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Articles |
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.
a 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 iss1{at}columbia.edu
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Abstract |
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 Top Abstract Bladder CancerBladder Cancer BiomarkersPractical GuidelinesConclusionReferences |
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Approach: We undertook a comprehensive literature search using Medline to identify all publications from 1980 to 1999. Articles that discussed potential biomarkers for TCC were screened. Only compounds that demonstrated high sensitivity or specificity, significant correlation with TCC diagnosis and staging, and extensive investigation were included in this review.
Content: Potential biomarkers of disease progression and prognosis include nuclear matrix protein, fibrin/fibrinogen product, bladder tumor antigen, blood group-related antigens, tumor-associated antigens, proliferating antigens, oncogenes, growth factors, cell adhesion molecules, and cell cycle regulatory proteins. The properties of the biomarkers and the methods for detecting or quantifying them are presented. Their sensitivities and specificities for detecting and monitoring disease were 54–100% and 61–97%, respectively, compared with 20–40% and 90% for urinalysis and cytology.
Summary: Although urine cytology and cystoscopy are still the standard of practice, many candidate biomarkers for TCC are emerging and being adopted into clinical practice. Further research and better understanding of the biology of bladder cancer, improved diagnostic techniques, and standardized interpretation are essential steps to develop reliable biomarkers. It is possible that using the current biomarkers as an adjuvant modality will improve our ability to diagnose and monitor bladder cancer.
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Bladder Cancer |
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TopAbstractBladder CancerBladder Cancer BiomarkersPractical GuidelinesConclusionReferences |
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Staging and grading currently are the most reliable variables for recurrence and progression. Patients with Ta disease progress in 4% (risk of recurrence, 52%), whereas progression in patients with T1 lesions is 30% (recurrence, 77%). Carcinoma in situ (CIS)1 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).
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Bladder Cancer Biomarkers |
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TopAbstractBladder CancerBladder Cancer BiomarkersPractical GuidelinesConclusionReferences |
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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.
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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® 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® 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)(23)(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
physician’s 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 G1;
cytology, 20–40%), but the sensitivity for high-grade lesions is
lower than cytology (BTA stat, 29–66% for
G2, 40–83% for G3;
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
G2/G3 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 (Ta, T1). 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 S-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.
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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 Ta and T1 tumors in regard to their fraction of detectable aberrations (49)(50)(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 (Lea,
Leb, and Ley) 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 (Lex) antigen expression
(58)(59). Whereas ABH antigen
deletion correlates with increased recurrence rates and the development
of invasive bladder cancer (60), the
Lex 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 Lex antigen over ABH antigen
is the enhanced Lex antigen expression in
positive patients, rather than a decrease similar to that measured in
the ABH antigen assay. Currently, Lex 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 Lex 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 Ta and T1
tumors, whereas the expression decreases with increasing tumor stage
and grade (25% for Tis, 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 Ta and T1 tumors, 60% of Tis 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 Ta and T1 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)(68)(69)(70)(71)(72)(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)(94)(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)(97)(98).
Growth factors [EGF, transforming growth factor-ß (TGF-ß),
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-
. EGF and TGF- 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)(101)(102). However, no correlations between EGF concentrations and stage, grade, and survival rate of bladder cancer have been reported (101)(102)(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 T1 and T2 tumors did not detect differences in the mean urinary VEGF concentrations, Ta tumors showed lower VEGF expression. Grading showed no differences between G2 and G3 tumors, but expression of VEGF in G1 tumors was lower. High concentrations of VEGF correlate with a high rate of bladder cancer recurrence (104)(105).
Among several other capacities, TGF-ß induces and inhibits proliferation, depending on the cell type. The antimitogenic activity of TGF-ß 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-ß expression showed slower proliferation, and indolent TCCs had significantly lower TGF-ß1 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
-2 and ß-4 integrin chains are related to tumor progression in
bladder cancers. -2 and ß-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 -2 integrin, and 100%
showed aberrant expression of ß-4 integrin (112). A
reduction in the amount of ß-4 integrin correlated with grade and
stage of bladder tumors, whereas -2 integrin does not show a
correlation. Furthermore, studies reported that a decrease in ß-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 G1 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-G1 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)(117)(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.
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Practical Guidelines |
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TopAbstractBladder CancerBladder Cancer BiomarkersPractical GuidelinesConclusionReferences |
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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.
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Conclusion |
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TopAbstractBladder CancerBladder Cancer BiomarkersPractical GuidelinesConclusionReferences |
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Footnotes |
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1 Nonstandard abbreviations: CIS, 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; ICM, 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. 
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TopAbstractBladder CancerBladder Cancer BiomarkersPractical GuidelinesConclusionReferences |
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