip‚ Fremont, CA). The versatility of this technology lies within the chemistry of the chip, which may be customized to capture a unique subset of proteins from a sample. The microchemistry of the chip surface can be hydrophobic, hydrophilic, cationic, or anionic, with broad specificity, so that it catches whole classes of proteins at the same time. Alternately, the ProteinChip array surface can be made immunospecific, with monoclonal antibodies ligand-specific for classes of receptors that are selective for only a few proteins. Once bound, the samples are washed, using a buffer to reduce nonspecific binding, and the retained proteins are then analyzed by mass spectrometry (Figure 1A). This analysis begins by coating the samples with a light-absorbing material. Once in place, a pulsed ultraviolet laser (typically nitrogen) provides the desorption energy to liberate gaseous ionized peptides from each sample on the chip. These ions are accelerated through an electrical field to a detector; their peptide mass is derived through measurement of time-of-flight (TOF) of the ions through the electric field to the detector, and is related to the ion analyte mass-to-charge ratio. A peptide mass map, referred to as a retentate map (retained proteins on the chip), is generated and displays individual proteins as separate peaks on the basis of their mass and charge (Figure 1B). The broad, ligand-specific binding Surface Enhanced Laser Desorption and Ionization (SELDI) properties of these chips form the core of SELDI technology. These surfaces provide for a high degree of enrichment of captured analytes because of the high specificity of biomolecular interactions with the unique chip surface chemistries. Manipulation of the microchemical environment, combined with the unique chip surface chemistry, can capture classes of peptides with specific characteristics from very crude samples without the need for elaborate preprocessing. In addition, the creation of a chip surface with known chemically unique characteristics may reveal the biochemical and chemical properties, such as charge, hydropathicity, and composition of the analyte, that further illustrate the potential of SELDI not only to contribute to biomarker discovery, but also to the study of biomolecular interactions (Figure 2). Data Analysis The task of interpreting the data produced by proteomic technologies represents an emerging field known as bioinformatics.18 This field entails the use of protein databases and analysis software to analyze the data generated from proteomic research. Chemical Surfaces + NR 3 My+ Hydrophobic Cu(II) IMAC Ionic Mixed Biochemical Surfaces Antibody DNA Enzyme Receptor Drug Figure 2. The ProteinChip Array. A variety of arrays are available. Each consists of 8 to 16 spots comprised of a specific chemically or biochemically modified surface. Chemically modified surfaces are used to retain groups of proteins based on specific chemical properties such as hydrophobicity and charge. Biochemical surfaces are used to isolate a specific protein or class of proteins. IMAC, immobilized metal affinity capture. Data from Ciphergen Biosystems website (http://www.ciphergen.com). these proteins is beyond the scope of this review, but will be described briefly. This process begins with isolating and purifying the protein(s) of interest using classical methods of protein purification that would focus on the charge and size properties indicated in the SELDI-TOF results. The purified protein(s) of interest can be sequenced using endoproteases that can be directed to specific amino acids. The resulting peptide products The microchemistry of the chip surface can be hydrophobic, hydrophilic, cationic, or anionic, with broad specificity, so that it catches whole classes of proteins at the same time. Such software permits the analysis of multiple ProteinChips along with the ability to simultaneously compare the data from different samples.37-40 Once a protein or set of proteins has been identified as either unique or significantly differentially expressed, compared to other samples, these proteins may then become potential biomarker candidates. The complex task of identifying – SO4 may then be further evaluated, using substances such as dephosphorylating or deglycosylating enzymes that affect known chemical groups on the peptides, to give information about changes that have occurred after the protein’s initial translation. To maintain control over the identity of the resulting changes during this treatment process, MS detection is performed to monitor for any changes in the mass-over-charge profile. Changes in the mass-over-charge profile are correlated with the mode of action of the enzymatic or chemical treatments in order to provide structural insight. The generated peptides are then compared to digests of proteins in databases for identification.41 Applications of SELDI in Urology The diversity of SELDI is appreciated because of its ability to detect potential biomarkers from crude samples such as serum, cellular extracts (both from microdissected cells and cell culture), seminal plasma, and urine.33,42-49 A challenge of proteomics has been the development of techniques to reduce the heterogeneity of the microenvironment of the tumor and to enhance the acquisition of protein profiles from specific cell populations. To this end, the introduction of laser capture microdissection has aided in the sampling of specific cell populations for proteomic analysis.50,51 During this procedure, a thermosensitive polymer film is applied over the specimen containing the cells of VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY 85 Surface Enhanced Laser Desorption and Ionization (SELDI) continued A B C D N BPH PIN PCA Figure 3. Differential protein expression among prostate tissue. This is a representative example comparing a small segment of the mass spectra of nondiseased (N) epithelial cells, benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCA) obtained from a surgical specimen, using laser capture microdissection. The top four panels are mass spectra views, and the bottom four panels represent gel views. The arrows identify a protein that is overexpressed in PIN and PCA. Reproduced with permission from Wright et al.36 interest. Once in place, an infrared laser melts the film over the target cells, forming a solid composite that may be easily lifted away from the rest of the specimen. Since its introduction, SELDI has been put to constructive use by many laboratories to determine the specific protein-profile patterns that characterize cellular progression from healthy to diseased states for prostate, bladder, and renal cell cancer.36,42-49,52,53 Studies by Wright and colleagues36 and Paweletz and associates46,47 have illustrated this changing protein profile within the prostate. Using a pure population of prostate cells from areas of normal, benign prostatic 86 VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY Figure 4. Detection of five transitional cell carcinoma (TCC)-associated protein peaks in urine. Mass spectra (top) and respective gel views (bottom) of urine samples from four different TCC patients (C1–C4), two normals (N1 and N2), and two patients with other urogenital diseases (B1and B2). The average molecular mass of the five proteins identified to be unique or overexpressed in the TCC specimens is UBC1, 3.352/3.432 kD (A, arrow); UBC2, 9.495 kD (B, arrow); UBC3, 44.647 kD (C, arrow); UBC4, 100.120 kD, and UBC5, 133.190 kD (D, arrows). Reproduced with permission from Vlahou et al.49 hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and adenocarcinoma tissue that were procured using laser capture microdissection, these investigators have identified patterns of proteins unique to each population of cells. This differential expression of proteins among cancer generated with SELDI-TOF of 182 serum samples from men with and without prostate diseases. Using the ProPeak software package (3Z Informatics, LLC, Mt. Pleasant, SC) to analyze the SELDI data, this group was able to identify differences between the protein profiles of An examination of the novel protein peaks and clusters in the urine of patients with cancer revealed enhanced detection rates up to 78% compared to 33% for urine cytology. cells, when compared to populations in normal, BPH, and PIN tissue, reveals the utility of SELDI in providing a protein profile of the changes that occur from premalignant to malignant lesions (Figure 3). In a recent abstract, Li and colleagues53 analyzed the protein profiles the men with and without prostate cancer. A limiting factor of protein profiling has been the interpretation of which of the protein peaks represents differences in expression among different samples and which are the result of artifacts unrelated to the disease process. The software employed Surface Enhanced Laser Desorption and Ionization (SELDI) Vlahou and associates49 discovered unique protein fingerprints among patients with bladder cancer. Protein changes were detected within urine samples of patients with known transitional cell carcinoma compared to healthy controls (Figure 4). An examination of the novel protein peaks and clusters in the urine of patients with cancer revealed SELDI mass spectroscopy identified the presence of a protein peak that was differentially expressed among all of the cancer samples and not within the control samples (Figure 6). After investigators measured the serum levels of PSA among these men, 24% of the men with cancer had normal PSA levels under 4.0 ng/dL. The protein biomarkers discovered by using In addition to the discovery of biomarkers and protein profiling, SELDI may also be used as an immunoassay platform. Figure 5. Comparison map for renal cell carcinoma (RCC). Protein profiles rendered in the gel view were obtained from cell extracts (peripheral and central tumor tissue) and compared with those from extracts of the respective normal control tissues. In the marked region in P and C, a double peak is present, which is scarcely detectable in N. In the comparison map at the bottom (normal versus cancer) the summarized differences between normal and peripheral/central tumor tissue are shown. In this map, a line in the negative direction indicates higher expression in the tumor. The line at 11951.1 and 12019.9 in the negative direction displays an overexpressed protein peak in the tumor tissue. Reproduced with permission from von Eggeling et al.52 by this group is unique; instead of looking at overall differences in all peaks from sample to sample, this software evaluates a panel of protein peaks selected by multivariate logistic regression. Although profiling software is new and possesses limitations discussed below, this study indicates the potential that SELDI, combined with bioinformatic tools, may offer to the discovery of new biomarkers. SELDI technology is also contributing to the search for biomarkers that have more improved sensitivities than urine cytology for detecting all grades of bladder cancer.49 Given the molecular heterogeneity of bladder cancers, SELDI offers the ability to simultaneously evaluate a panel of potential biomarkers directly from urine. Using this new protein-profiling technology, enhanced detection rates up to 78% compared to 33% for urine cytology. This promising result illustrates SELDI’s potential for both biomarker discovery and early diagnosis in bladder cancer. Work by von Eggeling and colleagues52 has applied SELDI technology to investigate the changes at the protein level in renal cell cancer. Protein profiles were generated from cell extracts isolated from tumorous tissue and compared to extracts of respective normal control tissues. As with the above studies, differences in protein expression were recognized while progressing from normal to malignant tissue. Although this work is preliminary, it illustrates the effectiveness of SELDI in identifying the differences between the protein profiles of tumor and normal tissues (Figure 5). The discovery of new biomarkers may also aid in improving the accuracy of our current screening regimen for prostate cancer. Using SELDI, Hlavaty and associates45 identified prostate cancer-specific proteins in the serum of patients with confirmed prostate cancer. These investigators examined serum samples in men diagnosed with prostate cancer prior to surgery and compared these results to age-matched controls. SELDI would thus aid in diagnosing cancers not identified by using PSA testing alone. In addition to the discovery of biomarkers and protein profiling, SELDI may also be used as an immunoassay platform.48 By applying an antibody to the protein chip, investigators may capture and assay specific proteins. For example, investigators have described the successful use of SELDI Figure 6. SELDI profiles from fractionated serum samples of three prostate cancer patients (A-C) and three controls (D-F). A single protein peak appearing at 50.8 kD was found in all 36 men with prostate cancer and in none of the 20 clinically determined prostate cancer-free controls. Reproduced with permission from Hlavaty et al.45 VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY 87 Surface Enhanced Laser Desorption and Ionization (SELDI) continued to develop immunoassays for the detection and measurement of PSA and prostate-specific membrane antigen in body fluids. Using SELDI to quantitate prostate-specific membrane antigen levels in serum, Xiao and colleagues48 accurately discriminated prostate cancer patients from those with BPH, whose PSA values from 4 to 10 ng/dL have been associated with a poor disease specificity. Limitations of SELDI There are some limitations to consider with the application of this technology to the biomarker discovery process as well as its potential use as a diagnostic instrument. One would be the stability of this complicated engineering platform when constantly applied in a diagnostic mode. Another would be the lot-to-lot reproducibility of the chip surface chemistry to assure the validity of comparative observations in complex clinical material such as serum, plasma, and urine. In addition, different underlying disease conditions in control and disease populations used for discovery or diagnosis might generate erroneous results. Finally, it cannot be assumed that current SELDI-TOF technology is ready for routine clinical diagnostic applications in the clinical chemistry laboratory. The technology should provide direction for the searching process, to identify and isolate disease-specific serum biomarkers that may be combined to improve detection, diagnosis, and prognosis of urologic cancers. Future engineering and software improvements to refine potential clinical laboratory applications are anxiously awaited by the medical community. Conclusions and Future Directions Although SELDI is still early in its development, modifications to SELDI proteomic technology are necessary to fully capture its clinical value in medicine. In order to identify the presence of low-abundance, differentially expressed proteins that may become masked within a sample, sample preprocessing and a more diverse array of capture surfaces will be required.18-24 Vital biomarker information may be unrecognized as there is no current method to amplify such proteins. The development of highly stable engineering platforms, more novel surface chemistry enhancements, improved manufacturing methods to maintain the reproducibility of mass-produced chips, and enhanced sample preparation techniques will enable the expanded detection of more cancer biomarkers in various disease states and the potential for new diagnostic instrument applications. Current developments such as SELDI-TOF enable the rapid analysis of small, crude samples from a variety of biological materials for biomarker detection and identification, and for the study of biomolecular interactions. Continued application of this technology to the research problems that remain for prostate, bladder, and renal cell cancer will result in the identification of unique proteins and protein profiles that characterize cancer progression from a healthy state to more aggressive malignant states. Further identification and characterization of these proteins should enable investigators to develop novel diagnostic and therapeutic modalities for cancer research. The laboratory of Daniel W. Chan, MD, has received support from Ciphergen Biosystems for SELDI research. References 1. 2. 3. 4. 5. 6. 7. Polascik TJ, Oesterling JE, Partin AW. Prostate specific antigen: a decade of discovery—what we have learned and where we are going. J Urol. 1999;162:293–306. Catalona WJ, Richie JP, Ahmann FR, et al. Comparison of digital rectal examination and serum prostate specific antigen in the early detection of prostate cancer: results of a multicenter clinical trial of 6,630 men. J Urol. 1994;151:1283–1290. 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Current bladder tumor tests: does their projected utility fulfill Main Points • During the last two decades, biomarker research has progressed with the introduction of new proteomic analytical techniques. • Previously, the use of two-dimensional electrophoresis dominated this field and provided numerous urologic candidate biomarker discoveries. • Surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) represents an emerging proteomic technology in biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures. • Continued application of SELDI-TOF to research problems for prostate, bladder, and renal cell cancer will aid in the discovery of unique protein profiles that characterize the progression of cancer from healthy to more aggressive malignant states. 88 VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY Surface Enhanced Laser Desorption and Ionization (SELDI) 8. 9. 10. 11. 12. 13. 14. 15. 16 17 18 19 20. 21. 22. 23. 24. clinical necessity? 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Abstract presented at: Annual Meeting of the American Urological Association; May 25-30, 2002; Orlando, FL. Abstract 1131. VOL. 5 NO. 2 2003 REVIEWS IN UROLOGY 89