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Current concepts in biomarker technology for bladder cancers.

Bladder Cancer

More than 50 000 new cases of bladder cancer were diagnosed during 1999, and this disease was responsible for ~12 500 deaths in the United States (1). The incidence of bladder cancer increased by 36% between 1984 and 1993, and this tumor now represents the 4th most common malignancy in males and the 10th most common in females (2). Transitional cells are the origin of >90% of diagnosed bladder cancers. Approximately 75% of patients have a superficial ([T.sub.a], [T.sub.1]), 20% have an invasive (higher than [T.sub.1]), and 5% have a metastasized tumor at time of diagnosis (3). The recurrence rate of superficial disease is >60%, and tumor progression to a higher stage or grade develops in 42% of patients by 10 years (4).

Staging and grading currently are the most reliable variables for recurrence and progression. Patients with [T.sub.a] disease progress in 4% (risk of recurrence, 52%), whereas progression in patients with [T.sub.1] lesions is 30% (recurrence, 77%). Carcinoma in situ (CIS) [3] is a flat epithelial, mostly high-grade tumor within the lamina propria, and it is a particularly virulent prognosis in up to 60% of cases (5). Grading also is a very important factor for prognosis. Progression in grade 1 tumors is 2-10% (recurrence, 63%), in grade 2 progression is 11-19% (recurrence, 67%), and in grade 3 progression is 33-45% (recurrence, 71%) (6).

Bladder Cancer Biomarkers

The main problems with bladder cancer are detection, recurrence, and progression. Dipstick urine analysis for the detection of microscopic hematuria, cystoscopy, and urine cytology are established modalities for detection and monitoring of bladder cancer. The diagnosis can be problematic because of the unspecific nature of the most common symptoms: irritative voiding and painless hematuria. Patients with these symptoms may undergo extensive urological examination to identify only a relatively small number of malignancies. For example, hematuria, the most prevalent symptom, is found in only 4-10% of cases of bladder cancer (7). Reagent strips for hemoglobin and erythrocytes are used to diagnose hematuria. Their accuracy for the detection of microhematuria is high, but their sensitivity and specificity for bladder cancer are low.

Bladder tumors are classically diagnosed by cystoscopy. This procedure presents the highest valuable standard for detection and monitoring. The sensitivity has been established at ~70%, and the technique allows characteristic information about the tumor, such as multifocality, appearance, and size (8). Cystoscopy is indispensable for resection and provides specimens for the most important pathological prognostic factors. However, cystoscopy is an invasive and costly procedure.

Urine cytology detects exfoliated malignant cells microscopically. These cells have characteristically large and eccentric nuclei with an increased nuclear-to-cytoplasmic ratio and irregular coarse chromatin. The cost for voided urinary cytology is approximately $100. Considering the low incidence of bladder cancer in patients with hematuria (only 4-10%), this procedure cannot be regarded as cost-effective. Nevertheless, urine cytology is a screening method for bladder cancer with a specificity >90% for true transitional cell carcinoma (TCC) diagnosis (9). In addition to voided specimens, urine can be obtained by catheterization of the patient and bladder washings in an attempt to increase sensitivity. The shortcomings of urine cytology include subjective variances between different pathologists and the limited rapid availability of the results. The main negative aspect is the poor sensitivity of 20-40% in the most common low-grade lesions irrespective of manner of collection. This phenomenon is based on the fact that cells from the most common well-differentiated tumors (low grade) do not appear diseased, are more cohesive, and are not readily shed into the urine. Urine cytology produces false-positive results in 1-12% of cases because of inflammation, urothelia atypia, and most important, because of changes caused by chemotherapy or radiation therapy. This procedure also can be falsely negative in 20% of cases, even those with high-grade tumors. Despite high sensitivity, the low overall accuracy allows urine cytology to be used only as an adjunct to endoscopic diagnosis.

The traditional approaches to detect and monitor bladder cancer are not sufficiently predictive in the individual patient, and a strong need exists for improved methods to predict superficial bladder cancer behavior and prognosis.

RATIONALE FOR BIOMARKERS

Identification of biomarkers may improve the screening and diagnosis of TCC, characterize the malignant potential, and determine the prognosis. Biomarkers should be noninvasive; rapid; easy to obtain, use, and interpret; inexpensive; and, most important, accurate with high sensitivity and specificity. The target groups of these markers could be high-risk patients with a history of smoking; patients with symptoms of bladder cancer, such as hematuria or irritative voiding symptoms; and patients after a bladder cancer diagnosis or treatment. Furthermore, if the incidence of bladder cancer continues to increase and such a biomarker can be developed, screening for bladder cancer in the aging population, similar to prostate-specific antigen in men for prostate cancer, might be evaluated.

The validity of biomarkers has to be measured by their ability to differentiate pathologic stage and grade. Currently, a variety of markers have been developed. Among these, Food and Drug Administration (FDA) approval for monitoring of patients with bladder cancer has been given to nuclear matrix protein (NMP) 22, fibrin /fibrinogen degradation product (FDP), and bladder tumor antigen (BTA; Table 1). Recently, NMP22 was approved as a test for bladder cancer screening.

FDA-APPROVED BIOMARKERS

NMP22. The nuclear matrix is a three-dimensional web of RNA and proteins that provides the structural foundation for a cell's nucleus (10). By serving as an anchoring point for enzymatic machinery, the nuclear matrix participates in DNA replication, transcription, RNA processing, and gene expression (10,11). Several of the NMPs are organ specific. Cancer-specific NMPs have been identified in breast, colon, bone, and urothelium, and NMP22 has been recognized as a potential urothelial-specific cancer marker (12).

The NMP22 test (NMP22[R] Test Kit; Matritech, Inc.) is an enzyme immunoassay that detects the NMP complexes, specifically NMP22. In malignancy, these NMPs are shed from the cell nucleus into the urine by apoptosis. The use of monoclonal antibodies specific for NMP22 in the assay allows a quantitative assessment of NMP22 concentrations in voided urine. Clinical research has demonstrated significant differences in NMP22 concentrations in urine of healthy volunteers compared with concentrations in patients with active TCC. One study measured NMP22 concentrations in the urine of 667 patients who suffered from TCC or benign bladder diseases, or who were disease free (13). Patients with active TCC had significantly higher median urinary NMP22 concentrations than those with no evidence of disease.

In 1996, the FDA approved the NMP22 assay for the detection of occult or rapidly recurring disease after transurethral resection. Several studies have reported high sensitivity of the NMP22 test, ranging from 68% (11) to 100% (14). This is greater than twice the sensitivity of cytology (20-40%). Conversely, the specificity of this assay differs significantly among authors. Several studies have reported specificities of 61% (15) and 85% (14), depending on the cutoff values for units of detected NMP22 (6.4 units/mL for patients previously diagnosed with bladder cancer, 10 units/mL for screening of patients with micro- and macrohematuria and voiding symptoms) that mark the difference between benign and malignant bladder disease. Others have reported high false-positive rates for urolithiasis (50%), benign prostatic hyperplasia (15.6%), and other benign urological diseases (25.6%) (16). One group was able to increase the specificity of this assay to 95.6% by excluding patients with a history of these diseases (17). Interestingly, stage and grade of the disease do not affect sensitivity and specificity of the NMP22 assay.

FDP. The FDP test (the old AuraTek[R] FDP was replaced with the new label, Accu-Dx; Intracel Corp.) recognizes increased FDP concentrations in the urine, which are associated with the presence of malignant bladder tumors (18,19). The test consists of a lateral flow immunoassay device that uses monoclonal antibodies to qualitatively detect urinary FDP. The colorimetric FDP test is a simple, rapid, point-of-care dipstick assay with an overall sensitivity over the entire range of superficial and invasive bladder cancers of 82.1% (20), considerably higher than cytology (38%). The sensitivity was 63.2% for grade 1, 88.2% for grade 2, and 95.0% for grade 3 disease. The specificity is reported to be 96% for healthy patients, 86% for subjects with urological disease other than bladder cancer, and 80% for patients under surveillance for bladder cancer with a negative cystoscopic finding at the time of the assay (18). The manufacturer has stopped producing this product; the lack of stability was related solely to manufacturing issues (21).

BTA. The BTA test (C.R. BARD Inc) is a latex agglutination test that quantitatively detects the presence of basement membrane complexes in the urine (22-24). These basement membrane complexes have been characterized in urine as a means of detecting bladder tumors, and the loss of these basement membrane proteins correlates to tumor stage and grade (25). In the BTA test, urine samples are mixed with latex particles coated with human IgG and blocking agents. If the proteolytic degradation products measured by the BTA test are present, the complexes combine with latex particles to form an agglutination reaction. This formation produces a visual color change, which differentiates positive from negative results by the use of a test strip. After the original BTA tests (sensitivity ~40%) did not perform any better than cytology, several modifications of the BTA assay were introduced, the BTA stat (26) and BTA TRAK (27,28). These assays detect a human complement factor H-related protein (hCFHrp), which is produced in vitro by several human bladder cancer cells but not other epithelial cell lines (28,29). The function and structure of hCFHrp is similar to human complement factor H (hCFH). hCFH inhibits the alternative complement pathway and therefore inhibits lysis of cells that are foreign to the host. Like hCFH, BTA interrupts the complement cascade and may confer a selective growth advantage to cancer cells in vivo by allowing the cells to evade the host immune system. The qualitative, single-step immunochromatographic BTA stat test is a dipstick test that can be done in the physicians office and provides immediate results after 5 min. The sensitivity of the BTA stat in low-grade lesions is higher than cytology (BTA stat, 50% for [G.sub.1]; cytology, 20-40%), but the sensitivity for high-grade lesions is lower than cytology (BTA stat, 29-66% for [G.sub.2], 40-83% for [G.sub.3]; cytology, 70-100%) (18, 24). The specificity, however, is lower than cytology (BTA stat, 72-95%; cytology >90%). The newest of the BTA tests, the BTA TRAK, is a quantitative immunoassay that measures the hCFHrp concentrations and must be sent to a reference laboratory. The limitation of these BTA tests is false positivity attributable to benign disease such as inflammation, stones, urothelial trauma, and other genitourinary malignancies.

OTHER POTENTIAL BIOMARKERS

Telomerase. Telomeres are short ends of chromosomes that undergo degradation with aging in somatic cells. With every round of replication, the cell continuously loses more of these telomeres (30, 31). The cell can afford to lose only a certain number of telomeres before important sequences of the parent DNA are lost, which leads to chromosomal instability and finally in cell death (32). However, germ cells survive these rounds of replication by producing an enzyme, telomerase, which maintains their telomeres (33). Telomerase activity is present not only in germ cells but in cancer cells as well (34,35). Urothelial tumors of all grades express telomerase activity in voided urine (36). One study described a sensitivity of 100% for grade 1, 92% for grade 2, and 83% for grade 3 tumors (37). Other initial reports utilizing telomeric repeat amplification protocols found a >85% detection rate of bladder cancers (38, 39). However, subsequent investigations have revealed sensitivities in voided urine in the 60-70% range (40). The specificity of the telomerase assay has been reported as 80% (36). False-positive results in 23.3% of cases were attributable to stones, inflammation, benign prostatic hyperplasia, and other benign urologic diseases (37).

Hyaluronic acid and hyaluronidase. This test is based on the knowledge that metastatic cells produce enzymes and enzyme products that help to dissolve the cellular matrix for malignant cells on their way toward blood vessels (41). These substances are identified as hyaluronic acid and hyaluronidase and have been detected in the urine by an ELISA-based assay. Studies have reported that hyaluronic acid was increased five- to sevenfold in all bladder cancer patients regardless of tumor grade, whereas hyaluronidase was increased only in higher grade bladder cancers (four- to sevenfold in [G.sub.2]/[G.sub.3] tumors) (42). The reported sensitivities and specificities of both hyaluronic acid and hyaluronidase have ranged from 86% to 92% (43).

Cell surface antigen (ImmunoCyt). ImmunoCyt (Diagnocure, Inc.) is a combination assay that uses three monoclonal antibodies to detect cell surface antigens (M344, LDQ10, and 19A211) of TCC. In this test, immunofluorescence and cytology can be performed on one slide. ImmunoCyt had an initial reported sensitivity of 95% in low-grade lesions ([T.sub.a], [T.sub.1]). Recent studies have shown 86.1% sensitivity for ImmunoCyt alone and 89.9% sensitivity when ImmunoCyt is used in combination with cytology (44), implying that the high sensitivity of ImmunoCyt alone does not need additive cytology. However, ImmunoCyt gives a significant number of false-positive results and therefore has a low specificity, and high specificity is the domain of cytology (>90%).

DNA ploidy and S-phase fraction [flow cytometry, image cytometry (ICM), laser scanning cytometry, and fluorescence in situ hybridization (FISH)]. Flow cytometry evaluation of urine is an automated measurement of cellular DNA (Table 2). It determines the DNA ploidy and estimates the 5-phase fraction (DNA synthesis). Because neoplastic cells display nuclear enlargement and hyperchromatism, reflecting a decreased DNA content, flow cytometry samples are compared with healthy control cells and the amount of DNA is displayed in a histogram as diploid, tetraploid, and aneuploid. Aneuploid cell populations and those samples with a higher percentage of cells in the S phase suggest the presence of higher grade cancers or CIS. Diploid tumors generally tend to be of low grade and low stage, with a better prognosis (45,46). Flow cytometry is especially accurate in patients with CIS or high-grade malignancies, of which 80-90% can be correctly identified (47,48). This technique is difficult and expensive to perform and requires trained personnel. Other problems include the lack of standardization of this assay and the need for large tumor sections. Furthermore, the analysis can be compromised if the sample includes noncancerous cells.

An alternative method to detect aberrations in cellular DNA content is FISH. FISH, which allows visualization and quantification of chromosomes and genes on a cell-by-cell basis, is easy to perform and requires no specialized equipment. Recent publications concerning this procedure have revealed its ability to differentiate between [T.sub.a] and [T.sub.1] tumors in regard to their fraction of detectable aberrations (49-51). Interestingly, application of the FISH procedure on bladder washings after therapy with bacille Calmette-Guerin showed that loss of chromosome 9, the most common chromosomal abnormality found in bladder tumors, correlates significantly with tumor recurrence and failure of bacille Calmette-Guerin therapy (52). With the growing interest in the ability to better classify between the different low-grade tumors, this procedure has gained in importance.

ICM uses a computer-controlled fluorescence microscope that analyzes smears of cells on a microscope slide and quantitatively measures the DNA content in each cell, which is directly proportional to the emitted fluorescence (53). Because individual cells can be examined, this technique can more easily use voided urine than flow cytometry, which requires a large cell population. ICM is more sensitive, especially for the detection of low-grade bladder cancer, than either flow cytometry or cytology (54).

Laser scanning cytometry combines the advantages of flow cytometry and ICM by using a laser scanning cytometer to measure the fluorescence of individual cells rapidly and accurately (55).

Blood group-related antigens (ABH and Lewis antigen). The ABH and Lewis blood group-related antigens ([Le.sup.a], [Le.sup.b], and [Le.sup.y]) are present on the surface of healthy urothelium. Most of these antigens are present in 75-80% of the population. Changes in cellular morphology, differentiation, and proliferation often are associated with alteration in the expression of these cell surface molecules (56). Malignant transformation of urothelial cells appears to be associated with loss of ABH blood group antigen (56,57) and enhanced Lewis X ([Le.sup.x]) antigen expression (58,59). Whereas ABH antigen deletion correlates with increased recurrence rates and the development of invasive bladder cancer (60), the [Le.sup.x] antigen seems to be expressed regardless of bladder cancer grade or stage (61). However, certain difficulties impede the clinical application of ABH antigens. The deletion of ABH antigen expression can be reliably determined only in secretory individuals (20% lack ABH expression). Furthermore, earlier promising studies with respect to loss of ABH antigen expression were not reproducible in a large well-controlled study (59). One of the advantages of [Le.sup.x] antigen over ABH antigen is the enhanced [Le.sup.x] antigen expression in positive patients, rather than a decrease similar to that measured in the ABH antigen assay. Currently, [Le.sup.x] remains the only blood group antigen with potential prognostic value. Recent studies have reported an 85% overall sensitivity and 85% specificity of bladder cancer detection of [Le.sup.x] immunocytology (62). High- and low-grade TCCs were detected with equal efficiency (61).

Tumor-associated antigen (M344, 19A211, T138, and DD23). Several TCC-associated antigens can be identified by monoclonal antibodies and are currently under investigation as potential tumor markers. These markers are mostly absent in healthy transitional epithelium. M344 antigen is a cytosolic protein that is detectable in ~70% of [T.sub.a] and [T.sub.1] tumors, whereas the expression decreases with increasing tumor stage and grade (25% for [T.sub.is], 15% for invasive disease) (63,64). The combination of monoclonal antibodies against M344, 19A211, and LDQ10 are currently under investigation as an immunocytochemical test in voided urine (ImmunoCyt) (44).

19A211 is a sialoglycoprotein that is expressed in 70% of [T.sub.a] and [T.sub.1] tumors, 60% of [T.sub.is] tumors, and 50% of invasive TCCs (60). Unfortunately, this antigen is found in 25% of healthy umbrella cells. 19A211 predicts a lower tumor recurrence, whereas detection of T138 predicts a significantly higher chance of recurrence (65). T138 is a glycoprotein that is expressed in only 15% of [T.sub.a] and [T.sub.1] TCC disease and 60% of invasive cancers (63).

These antigen markers appear to be promising as future biomarkers, but they require further evaluation. The combination of these antigens with cytology shows a higher sensitivity and specificity. For example, the combination of the novel antibody DD23 (UroCor) and cytology has 94% sensitivity and 85% specificity.

Proliferating antigens (Ki-67 antibody, proliferation cell nuclear antigen). The two most promising immunohistochemical markers of cellular proliferation are Ki-67 and proliferation cell nuclear antigen (PCNA). The murine monoclonal antibody Ki-67 reacts with nuclear antigen that is related to cell proliferation (66). Studies have reported increased Ki-67 immunostaining in TCCs with higher tumor grade, stage, and recurrence (67-73). Ki-67 also seems to correlate with progression and reduced survival rate (74). The PCNA monoclonal antibody is incorporated into the cellular nuclei at the time of DNA synthesis. Therefore, 95.8% of TCCs stain positive for PCNA. The mean labeling index was higher in invasive and high-grade tumors (75).

Oncogenes (c-erb-b2, c-ras, c-myc, c jun, mdm2). Malignant transformation can be the result of genetic changes. One mechanism of these genetic changes is the mutation of normal genes to oncogenes, which allows cells to escape the regulation of cellular growth control.

Studies have reported that mutation in the ras gene family (c-H-ras, c-K-ras, p21 ras) is associated with the development, progression (76), grade, and recurrence (77) of bladder cancer. However, among the ras gene family, mutations of the c-H-ras gene reportedly are the most common, especially point mutations at codons 12, 13, and 61. The alteration of c-H-ras has been reported in only 10-36% of bladder cancers (78,79).

The major alteration of the c-myc gene is hypomethylation, which appears in the gene's flanking and promoting regions (80). Although some authors have reported that recurrence and progression of superficial bladder malignancies are associated with the detection of the c-myc oncogene (81), other authors were not able to report any independent prognostic value for c-myc (82).

mdm2 is a protein that can bind to p53 protein in the nucleus. This binding inactivates p53 function by exporting it (as part of a mdm2/p53 complex) out of the nucleus into the cell cytoplasm where it can no longer influence transcription. Additional proteins such as RanGTP and CRM1 are essential mediators for this "nuclear-cytoplasmic shuttling" of p53 protein (83). This shuttling of the mdm2/p53 complex to the cytosol indirectly promotes cell proliferation because p53 is active mainly in nuclear cell cycle regulation.

Some investigators have demonstrated mdm2 amplification in 20-30% of TCCs (84); others reported rather infrequent results (85). No correlation between staging and grading and mdm2 expression has been found (86). Nevertheless, recent reports have shown promising results with simultaneous evaluation of p53 and mdm2 immunostaining for prognosis of bladder cancer.

c-jun, another nuclear gene, encodes the main component of a major transcription factor (AP-1), which plays an important role of growth regulation (87, 88). Therefore, alterations of c-jun cause insufficient cell cycle control. Overexpression of the protooncogene c-jun correlated with invasive stages (89) and increased expression of the epidermal growth factor receptor (EGF-R) (90). Furthermore, structural (similar to tyrosine kinase activity) and functional (stimulation of cellular growth) homology is reported between EGF-R and another protooncogene, c-erbB-2 (91, 92). C-erbB-2 is a transmembrane glycoprotein that is overexpressed in high stage and grade bladder tumors (93-95). However, conflicting results regarding increased tumor progression and prognosis have been reported, and it is questionable whether this biomarker provides additional prognostic information to previously known staging and grading (96-98).

Growth factors [EGF, transforming growth factor-[beta] (TGF-[beta]), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF)]. EGF-R is a transmembrane glycoprotein that is activated by binding of either EGF or TGF-[alpha]. EGF and TGF-[alpha] induce cellular proliferation. In healthy transitional epithelium, EGF-R usually is located in the basal cell layer. In malignant tissue, the pattern of distribution of EGF-R involves all layers. Interestingly, this widespread distribution of EGF-R is also found throughout the healthy-appearing urothelium of bladder cancer patients. Several reports have demonstrated that up-regulated expression of EGF-R in bladder cancer tissue correlates with increased tumor stage and grade. The practical utility of EGF-R as a biomarker for progression of bladder malignancies is limited by the complicated method of detection: immunostaining of the EGF-R requires frozen sectioning rather than paraffin sections.

Currently, the prognostic significance of EGF is unknown. Some studies have demonstrated no difference between urinary EGF concentrations in patient with bladder cancers compared with controls (99), whereas others have shown significantly lower concentrations in patients with bladder neoplasm (100-102). However, no correlations between EGF concentrations and stage, grade, and survival rate of bladder cancer have been reported (101-103).

VEGF is a potent and specific inducer of angiogenesis by induction of endothelial cell proliferation and migration. It is well established that angiogenesis is an important factor of tumor growth. Because VEGF is highly expressed in several tumors, including bladder cancer, this protein could be a potential biomarker. Recent studies have reported that VEGF urine concentrations were higher in patients with cancer than in patients with benign conditions. Whereas analysis of stage [T.sub.1] and [T.sub.2] tumors did not detect differences in the mean urinary VEGF concentrations, [T.sub.a] tumors showed lower VEGF expression. Grading showed no differences between GZ and G3 tumors, but expression of VEGF in [G.sub.1] tumors was lower. High concentrations of VEGF correlate with a high rate of bladder cancer recurrence (104,105).

Among several other capacities, TGF-[beta] induces and inhibits proliferation, depending on the cell type. The antimitogenic activity of TGF-[beta] is mediated by modulation of p15 and p27, two nuclear proteins that inhibit the phosphorylation of the protein of the retinoblastoma gene (pRb) by various cyclin-dependent kinases. By preventing the inactivation of pRb and the uncomplexing of pRb and E2F through p15 and p27, TGF indirectly induces cell cycle control. Tumors with increased TGF-[beta] expression showed slower proliferation, and indolent TCCs had significantly lower TGF-[beta]1 expression than more aggressive stages (106).

Basic FGF (bFGF) stimulates endothelial cell migration and cellular motility. In vitro studies have demonstrated that bFGF-transfected human bladder cancer cell lines showed a higher drug resistance to cisplatin (107) and increased invasive potential (108). Several investigators have reported increased bFGF in TCCs compared with benign tissue. Tumor stage has been reported to correlate with detected bFGF concentrations in urine (109). bFGF was found to be increased in most tumors of high stage, and its presence correlated with the occurrence of early local relapses (110). A disadvantage of bFGF is its lack of specificity, with high false-positive tests in benign disease (111).

Cellular adhesion molecules (cadherins, integrins). Growing evidence suggests that alterations in the adhesion of malignant cells plays an important role in bladder cancer progression. Cellular adhesion molecules are the important factors for interaction between adjacent cells. Several families of these factors exist, such as cadherins and integrins. Cadherins are transmembrane glycoproteins that maintain cellular adhesion to neighboring cells. Their structure contains three parts: an intracellular, a transmembranous, and an extracellular component. Through catenins, the intracellular component is connected to the extranuclear cytoscelet of the cell. The loss of intracellular adhesion is the basis for metastatic spread of tumor cells. Obviously then, a decrease in cadherins promotes the lymphatic or blood-related expansion of bladder cancer cells. Recent studies reported that 62% of noninvasive bladder cancers stained positively for epithelial cadherin (E-cadherin). In contrast, 75% of invasive tumors showed aberrant expression of E-cadherin. E-Cadherin correlated with stage but not with grade (112).

Another group of cellular adhesion proteins are the integrins, which usually are located only on the basement membrane surface of the basal urothelial cell layer. However, bladder cancers often express integrins diffusely throughout the tumor tissue. Reductions in the amounts of [alpha]-2 and [beta]-4 integrin chains are related to tumor progression in bladder cancers. [alpha-2 and [beta]-4 integrins were strongly expressed in healthy urothelium, and 29-35% of noninvasive bladder tumors stained positively for integrins, depending on the subtype. However, 81% of invasive tumors showed aberrant expression of [alpha]-2 integrin, and 100% showed aberrant expression of [beta]-4 integrin (112). A reduction in the amount of [beta]-4 integrin correlated with grade and stage of bladder tumors, whereas [alpha]-2 integrin does not show a correlation. Furthermore, studies reported that a decrease in [beta]-4 integrin plays a role in intraepithelial spreading of CIS by enhanced migration on laminin, a component of the extracellular matrix (113).

Cell cycle regulatory proteins (p53, pRb, cyclins, cyclin-dependent kinases, p15, p16, and p21). Cell cycle regulatory proteins control proliferation and cell cycle progression of healthy, nonmalignant cells. Neoplasms are characterized by an uncontrolled cell growth. Loss of cell cycle control seems to be an early sign in malignant transformation and cancer progression. Several alterations of genes and protein products of cell cycle regulation are identified in bladder tumors and appear to be associated with development of TCC.

The wild-type tumor suppressor gene p53 regulates the checkpoint that mediates apoptosis or cell-cycle arrest in [G.sub.1] in response to DNA damage. Cell-cycle arrest allows cells to repair their DNA and prevents propagation of DNA defects. However, the regulatory role of p53 on the cell cycle is mediated through other genes, such as the retinoblastoma (Rb) tumor suppressor gene. In its hypophosphorylated state, pRb forms stable complexes with transactivation properties, such as E2F. This hypophosphorylated, active form of pRb has an inhibitory effect on cell cycle progression. Phosphorylation (and inactivation) of pRb in mid-[G.sub.1] liberates the bound transactivation properties, several of which are essential for DNA replication. Phosphorylation of pRb is mediated by cyclins, which form complexes with catalytic subunits, cyclin-dependent kinases. Cyclin-dependent kinases, on the other hand, are inactivated through stable complexes with p15, p16, and p21, for example. This, in turn, inhibits pRb phosphorylation.

Mutations of p53 are the most common genetic defects in human tumors (114), including bladder cancers. Nuclear accumulation of p53 detected by immunohistochemical staining correlates with increased p53 mutations in DNA sequence analysis (115). Recent studies have reported that increased p53 nuclear reactivity is associated with bladder cancer progression, increased recurrence, decreased overall survival, decreased responsiveness to chemotherapy (including therapy with bacille Calmette-Guerin), higher grade, and higher stage of TCC (116-118). However, p53 mutations are detected by manual immunohistochemistry, which presents a rather inaccurate technique for quantification because of a lack of reproducibility and standardization.

Because of the interaction between p21 and p53 in the cell cycle regulation, investigators have demonstrated that loss of p21 expression is believed to be one of the mechanisms by which p53 mutation and inactivation may influence TCC progression (119). The Rb gene plays a key role in the development and progression of many malignancies, including bladder cancers. Alteration or loss of Rb expression seems to be an important prognostic factor in TCC. It has been reported that patients with invasive bladder cancer showed altered Rb expression more often than those with superficial disease and had a significantly lower 5-year survival (120,121). Although promising tools as future bladder cancer predictors, cell cycle regulatory proteins have not been studied thoroughly enough for wide clinical application.

Practical Guidelines

Despite the recognized specificity of cytology, its low sensitivity prevents its use as a replacement for cystoscopy. Prospective standardized multicenter trials to confirm whether one of the new biomarker tests might (to some degree) replace some cystoscopic studies are necessary. Patients with TCC can be assigned into low- or high-risk groups based on well-established factors such as pathologic stage, grade, the risk for recurrence, single or multiple lesions, or the presence of CIS. Biomarkers can be used in combination with cytology or alone to increase or decrease the intervals between monitoring cystoscopies. For example, a normal NMP22 value would lead to increased time intervals between cystoscopy. In contrast, high NMP22 values would prompt cystoscopic evaluation sooner. This approach might improve a patient's compliance and hopefully reduce the cost of management. Furthermore, NMP22 concentrations in voided urine might determine the type of anesthesia needed in the cystoscopy. Whereas patients with low NMP22 values are monitored with office-based cystoscopies that utilize local intraurethral anesthesia, patients with high values would require a procedural visit for definitive treatment that needs more substantial anesthesia.

NMP22, the Accu-Dx test, the BTA tests, telomeric repeat amplification protocols, and ImmunoCyt are easy-to-use techniques. Currently, only NMP22 and the BTA tests are clinically available in the United States. In general, all of the biomarkers have higher sensitivity and lower specificity compared with cytology. These tests have an advantage in detecting low-grade superficial tumors. p53 and Rb are the only prognostic biomarkers that showed promising results for selecting patients for adjuvant chemotherapy and predicting disease progression in multivariate comparisons of p53 status, pathological stage, and histological grade. Bladder cancer biomarkers, specifically NMP22, may also be useful for screening in high-risk groups, just as colonoscopy, mammography, or prostate-specific antigen are used at present.

Conclusion

Research for new biomarkers for the early detection, monitoring, and prognosis of bladder cancers is an active area of interest. Current biomarker technology mainly provides a higher sensitivity, making established methods such as cystoscopy and cytology difficult to replace. None of the discussed markers has 100% sensitivity and specificity. In addition, it is still too early to say whether any one of them is an excellent predictor of the natural history of the disease and response to treatment. Furthermore, standardization and reproducibility are still major difficulties that hinder the clinical application of the majority of these biomarkers. However, their potential applicability cannot be ignored. Therefore, the most effective way of screening or monitoring patients for bladder cancer with current biomarker technology is in new adjuvant fashions that redefine bladder cancer standards. Further research in understanding cancer-related incidents such as loss of cell cycle control and loss of intercellular binding would hopefully lead to the recognition and utilization of bladder cancer markers in clinical practice.

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MARTIN BURCHARDT, [1,2] TATJANA BURCHARDT, [1] AHMAD SHABSIGH, [1] ALEXANDRE DE LA TAILLE, [1] MITCHELL C. BENSON, [1] and IHOR SAWCZUK [1] *

[1.] Department of Urology, College of Physicians and Surgeons of Columbia University, New York, NY 10032.

[2.] Department of Urology, Heinrich-Heine-Universitaet, 40225 Dusseldorf, Germany.

All authors contributed equally to this work.

[3.] Nonstandard abbreviations: ClS, carcinoma in situ; TCC, transitional cell carcinoma; FDA, Food and Drug Administration; NMP, nuclear matrix protein; FDP, fibrin/fibrinogen degradation product; BTA, bladder tumor antigen; hCFH and hCFHrp, human complement factor H and hCFH-related protein; FISH, fluorescence in situ hybridization; 1CM, image cytometry; Le, Lewis blood group-related antigen; PCNA, proliferation cell nuclear antigen; EGF and EGF-R, epidermal growth factor and EGF-receptor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; pRb, protein of the retinoblastoma gene; bFGF, basic fibroblast growth factor; and E-cadherin, epithelial cadherin.

* Address correspondence to this author at: Department of Urology, Columbia-Presbyterian Medical Center, Atchley Pavilion, 11th Floor, 161 Fort Washington Ave., New York, NY 10032. Fax 212-305-0106; e-mail issl@columbia.edu.

Received October 29, 1999; accepted February 28, 2000.
Table 1. Monitoring biomarkers.

Test Sample Quantitative Qualitative FDA
 approved

NMP22 Urine Yes Yes
FDP Urine Yes Yes
BTA Urine Yes Yes
BTA TRAK Urine Yes Yes
BTA stat Urine Yes Yes
TRAP (a) telomerase Urine Yes Yes
Hyaluronic acid/
 hyaluronidase Urine Yes
Cell surface antigen
 (ImmunoCyt) Urine Yes

Test Sensitivity, Specificity, Reference(s)
 % %

NMP22 68-100 61-85 10-17
FDO 82.1 86-96 18-21
BTA 54-83 72-95 22-25
BTA TRAK 54-78 95-97 27-29
BTA stat 80-90 72-95 26
TRAP (a) telomerase 85-100 80 30-37
Hyaluronic acid/
 hyaluronidase 86-92 86-92 41-43
Cell surface antigen
 (ImmunoCyt) 86-95 90 44

(a) TRAP, telomeric repeat amplification protocol.

Table 2. Prognostic biomakers. (a)

Test Sample Quantitative

DNA ploidy/S-phase
fraction
 Flow cytometry Urine Yes
 ICM Urine Yes
 LSCM (b) Urine Yes
 Fish Tissue/bladder
 washing Yes
Blood group-related
antigens
 ABH Tissue
 Lewis antigen Tissue
Tumor-assisted
antigens
 M344, 19A211,
 T138, DD23 Tissue/Urine
Proliferating antigens
 K1-67 antibody, PCNA Tissue Yes
Oncogenes
 c-erb-B2 Tissue Yes
 ras genes Tissue Yes
 c-myc Tissue Yes
 c-jun Tissue Yes
 mdm2 Tissue Yes
Growth factors
 EGF Tissue Yes
 TGF-[beta] Tissue Yes
 TGF Tissue Yes
 VEGF Tissue Yes
Cellular adhesion
molecules
 Cadherins Tissue Yes
 Integrins Tissue Yes
Cell cycle regulatory
proteins
 p53, pRb, cyclins,
 p15, p16, p21 Tissue Yes

Test Qualitative Reference(s)

DNA ploidy/S-phase
fraction
 Flow cytometry Yes 45-48
 ICM Yes 53-54
 LSCM (b) Yes 55
 Fish Yes 49-51
Blood group-related
antigens
 ABH Yes 56-61
 Lewis antigen Yes 56-61
Tumor-assisted
antigens
 M344, 19A211,
 T138, DD23 Yes 44, 63-65
Proliferating antigens
 K1-67 antibody, PCNA 66-75
Oncogenes
 c-erb-B2 91-98
 ras genes 76-79
 c-myc 80-82
 c-jun 87-90
 mdm2 83-86
Growth factors
 EGF 99-103
 TGF-[beta] 106
 TGF 107-111
 VEGF 104-105
Cellular adhesion
molecules
 Cadherins 112
 Integrins 112, 113
Cell cycle regulatory
proteins
 p53, pRb, cyclins,
 p15, p16, p21 114-121

(a) None of these biomakers have been approved for use by the FDA.

(b) LSCM, laser scanning cytometry.
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Publication:Clinical Chemistry
Date:May 1, 2000
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