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1 CE update [molecular diagnostics cytology chemistry] Urine-Based Assays for Bladder Cancer Robert H. Getzenberg, PhD Departments of Urology, Pathology, and Pharmacology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA DOI: /MRF2YWMVUAVFB8EC After reading this article, the readers should know which assays for bladder cancer have been approved by the FDA and which assays are in development. The reader should understand the mechanisms involved in all of these assays. Molecular Diagnostics questions and corresponding answer form are located after the Your Lab Focus section on p There are a large number of markers which have been identified in the urine of patients with bladder cancer which may provide both diagnostic as well as prognostic information regarding the disease. These markers may also provide for the earlier detection of bladder cancer, and therefore, the prevention of the development of life-threatening illness. According to the American Cancer Society, approximately 57,000 individuals will be diagnosed with bladder cancer in the Unites States this year, and 12,500 individuals will die from the disease. 1 Bladder cancer is more common in men, where it represents the fourth most common cancer in terms of incidence. In women, it represents the ninth most common form of cancer identified. As opposed to a number of other cancer types, it is more common in Caucasians than African Americans. Listed among the causes of bladder cancer are occupation exposures, spinal cord injuries, and cigarette smoking, which may cause as high as a 4-fold increased risk for developing the disease. 2,3 When bladder cancer is detected early, the 5-year survival rate is quite high, approximately 94%. When the disease is diagnosed at a later time point, either when there is regional or distant metastases, the 5-year survival rate goes down significantly to 48% and 6%, respectively. 1 The most common symptom that presents in patients with bladder cancer is hematuria or blood in the urine. This hematuria can take the form of micro- or macrohematuria depending on the quantities of blood found. Hematuria by itself is not a good predictor of bladder cancer, because more than 90% of the individuals with hematuria do not have bladder cancer. There are also a number of individuals with bladder cancer that do not possess either micro- or macrohematuria. Therefore, the presence or absence of blood in the urine cannot be utilized as a sole factor to identify individuals with bladder cancer. Urine Cytology and Cancer Markers The most common method by which bladder cancer is diagnosed today is urine cytology or examination of cells in the urine to determine if bladder cancer cells are found. Although urine cytology is frequently utilized, there are many limitations to this procedure. First, it is quite dependent on the ability of the individual cytologist to read the sample, and there is great variability from institution to institution. Second, and probably more importantly, is the fact that there are typically few cells found in the urine, even in patients with bladder cancer. Further, because of the harsh environment of the urine, when these cells are found, they are typically in poor shape, making the differentiation between a cancer cell and a normal cell difficult. When bladder cancer cells are positively identified in the urine, this almost certainly indicates the presence of bladder cancer. Therefore, the sensitivity of this assay is quite low, because it misses many individuals with bladder cancer. However, the specificity is high, and in some cases, it has been reported to be higher than 98%. There is a need to develop novel bladder cancer markers on several fronts. The first is for screening or detection. As stated previously, individuals with bladder cancer that is detected early have 5-year survival rates which are quite high. The early detection of the disease would appear to be important in preventing its subsequent mortality. Therefore, the utilization of markers for screening or early detection of bladder cancer would seem to be a high priority. The second area in which bladder cancer markers are needed is for diagnosis of the disease. Many individuals present with symptoms which may be indicative of bladder cancer but which most likely represent benign conditions of the bladder, kidney, or prostate. Therefore, it would be of great clinical value to have a test with a high specificity that would identify individuals with bladder cancer while differentiating them from the large majority of individuals with benign diseases. Finally, as many as 70% of the individuals with superficial bladder cancer that is resected end up having recurrent disease. 4 This appearance of recurrent disease requires frequent cystoscopy, certainly in the first several years after initial diagnosis and then annually throughout the patient s lifetime. Development of a noninvasive test which could identify the group of individuals that have recurrent bladder cancer would permit the cystoscopic analysis only of those individuals with the disease. Therefore, patients who do not have recurrence of the disease would not have to undergo the invasive procedure. Two types of bladder cancer markers have been identified and studied to date. The first falls into a family which could be considered adjuncts to cytology. These tests utilize the fixed cells which are obtained from urine or bladder washings. The goal for these assays is to increase the 613

2 614 sensitivity, which is typically missing for urine cytology alone. Therefore, identifying methods to aid in the detection of bladder cancer cells in the urine would add to the high specificity already present in cytology examination. These techniques provide opportunities to identify more individuals with bladder cancer utilizing these non-invasive approaches. In this review, we will not focus on these types of markers, but will instead focus on the larger group of assays that have been developed to identify urine-based markers of the disease. Urine-Based Markers Not only is bladder cancer an important clinical problem for which markers need to be developed, but bladder cancer represents, in some ways, an ideal model for identification of markers which may be specific for the disease. The bladder is one of the few organs in the body which is continually exposed to body fluids and can be sampled at regular periods of time. In addition, with the use of cystoscopy (visual imaging of the bladder), we can compare results obtained from urine markers to those actually seen within the bladder. Cystoscopic examination of the bladder remains the gold standard for the disease. Utilizing instruments that can visualize the bladder surface, suspicious lesions can be biopsied and pathologic determination performed to identify cancerous lesions. Cystoscopy is frequently used as the point of comparison for many of the urine-based markers which have been developed. While cystoscopy does identify most lesions found in the patients with bladder cancer, it can miss a number of areas of the bladder as well as some lesions that may not be grossly evident upon examination. 5 Therefore, there are a number of bladder cancers which are missed by cystoscopy alone. With this in mind, it is still considered to be the one technique that can be utilized to determine the sensitivity of bladder markers. Although bladder cancer may be in some ways considered to be an optimal system for which to develop tumor markers, in other ways it is less than ideal. The bladder is a frequent site of inflammation and other benign conditions that can cause elevations in many of the identified markers. In addition, the urine environment is not the friendliest for protein or nucleic acid detection techniques. However, this harsh environment has allowed for simplification of the protein and nucleic acid content within the urine, and in some ways, it has provided for a selection against some of the background factors which can sometimes complicate assay development. There are currently 2 tests which have been FDA-approved for the detection of recurrent bladder cancer. The first of these urine-based markers is known as bladder tumor antigen or BTA. The BTA test is currently in its second generation and represents a different marker than originally identified in the assay. The BTA test currently identifies a complement-related factor H molecule which can be found in the urine of patients with bladder cancer. This test is available in both quantitative (TRAK), as well as STAT forms. There have been extensive studies performed on BTA, and these have indicated sensitivities ranging anywhere from 24% to 89% with a specificity of almost the identical range, anywhere between 26% to 93% Recent studies of BTA STAT have demonstrated an increase in BTA with hematuria. 17 Overall, because its sensitivity is not high and the specificity appears quite low for BTA, its general utility has been quite limited in the urologic clinic. The second of the FDA approved urine-based markers for recurrent bladder cancer is NMP22. This product, which was developed by Matritech M (Newton Center, MA) is an assay which detects a nuclear protein known as NuMa, a protein component of the mitotic apparatus which is found in all eukaryotic cells. Since the marker is found in all mammalian cells, it is not bladder cancer-specific. However, a number of studies have demonstrated that the detection of NMP22 in the urine appears to identify individuals at higher risk for having bladder cancer. These studies have demonstrated a sensitivity ranging anywhere from 38% to 100% with a specificity of 61% to 95%. 14,18-23 Urine samples for NMP22 require stabilization in order to preserve the antigen and permit its identification. Recently, in addition to the quantitative assay to determine NMP22, Matritech has developed a rapid test which can identify this antigen. The third assay, which has previously been FDA approved, detects a protein called fibrinogen degradation protein (FDP). This antigen was detected based upon an immunoassay that was developed as a rapid test and actually resulted in reasonable sensitivities ranging from 76% to 81% and specificities from 75% to 86%. 9,24-26 Despite these encouraging results, the FDP assay has subsequently been taken off of the market and is not currently available in the United States. Assay Types There have been 2 major types of urine-based assays which have been developed. The first are assays which detect nucleic acids or nucleic acid alterations within the urine, and the second are protein-based assays for the disease. The assays to detect nucleic acid alterations have focused on several fronts. Microsatellite alterations have been studied in the urine sediment to identify DNA fragments which may exist. These examine loss of heterozygosity or instability which may be occurring within the genome. Twenty microsatellites have been examined in urine and compared with blood samples. Sensitivities of these studies have ranged from 83% to 100%, whereas specificity has been very close to 100% One of the limitations of these studies has been the difficulty in standardizing the assays from 1 institution to another, as well as the fact that the assays themselves are quite expensive to run. Despite this, there is a general enthusiasm for the potential utility of microsatellite alterations in identifying individuals with bladder cancer. Another area of increased enthusiasm is examining telomerase in the urine of patients with bladder cancer. Telomerase has been detected both at the RNA template level as well as the enzyme itself. When the htert mrna has been determined by RT- PCR, sensitivity ranges anywhere from 62% to 85%, and specificity ranges anywhere from 60% to 96%. 30 A recent study published in the Journal of Urology, indicated that the detection of the htert

3 mrna by PCR resulted in sensitivity of 75% and a poor specificity of 69%. 31 Detection of the enzyme itself, using a TRAPeze ELISA-based assay, has made a number of advancements. To perform this technique, the authors first detect circulating tumor cells in the blood using immunobead enrichment for epithelial cells. In a study designed to look at highgrade muscle invasive or metastatic bladder cancers, sensitivity was obtained as high as 90% where specificity was 100%. 32 Again, this was on a relatively small population of very advanced bladder cancer patients. In another recent study, in a larger patient population of 265 patients, 55% of which had recurrent disease, the sensitivity of detection of the telomerase protein was 46%, which was considered to be significantly lower than the other assays which were performed on the same population. 33 In general, despite the potential biologic importance of these nucleic acid alterations in bladder cancer, it has been difficult to translate these into urine-based assays that can be readily applied for the detection or prognosis of the disease. Cancer Markers in Development Over the last several years, there has been the development of a large number of urine-based markers for bladder cancer which are at various stages of development. We will attempt to briefly review these markers and identify their stage of development, as well as give the available sensitivity and specificity data regarding these markers. First, UBC is an assay developed to detect cytokeratin 8 and 18. It has been known for a number of years that cytokeratin fragments appear in the urine of patients with bladder cancer. Studies in 2001 on relatively small sample sizes, reveal sensitivities ranging from 72.3% to 86.7%, and specificities anywhere from 71.8% to 86.5%. 34 A recent study examining this test on a larger sample size, resulted in a sensitivity of 40% and a specificity of 72%. 35 In addition to the UBC assays, a number of other cytokeratin-based assays have been developed. An assay known as CYRFA-21-1, which detects cytokeratin 19, is a urine-based immunoassay that has been reported to have a sensitivity anywhere from 83% to 100% and a specificity of 74% to 100% Furthermore, cytokeratin 20 mrna levels have been detected by RT-PCR, resulting in sensitivity of 82% to 91% and a specificity between 67% and 97% Alterations in cytokeratins have been some of the first noted changes occurring in bladder cancer cells. From these recent encouraging results, it is possible that some of these changes in cell structure may be appropriate as bladder tumor markers. However, the translation of this into assays that can be utilized in the urologic clinic has not been successful. Perhaps one of the oldest assays to detect bladder cancer is the Lewis X antigen, which is a blood-based antigen that has been previously identified individuals with the disease. More recent studies revealed sensitivities ranging from 80% to 81% and a specificity of 86%. 45 Despite the fact that this assay has been around for quite sometime, it is not widely used to detect bladder cancer. Of the more recently identified markers that have significant potential are the hyaluronidase and hyaluronic acid assays developed by Lokeshwar and colleagues at the University of Miami. These assays detect GAGlinked proteins which appear to be quite specific for the identification of bladder cancer. In several publications, they have identified sensitivities ranging from 90% to 92% and specificities from 84% to 92% Another marker which seems to be quite exciting is the inhibitor of apoptosis, survivin, which has been identified in a number of cancer types. In an initial study, the results indicate a sensitivity of 100% but the specificity was not determined in this study. It is clear that although survivin has great potential, further studies are necessary to determine its clinical utility. 49 Mechanisms and Hallmarks Our laboratory has focused significant effort on the identification of bladder tumor markers. Our concentration has been on trying to understand and utilize some of the hallmarks of the bladder cancer cell in order to develop assays which detect protein components which are specific for the disease. One of the fundamental changes which occurs in a cancer cell is alterations in both cell and nuclear shape. These alterations are utilized by the pathologist to identify cancer cells on microscopic examination. All cancer cells undergo these characteristic changes, and they are considered to be defining aspects of the tumorigenic process. Since changes in nuclear shape are characteristic of the cancer cell, our work has focused on understanding nuclear structure as an underlying framework for the observed changes in nuclear shape. In addition, it is known that a number of processes altered within the cancer cell could be traced back to changes within the nuclear structure. In cancer cells, it is common to find rearrangements, translocations, and other chromosomal events that are atypical for normal cells. In addition, genetic instability (in that genes which may be differentiation-related may be turned off and embryonic or other types of genes may be turned on) is common place in the cancer cell. Our hypothesis is that not only are changes in nuclear structure reflective of the characteristic changes in nuclear shape observed by the pathologist in a cancer cell, but that these changes in nuclear structure may also result in some of the loss of fidelity of these nuclear processes which are known to rely upon nuclear structure for organization and function. We have performed proteomic analysis of the nuclear structural components, termed the nuclear matrix, in order to determine differences in these components between cancer and normal cells. We have previously identified a series of these nuclear structural alterations characteristic of bladder cancer that are not found in individuals that do not have the disease. 50 BLCA-4 Here we will discuss one of these markers, BLCA-4, which is found throughout the bladder in people with bladder cancer including both tumor and normal regions, but is not found in the bladder of individuals without the disease. Therefore, this marker may reflect a type of field effect that has been observed at the genetic level by a number of investigators. Recent studies from our group have revealed that this marker appears to be a 615

4 616 transcriptional regulator that may play an active role in the regulation of gene expression within bladder cancer. 51 We have developed an immunoassay to detect this marker in patients with bladder cancer. This assay requires no stabilization and can be run on straight urine samples. In our initial clinical trial of 106 individuals, using a perspectively defined cutoff based upon the first 3 tumor and normal samples, we demonstrated a sensitivity of 96.4% and a specificity of 100%. 50 Furthermore, we were able to demonstrate that this assay was able to detect almost all of the individuals that were not considered to be positive for bladder cancer by cytology. In order to examine the expression of this and other markers in a high risk population for the development of bladder cancer, we have been studying individuals with spinal cord injuries. It is known that individuals with spinal cord injuries have up to a 460-fold increased risk for developing bladder cancer; as many as 1 in 10 individuals with spinal cord injuries actually develop the disease. 52 In addition, this population represents a difficult group in which to utilize bladder cancer markers, since the presence of cystitis and other types of inflammation within the bladder, as well as irritation of the bladder, is quite high. In a recent study in this population, we demonstrated that the specificity of the BLCA-4 marker continues to be quite high and is not elevated with cystitis, smoking, or other kinds of conditions for which a number of the other bladder tumor markers have been shown to be falsely elevated. In addition, we have identified some individuals with bladder cancer who presented with no symptoms of the disease. 53 A large national clinical trial is currently underway to validate these studies and determine the utility of this marker in diagnosing bladder cancer. We are excited about BLCA-4 and its potential clinical utility and await the outcome of these studies. Further Comments on Markers Several other markers have been developed and have been studied, perhaps not as extensively as some of the others which have been identified. CD44 is a marker which has recently been studied. The message levels for CD44v6 were examined by RT-PCR and recently shown to impart a sensitivity of 77% and a specificity of 100%. In this same study, CD44v8-10 was examined as a prognostic factor in exfoliated cells. 54 Other potential markers include PAX5, matrix metalloproteinase, and matrix metalloproteinase inhibitors. 55 While the above described markers have principally been focused on the diagnosis of bladder cancer, a couple have been identified with potential prognostic implications. For example, p53 has been extensively studied and its ability to stain tissue samples has been shown in most of these studies to be able to impart prognostic implications in relationship to patient outcome. Recently, serum VEGF levels were shown to be a negative prognostic factor for bladder cancer resulting in a sensitivity of 88% and a specificity of 98%. 56 The ability to provide prognostic information to patients with bladder cancer would appear to be of high priority. Despite this, few markers are currently available that provide any information in this regard. 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