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Am J Transl Res. 2010; 2(4): 412–440.
Published online 2010 July 25.
PMCID: PMC2923865

Urine cytology and adjunct markers for detection and surveillance of bladder cancer

Abstract

Urine cytology coupled with cystoscopic examination has been and remains the standard in the initial evaluation of lower urinary tract lesions to rule out bladder cancer. However, cystoscopy is invasive and may miss some flat lesions, whereas cytology has low sensitivity in low-grade papillary disease. Additional lab-based or office-based markers are needed to aid in the evaluation of these lesions. Recently, many such markers have been developed for the purpose of improving the cytologic diagnosis of bladder malignancies. In this review, we will first discuss conventional cytomorphologic analysis of urine cytology followed by a discussion of markers that have been developed in the past for detection and surveillance of urothelial carcinoma. We will focus on how these markers can be used in conjunction with urine cytology in daily practice.

Keywords: Bladder cancer, urine cytology, tumor markers

Introduction

Bladder cancer, most commonly urothelial carcinoma, is the 4th most common cancer in males in the United States [1]. However its cost per patient is the highest of all the cancer types, reaching approximately 200,000 U.S. dollars per patient from diagnosis to death [2]. It has been estimated that in 2009 approximately 70,980 new cases of bladder cancer will be diagnosed with 14,330 deaths in this country [1]. Approximately 75% of patients present with superficial disease (Ta and T1) while 20% present with T2 or greater disease. The remaining 5% of patients present with metastatic disease. Overall, 70% of treated tumors recur, with 30% of recurrent tumors progressing to metastatic disease [3].

Roughly 60% of patients with newly diagnosed bladder cancer do not have muscle invasive disease and do not require cystectomy [4]. The majority of these patients have a recurrence after endoscopic resection, thus lifelong surveillance with cystoscopy is recommended. Unfortunately, cystoscopy, which is the “gold standard” for the detection of de novo and recurrent bladder cancer, is an expensive and invasive procedure. In addition, it may miss a flat lesion, especially carcinoma in situ which is considered a high grade malignant condition rather than a precancerous lesion as in other organ systems.

Voided urinary cytology is a useful noninvasive adjunct to cystoscopy because of its overall high specificity. Cytology also has a relatively high sensitivity at detecting high-grade lesions. Its sensitivity, however, is anywhere between 20 to 50% for low-grade papillary tumors. Of the non-muscle-invasive lesions, approximately 10% of low-grade papillary tumors subsequently develop muscle-invasive or metastatic cancer whereas roughly a third of high-grade tumors progress, if not already muscle-invasive at the time of diagnosis [4]. Therefore, close monitoring and early detection of all lesions are important for management, and noninvasive tumor markers with high accuracy for the detection of all grades of urothelial carcinoma will significantly reduce patient cost, anxiety and morbidity.

Urine cytomorphological analysis

Urinary cytology identifies malignant cells that have been exfoliated from the urothelium into the urine. The specificity of cytology is greater than 90% [5], while the sensitivity for high-grade disease and carcinoma-in-situ (CIS) can be as high as 80 to 90% [6, 7]. As indicated before, however, the main shortcoming of voided cytology is the low sensitivity (approximately 20-50%) for detecting low grade neoplasms including benign papilloma, urothelial carcinoma with low malignant potential (borderline), and low grade papillary urothelial carcinoma (Grade 1 to 2 of 3 of the WHO classification) [3] [4, 8]. There are two main reasons for such low sensitivity. First, tumor cells of the low grade tumors are not routinely shed into the urine because of their cohesive nature. Second, and probably more important, is the fact that low grade tumor cells by definition have similar cytomorphology to normal urothelial cells microscopically. While increased cellularity and presence of papillary fragments in “true” voided urine sample may be a hint for such a low grade lesion, one has to rule out the possibility of urothelial hyperplasia due to various reasons such as lithiasis, infection, and instrumentation.

Probably the most common reason for the presence of increased cellularity or papillary fragments in an otherwise morphologically normal voided urine sample is instrumentation as a result of cystoscopy, since many such samples are collected after the procedure is performed even though the requisition may incorrectly state the specimen is a “voided urine”. Thus, caution should be taken and clinical correlation should be advised in such a setting.

Common indications for urinary cytology

Urine cytology, as an “ancient” technique, has been used in following. First, it has been used as a screening tool to detect urothelial cancers in high risk populations, especially in populations exposed to chemical carcinogens through occupational means, for example the Drake cohort [9]. Second, it has been used as an initial test for patients presenting with hematuria to rule out (or rule in) the possibility of urothelial malignancy. Third, it has been used as a monitoring and follow-up tool for patients with a previous diagnosis of urothelial cancer to rule out tumor recurrence. Fourth, it has been used after transurethral resection for assessment of the completeness of tumor removal [10]. Finally, recently it has been applied as a test for detecting inflammation or infection, especially in kidney transplant patients where the presence or absence of polyoma virus infection may have significant clinical implications for rejection [11].

Type of urine samples

The most common type of urine specimen for cytologic analysis is voided urine. Again, keep in mind that although the submitted sample is marked as a “voided urine”, it is important to determine whether a cystoscopy has been performed, and if so, whether the sample is collected before or after the procedure. In collecting “true” voided urine, one should avoid a “first morning” specimen and collect the “second morning” voided sample, since the overnight urine often contains many degenerated urothelial cells complicating both morphologic and marker analysis. Although there are data to suggest that three specimens of “second morning” voided urine collected over three consecutive days may optimize the detection of urothelial malignancy [12], this is not a common practice, likely because of cost and convenience.

It is important to remember that, unlike wash or brush samples as discussed below, a true voided urine has the so-called “funnel” effect, i.e., it samples the entire urinary tract system from renal pelves (bilateral) to ureters (bilateral), bladder, and urethra. Considering the fact that urothelial cancer is often a “field” disease, the funnel effect ensures detection of lesions in the entire urinary tract, especially high grade lesions. Thus, at least in theory, voided urine should have a higher sensitivity for detecting urothelial malignancies of the entire urinary tract. However, the trade-off is that often the exact location of the lesion may be difficult to find, especially if the lesion is in the upper urinary tract (ureters and renal pelves). This may result in a so-called “false” false positive urine cytology. Last but not least, in female voided-urine samples, most of the epithelial cells present on the slide are squamous cells contaminated from the female genital tract. Thus, for any type of molecular marker analysis, especially PCR-based rather than image-based analysis, the contaminated squamous cells will be problematic. Unfortunately, the contamination factor is often ignored in many molecular based studies.

Another common urine sample is catheterized urine, which is usually more cellular than true voided urine but is otherwise identical. Genital contamination may be less of a problem compared to a true voided urine. The wash and brush samples from bladder, ureter, or pelves provide a complement to voided urine samples for the evaluation of urinary tract lesions where cystoscopy (or retrograde ureterocystoscopy) is performed at the same time. Depending on whether a suspicious lesion is seen, a washing or brushing may be performed at the same time as well. The advantages of washing and brushing samples include greater cellularity and a more targeted and homogeneous population of urothelial cells to be analyzed. For low grade lesions, cytomorphologic analysis alone for washing or brushing samples can be extremely challenging. In such a setting, correlating the cytology with the cystoscopy finding is essential. In contrast, for high grade lesions, especially carcinoma in situ, there may be many single or loosely cohesive, highly atypical cells on cytology while cystoscopy may or may not show a visible lesion. Biopsy may show only a few tumor cells on the surface (the “clinging” type of carcinoma in situ). A positive cytology coupled with what appears to be a negative cystoscopy or biopsy is another source of the so-called “false” false positive diagnosis. More discussion on this point will be provided in conjunction with the discussion on tumor markers below.

Other specimen types include ileal conduit or neobladder urine, which are often characterized by the presence of many degenerated columnar epithelial cells and inflammatory cells. On occasion, recurrent urothelial carcinoma may be seen, and diagnosis of such lesions can be extremely difficult since many biomarkers (as discussed below) may not be helpful in such a case.

Cells and other materials

Cells and other materials are summarized in Box 1.

Box 1

  • Cells normally occurring in urine (Figures 1A-B):
    Figure 1
    Normal cell components present in urine. (A) Basal urothelial cells have moderate dense cytoplasm with well-defined borders. Nuclei are centrally-placed with small nucleoli and smooth nuclear contours. (B) Superficial urothelial cells (umbrella cells) ...
    • Urothelial cells - basal, intermediate, superficial (umbrella) cells
    • Squamous cells - trigone, distal urethra, vagina, squamous metaplasia
  • Other cells in urine:
    • Glandular cells - prostate, endometrium, cystitis glandularis, paraurethra
    • Renal tubular cells
    • Lymphocytes, leukocytes, RBC's
    • Seminal vesicle cells (Figure 2)
      Figure 2
      Seminal vesicle cells may occasionally be seen in urine. The cytoplasm may have a golden-brown pigment. Nuclei tend to be hyperchromatic with degenerative chromatin. Note the associated spermatozoa. (Papanicolaou stain, 600×)
      • Sporadically seen in urine specimens of older patients
      • Bizarre appearance with greatly enlarged nuclei and foamy fragmented cytoplasm
      • Golden-brown lipofuscin pigment
      • Spermatozoa accompany cells
      • Abnormal DNA ploidy
        -Eosinophilic inclusion bodies - giant lysosomes
      • Hyaline inclusion bodies
  • Non-cellular components:
    • Crystals, casts, spermatozoa, corpora amylacea, mucus, fibrin, lubricant, pollen, and rarely Alternaria spp. and microconidia

Urine sample processing

Sample fixation: The urine specimen should be processed immediately or refrigerated at 4 degrees Fahrenheit for no longer than 24 hours. If a delay of greater than 24 hours is anticipated, the specimen should be fixed with an equal volume of 50% ethanol, or the specimen should be centrifuged and the sediment mixed with an ethanol-based fixative for liquid-based cytology or with 50% isopropyl alcohol or denatured alcohol. Low pH appears to favor preservation of urothelial cells.

Specimen processing: Specimens should be processed by cytocentrifugation or by a liquid-based preparation. Fifty milliliters of specimen are transferred to individual centrifuge tubes and spun down at 10 min / 1500 rpm. The supernatant is aspirated off and the sediment is resuspended in a balanced salt solution. Most commonly used slide preparation methods include cytocentrifugation and SurePath or Thin-Prep liquid-based techniques. The traditional membrane filter technique is rarely used currently.

Specimen adequacy

Unlike cervical specimens, exact adequacy guidelines for urine specimens have not been established. In general, slides should contain at least fifteen well-visualized basal and intermediate cells to be classified as adequate. Specimens with abnormal cells are by definition satisfactory.

Diagnostic format and categories for urine cytology specimens

The format shown in Box 2 is recommended for urinary cytology diagnosis.

Box 2. Diagnostic format and category recommendations for urine cytology specimens:

Adequacy Statement (optional)

Satisfactory for evaluation

List any quality factor affecting specimen

Unsatisfactory for evaluation (give reason)

General Categories

Negative for epithelial cell abnormality (see descriptive diagnosis)

Epithelial cell abnormality present (see descriptive diagnosis)

Negative for epithelial cell abnormality:

Infectious agents

Bacterial organisms

Fungal organisms

Viral changes (CMV, herpes, polyomavirus, adenovirus)

Nonspecific inflammatory changes

Acute inflammation

Chronic inflammation

Changes consistent with xanthogranulomatous pyelonephritis

Cellular changes associated with:

Chemotherapy/radiation

Epithelial cell abnormality present:

Atypical urothelial cells (further comment - optional)

-Favor reactive

-Favor urothelial carcinoma

Suspicious for urothelial carcinoma

Low-grade urothelial tumor versus hyperplasia

High-grade urothelial carcinoma

(including invasive carcinoma vs. carcinoma in situ)

Atypical squamous cells

-NOS

-HPV related changes/condyloma

-Squamous cell carcinoma

Atypical glandular cells

-NOS

-Adenocarcinoma, NOS

-Adenocarcinoma of prostate

Other malignant neoplasms (specify type)

Other (Specify)

For samples that are negative for an epithelial cell abnormality, the type of inflammation (acute versus chronic mixed), if present, should be characterized since the information may help the urologist to determine potentially treatable conditions for the patient's urological symptoms. The presence of specific organisms, if observed, should be specified as well. Figure 3A shows polyoma virus-infected cells. Occasionally SV40 immunocytochemistry may be added to confirm the diagnosis (Figure 3B).

Figure 3
Polyoma virus-infected cells. (A) Infected cells have an increased nuclear to cytoplasmic ratio mimicking carcinoma in situ (“decoy cells”), a ground glass nucleus with marginated chromatin, and occasionally short cytoplasmic tails (“comet ...

Therapeutic changes are the major source of ”true” false positive diagnoses. However, as a general rule of thumb, therapeutic changes are characterized by large cells with abundant cytoplasm, low nuclear-to-cytoplasmic ratio, and smudgy nuclear chromatin (Figures 4A-C). Cellular-based markers, especially uCyt+ (ImmunoCyt)™, may be useful in assistingthe evaluation.

Figure 4
Urothelial cells with therapeutic changes. Bacille Calmette-Guerin (BCG), an attenuated bovine mycobacterium, is used to treat carcinoma in situ. It also induces an inflammatory reaction, especially submucosal granulomas, and urothelial atypia. Urine ...

Needless to say, the category of so-called “atypical urothelial cells” is probably the most controversial diagnosis in terms of patient management. In our experience, about 20 to 25% of all samples will be signed out as “urothelial cell atypia” (this does not include the “suspicious for malignancy” category - unpublished data). This category will probably benefit the most from marker analysis. As discussed below, we have applied marker analysis (mainly uCyt+ and sometimes cytokeratin 20 immuno-cytochemistry) as a reflex test for cases signed out as urothelial cell atypia.

As discussed before, urine samples for low grade lesions (papilloma, borderline, and low grade urothelial carcinoma) are more cellular than normal, with cohesive or papillary fragments and subtle morphologic changes that overlap greatly with hyperplastic urothelial lesions (Figures 5A-C). Therefore, these lesions are grouped together.

Figure 5
Low grade urothelial neoplasia versus hyperplasia. The distinction may be difficult as both entities form papillary clusters with cellular crowding. The nuclear changes in low grade neoplasms (increased nuclear to cytoplasmic ratio, irregular nuclear ...

High grade tumor (including carcinoma in situ) usually shows many atypical single cells and loosely cohesive groups, many of them degenerated. The background may be necrotic, bloody, inflammatory, or clean. It is important to find viable cells and carefully evaluate the nuclear-to-cytoplasmic ratio, nuclear membrane, and chro-matin. Examples of high grade urothelial neoplasms are shown in Figures 6A-C.

Figure 6
High grade urothelial carcinoma. The cytologic changes are more apparent in high grade lesions and include a very high nuclear to cytoplasmic ratio, dark coarse chromatin, irregular nuclear borders, and occasional prominent nucleoli seen in large, often ...

Squamous cell lesions may be seen either with or without human papilloma virus (HPV) effect. The HPV-related changes are relatively rare in specimens from male patients and more common in female patients, mostly due to contamination from the gynecologic tract (Figure 7). Squamous cell carcinoma (Figure 8) in the United States is mostly associated with diverticulitis while elsewhere, especially in Egypt, it is associated with schistosomiasis.

Figure 7
HPV changes of squamous cells in urine. The changes may be seen as primary infection of squamous cells of the urinary tract (for example, urothelial cells with squamous metaplasia or squamous cells lining the distal urethra) or in female urine, as contaminations ...
Figure 8
Squamous cell carcinoma. Malignant cells have abundant dense cytoplasm that can be intensely orangeophilic. Nuclei are large and hyper-chromatic with irregular nuclear borders. Note the granular, necrotic background. (Papanicolaou stain, 400×) ...

Adenocarcinoma can be either primary (mostly derived from glandular metaplasia or from urachal remnants) or metastatic. Considering the high incidence of prostate carcinoma, it is rare to see the shedding of prostate cancer cells, characterized by loosely cohesive or single oval-to-low columnar cells with amphophilic cytoplasm and prominent nucleoli into urine (Figure 9). This is because most prostate cancers arise from the peripheral zone. Only a large tumor that extends to the urethra or a primary central zone tumor (the so-called ductal/ endometrioid type of prostate carcinoma) may shed into urine. Renal cell carcinoma may also be seen in voided urine, although this is uncommon (Figure 10). For mucinous tumors, it may be impossible to distinguish between a tumor of primary urothelial origin, urachal origin, or metastatic from the gastrointestinal tract based solely on cytology alone or even on a small biopsy. Currently no specific markers exist to make this distinction, and clinical or radiologic correlation is the only way to determine the origin of the tumor.

Figure 9
Prostate adenocarcinoma. Sheets or small aggregates of uniform glandular cells have large round nuclei, open chromatin, and prominent single or double nucleoli. (Papanicolaou stain, 600×)
Figure 10
Renal cell carcinoma. Renal cell carcinoma cells rarely shed into urine. This air-dried smear demonstrates the abundant, vacuolated cytoplasm characteristic of renal cell carcinoma. Atypical, centrally-placed nuclei are also noted. (Diff-Quik stain, 600×) ...

Potential sources of misdiagnosis

While it is uncommon to have a false positive urine cytology, especially in the case of low grade lesions, occasionally one may be encountered. Potential causes include the so-called “false” false positive diagnosis, instrumentation effect (Figure 11), stones, therapeutic changes, including chemotherapy and radiation, and viral infection (e.g., polyoma or CMV). The so-called “false” false positive finding is a positive urine cytology with lesions that are not detected clinically at the time of urine examination. Most commonly this is a flat dysplastic or malignant lesion or occasionally an upper tract lesion that may be missed by cystoscopy and biopsy. There has been no specific study to determine the exact proportion of such a lesion in urine cytology.

Figure 11
Instrumentation effect. Bladder washing or voided urine after cystoscopy will contain tissue fragments with smaller basal cells surrounded by large binucleated umbrella cells. Note that each cell has a distinct outline and that the tissue fragment boundaries ...

Adjunct markers to urine cytology

Due to its ease of accessibility, the bladder represents an ideal model for studies in risk assessment, early detection, and the investigation of biomarkers. The ideal biomarker should be noninvasive, provide rapid results, be easy to interpret with little or no variability amongst users, be cost-effective, and most importantly, have a high sensitivity and specificity [3, 4]. Potential roadblocks in identifying the ideal marker include the need to obtain consistent samples, to standardize methods of fixation, to assure quality control of assay methods, and to optimize interpretation of the data in the context of the clinical question at hand [8]. The selection of a biomarker depends on whether the objective is prevention, screening, surveillance, or predicting the biological behavior (i.e., risk of progression) of the neoplasm [8].

We will briefly highlight markers that are currently available or under investigation for the detection and monitoring/surveillance of bladder cancer (Tables 1 and and2).2). As such, markers that are useful in predicting recurrence are beyond the scope of this review, although many of the markers herein discussed will cross over into the other categories. We will first briefly discuss markers currently used in clinical practice, some of which have been approved by the Federal Drug Administration (FDA). We will follow with a preview of markers that are more investigational but may potentially be integrated into clinical practice in the near future.

Table 1
Adjunct markers for urine cytology
Table 2
Strip-based Adjunct Markers for Urine Cytology (all FDA-approved)

Nuclear matrix protein 22 (NMP-22) - FDA approved

Nuclear matrix proteins (NMP) consist of a three -dimensional web of RNA and proteins that supports the nuclear shape, organizes DNA, and coordinates DNA replication, transcription, and gene expression [3, 13]. NMP-22 is released from the nuclei of tumor cells during apoptosis. NMP released into the urine may be detected by an FDA-approved NMP-22[14] enzyme-linked assay kit (Matritech, Newton, Mass).

NMP-22 is a 238-kDa protein that may be detected at up to 25-fold greater concentration in tumor than normal urothelium [15, 16]. The enzyme-linked immunoassay uses two monoclonal antibodies to measure the levels of complexed and fragmented forms of the mitotic apparatus in urine [15]. A cut-off of 10 u/ml is endorsed by the manufacturer and initial study for recurrence [17]; however, there is no universally accepted cut-off point. Other studies have suggested cut-offs ranging from 5 to 20 u/ml [17-21].

Some suggest NMP22 may be useful as a screening tool [22]. Surveillance studies either alone or as an adjunct to cytology have estimated sensitivities ranging from 32-100% and specificities from 56-95% [23]. In a 50 study meta-analysis, Lotan et al reported a median sensitivity and specificity of 73% and 80% respectively, superior to a voided urine cytology sensitivity of 34% [24]. Major sources of false positivity are hematuria and pyuria [25]. This is a serious problem since many benign urologic conditions such as stone disease and infection present with hematuria [26]. In general, NMP-22 has a higher sensitivity than cytology, especially in detecting low grade and low stage tumors.

Multiple studies have evaluated the usefulness of NMP-22 as a marker of tumor recurrence. Soloway et al used NMP-22 to predict the likelihood of recurrence after transurethral resection at subsequent cystoscopy in ninety follow-up patients [17]. Levels less than 10 U/mL were predictive of a low likelihood of recurrence while levels greater than 10 U/mL were predictive of recurrence (overall sensitivity of 69.7% and specificity of 78.5%). Subsequent studies, however, were less impressive. Boman et al reported a sensitivity and specificity of 45% and 65% respectively [27]. Miyanaga et al reported a sensitivity and specificity of 18.6% and 85.1% respectively. Both studies concluded that the low sensitivity was due to the small size of recurrent tumors [18].

A new point-of-care test for NMP22 (Bladder-Chek test) was shown to have sensitivities ranging from 50-85% and specificities ranging from 40-90% [28-32]. Advantages include on-site testing with immediately available qualitative results, making the test an attractive adjunct for cystoscopy.

Overall analysis of the data shows that the NMP-22 test has superior sensitivity over cytology for detection of low grade bladder cancers and may be used to predict increased recurrence risk in patients with elevated levels after transurethral resections. Because of the low specificity, using NMP-22 routinely as a primary detector of bladder cancer is not recommended. The specificity, however, can be improved if patients with benign inflammatory conditions (infections, etc), renal or bladder calculi, foreign bodies (stents or nephrostomy tubes), bowel interposition, other genitourinary cancer, and/or instrumentation are excluded [25, 33].

Bladder tumor antigen (BTA) -FDA approved

The term BTA actually describes three separate tests: 1) BTA, 2) BTA stat, and 3) BTA TRAK. Since the BTA tests depend on the disruption of basement membrane, their sensitivity improves with more invasive cancer [34]. Advantages include increased sensitivity for invasive tumors. Disadvantages include a high rate of false-positive readings secondary to patients with inflammatory conditions secondary to benign prostatic hypertrophy (BPH) and a low overall sensitivity for detection of all bladder tumors.

The original BTA test was a latex-agglutination test that measured levels of basement protein antigen released into urine as a result of tumor invading into the stroma [35]. In a review of over 1000 patients (seven series), the sensitivity of the original BTA test was only 52.3%, while the specificity was 84.6% [20] [36-41].

BTA stat and BTA TRAK detect human complement factor H-related protein (hCFH) which is produced and secreted by several bladder and renal cancer cell lines. The qualitative BTA stat test costs only five dollars and is easily performed in the office with a dipstick format [42]. The overall sensitivity ranges from 9.3% to as high as 89% with higher sensitivity in higher grade tumors [43-50]. The specificity of the BTA stat among healthy individuals is greater than 90%. However, it has low specificity (about 50%) among patients with urinary tract infections, urinary calculi (90% positive using BTA stat [51]), nephritis, renal stones, cystitis, BPH, he-maturia, and 2+ to 3+ protein on urine dip stick [49, 52-54]. The low specificity in these conditions is secondary to the test's ability to detect both complement factor H-related protein and complement factor H. Complement factor H is present in human serum at high concentrations and therefore BTA-Stat testing may be falsely positive in benign, hematuria-causingconditions [13].

BTA TRAK is a quantitative test that has a slightly improved sensitivity over its two BTA predecessors [55-57] but has high false positive rates for similar reasons (e.g., inflammation and trauma) which therefore leads to low specificity [57-59]. Moreover, multi-center studies and cohort studies have shown that the sensitivity of the BTA TRAK also varies depending upon the cut-off limit used for the test [57, 59-62].

Overall, the three BTA tests lead to an improved sensitivity compared to cytology but lower specificity due to high false positive rates associated with recent instrumentation, stones, inflammatory conditions, BPH, and hematuria.

Fibrin-fibrinogen degradation products (FDP) - FDA approved

Since bladder tumor cells induce vascular permeability, cellular proteins such as plasminogen and fibrinogen leak into the urine. Urokinase subsequently converts plasminogen into plas-min which then converts fibrinogen into fibrin-fibrinogen products (FDP) [63]. Thus, patients with bladder cancer may have increased levels of FDP in their urine. In a review of four series, the sensitivity and specificity of the AccuDx-FDP assay ranged from 68-83% and 68-100%, respectively [38, 64-66]. Advantages include high-yield with invasive tumors presumably because of increased leakage of FDP. Disadvantages include poor sensitivities for low-grade disease and poor specificities due to reasons previously mentioned in association with BTA tests. The test costs about fifteen dollars and takes less than 10 minutes to complete [42]. However, the assay is currently not being produced due to issues regarding test formulation [4].

uCyt+ (ImmunoCyt)™ and tumor associated antigens -FDA approved

uCyt+ (ImmunoCyt)™ is based on the detection of tumor-associated antigens, mostly mucingly-coproteins, in transitional or urothelial carcinoma using monoclonal antibodies. The uCyt+™ assay is the most frequently used immunocytological test today. Three antibodies, fluorescein-labeled M344 and LDQ10 (directed against sulfated mucinglycoproteins), and Texas -red linked antibody 19A211 (directed against glycosylated forms of high molecular carcinomaembryonic antigens, i.e., CEA) are used.

Many studies have evaluated the performance of the test since 1997. Using a threshold of any single cell positive as positive, uCyt+™ has a sensitivity and specificity ranging from 67-100% and 62-84% [67-76] for all tumors and is a promising diagnostic marker for bladder cancer. Since one of the antibodies, M344, appears to be quite sensitive for low grade tumor cells, this test offers an important advantage for detecting low grade tumors. Indeed, a recent split sample study comparing cytology, uCyt+™, and UroVysion™ in 100 urine samples collected from 100 bladder cancer patients (monitoring population) showed that the sensitivity of uCyt+™ outperforms cytology and UroVysion™ for detecting bladder cancer, especially in low grade tumors [77]. The sensitivities of all tumors for UCyt+™, cytology, and UroVysion™ were 76%, 21%, and 13%, respectively. The drawback of the test, however, is low specificity compared to cytology or UroVysion™ (63% for UCyt+™ versus 97% for cytology and 90% for UroVysion™). Piaton et al [78] demonstrated that patients who had a positive UCyt+™ but negative cystoscopy (so called “false” positive) had a much higher risk of tumor detection within a 12 month-follow up (Table 3). The findings suggest that many of these so-called “false” positive samples may actually be “false” false positive, i.e., the test detects early lesions, or lesions that may not be seen by cystoscopy at the time of examination. Figure 12 shows UCyt+™ staining in a urine sample that had atypical urine cytology and subsequent low grade tumor on cytology six months later. Exactly how many of the false positives belong to the “false” false positive category versus the true false positive category due to benign conditions such as urinary tract obstruction is not clear.

Figure 12
UCyt+™ (ImmunoCyt) in a sample with atypical urine cytology. Fluorescein- and Texas-red-labeled antibodies detect tumor-associated antigens in urothelial carcinoma. Positive tumor cells are both green and red under fluorescence microscopy. (400×) ...
Table 3
Tumor Recurrence in Patients with Negative Cystoscopy [78]

In addition to a relatively high sensitivity in detecting malignancy, uCyt+™ is technically simple (about a 30 minute incubation on either Thin-Prep or filtered slides) and relatively inexpensive. However, the disadvantage is the false positive result occasionally seen associated with urinary tract obstruction due to either stones or BPH. Also, the interpretation of the test using fluorescence microscopy may be difficult for many cytologists, since determination of a positive cell may not be as easy as one might expect in some cases. Further, because the antigens detected are mucinglycoproteins or glycosylated CEA, colonic mucosa cells are positive for the test. As such, the test is not suitable for loop or neobladder urine samples. Another important point worth noting is that the FDA approved this test with a threshold of any single cell positive as positive. However, in our experience (data not shown), and perhaps as expected, samples that have less than or equal to 5 cells positive. Thus, in our report, we not only provide the overall positive or negative finding of the test, but also report samples with 0-5 cells positive as borderline and specify the number of fluorescein- or Texas-red-labeled positive cells.

Multi-target multicolor FISH assay (UroVysion™ test) - FDA approved

Urothelial carcinomas have a number of associated cytogenetic abnormalities involving chromosomes 1, 3, 4, 7, 8, 9 11, 17, etc.[79-81]. These chromosomal abnormalities can be detected by fluorescent in situ hybridization (FISH) using DNA probes to chromosome centromeres or unique loci that are altered in tumor cells. Hybridization is detected by fluorescent microscopy. By utilizing multicolored probes (i.e., different DNA probes labeled with different fluorescent dyes), sensitivity is improved compared to using a single probe.

The UroVysion™ test is a multi-target, multicolored FISH assay that utilizes peri-centromeric fluorescent probes for chromosomes 3, 7, 17, and a locus-specific probe to the 9p21 (p16 locus) region. Exfoliated cells from urine specimens are fixed into 12-well slides and incubated with denatured Chromosome Enumeration Probe (CEP) 3 (spectrum red), CEP7 (spectrum green), CEP 17 (spectrum aqua), and Locus Specific Identifier (LSI) 9p21 (spectrum gold). The slides are counterstained and observed under a fluorescent microscope (UroVysion™/Abbott Laboratories). Suggested criteria for a positive assay include finding 5 or more urinary cells with gains of 2 or more chromosomes, or 10 or more cells with gain of a single chromosome (e.g. trisomy 7). Also homozygous detection of 9p21 locus in greater than 20% of epithelial cells is considered a positive test [48, 82]. However, consensus criteria for a positive FISH test have not been determined, and the studies evaluating the sensitivity and the specificity of UroVysion™ utilize varying criteria for positivity.

Initial case-control cohort studies showed that the sensitivity of UroVysion™ to detect bladder cancer was 81-84% [83, 84]. In more recent studies, the sensitivity ranged from 30-86% [85-88]. The assay has increased sensitivity for detecting higher grade and higher stage tumors, however, the sensitivity for detecting low grade tumors is not clear. Low grade tumors are the most difficult to diagnose by cytology. In fact, our split-sample study of 100 bladder cancer monitoring urine samples showed that the sensitivity for UroVysion™ test is substantially lower compared to the uCyt+™ test (13% versus 76%, respectively) [77]. The specificity for UroVysion™, however, is high in our study (90%, similar to cytology) and varies between 75% and 100% by others [48, 85, 89-94]. Notably, the test appears to have high specificity among patients who have a variety of benign genitourinary conditions, including microhematuria, BPH, infections, and inflammation [48, 84, 91].

Some studies also suggest that UroVysion™ can predict recurrence. Skacel et al in a retrospective cohort study reported that 8 out of 9 FISH positive patients with atypical cytology but negative biopsy had biopsy proven bladder cancer within 12 months [95]. In another study, Buben-drof et a l reported that 4 of 5 so-called “false-positive” UroVysion™ tests had recurrence within 8 months; none of the true negative cases recurred within 18 months. However, the criteria used for a positive test in this study differed from those suggested by the manufacturer. Moreover, the authors concluded that not all FISH aberrations were equally important [96].

In summary, UroVysion™ seems to have high specificity for the detection of bladder cancer and for the ability to detect bladder tumor recurrence prior to clinical detection. Thus, it may be used as a confirmatory test for either cytology or uCyt+™ test. One major limitation to the assay is the lack of consensus on the criteria used to evaluate abnormal cells. Additionally, the test has relatively low sensitivity in the detection of low-grade bladder tumors as discussed before and therefore may not improve the sensitivity as an adjunct for cytomorphologic analysis.

BLCA-4

Konety and Getzenberg have described several specific nuclear matrix proteins which are present only in patients with bladder cancer (BLCA 1-6) and three proteins that are present in normal bladder tissue (BLNL 1-3) [97]. One of these markers, BLCA-4, is found throughout the bladder in patients with bladder cancer, including both tumor and normal regions. The marker is hypothesized to reflect a type of “field effect”, which has been described by several investigators at the genetic level. Studies by Getzenberg et al have shown that this marker appears to be a transcriptional regulator that may play a role in regulating gene expression in bladder cancer [98]. In initial studies using an indirect enzyme linked immunosorbent assay (ELISA), they report that BLCA-4 levels were significantly higher than those found in normal controls and that 53 of 55 (96% sensitivity) samples had BLCA-4 expression [99]. Subsequent trials utilizing a sandwich-based immunoassay examined BLCA-4 expression in a variety of patients including those with biopsy proven bladder cancer, benign urologic conditions, prostate cancer, and normal individuals. The results of this trial demonstrated a sensitivity of 89% and a specificity of 100% [100].

Similar proteins, namely BLCA-1, have also been found to be potentially useful. Unlike BLCA-4, BLCA-1 is expressed in tumor areas only and is not seen in adjacent normal tissue or tissue from normal individuals. An immunoassay detecting BLCA-1 in urine samples has been developed and demonstrates relatively high sensitivity and specificity [101]. However, additional independent studies will be needed to validate the findings.

Telomerase

Telomeres are nucleotide sequences on the ends of chromosomes that are important in maintaining the integrity of DNA. With each replication cycle, a portion of the telomere is lost, and complete loss of telomeres is associated with cell death. Telomerase is an enzyme that lengthens telomeres; thus, increased levels of telomerase allow tumor cells to maintain immortality [102].

The telomeric repeat amplification protocol (TRAP) assay is a polymerase chain reaction (PCR)-based test that detects increased levels of telomerase secreted into the urine by bladder cancer cells. Other telomerase assays are available that detect human telomerase reverse transcriptase and its RNA component. However, for sake of brevity, discussion will be limited to the TRAP assay. It detects telomerase reaction products in vitro, has a 10 hour turnaround time, and costs around seventeen dollars [4, 102]. Generally, the TRAP assay has better sensitivity than cytology with slightly lower specificity [20, 103]. Recent studies show the sensitivity to range from 70-90% at a 50 arbitrary enzymatic unit cutoff value. Specificity is slightly lower, ranging from 66-88% [13, 20, 104-107]. The lower specificity may be explained by contamination of benign cells with telomerase activity (e.g., lymphocytes). The sensitivity was slightly increased with bladder washings compared to voided urine [103]. In detecting recurrent tumors, however, TRAP has a low sensitivity (35%) [108].

Difficulties associated with the telomerase test have limited its widespread use. Urine must be processed within a 24 hour period [13]. At least 50 cells must express telomerase for the assay to detect telomerase reliably [13, 109]. Finally, false negative results may occur depending on sample collection, processing, and the presence of PCR inhibitors or ribonucleases [110]. Currently the TRAP assay is not recommended in the clinical setting because of complicated laboratory procedures and the lack of standardized sample processing to reduce false positive and false negative results.

Cytokeratins

Cytokeratins (CK) make up a large component of intermediate filaments that are found in epithelial cells [111]. Twenty cytokeratins have been identified in human cells, and their expression varies depending on epithelial cell type and state of differentiation [112]. Expression of cytokeratins 8, 18, 19, and 20 has been evaluated as potential bladder cancer markers.

UBC-Rapid and UBC-ELISA tests (manufactured by IDL Biotech, Börlabger Sweden) detect the presence of cytokeratin 8 and 18 in the urine of bladder cancer patients. UBC-Rapid is a point-of-care test, and UBC-ELISA is a 2-hour sandwich ELISA test. Several studies show that the sensitivity of the UBC tests to detect both primary and recurrent bladder cancers varies from 12-79% with a specificity ranging from 63-97% [46, 57, 113-121]. Several studies have also reported lower sensitivities for the detection of low grade and low stage tumors. The sensitivity of UBC to detect grade 1, 2, and 3 bladder tumors is 13-60%, 42-79%, and 35-75% respectively [114, 116, 117, 122]. Retrospective studies report a 21-25% sensitivity of UBC- Rapid to detect stage Ta tumors and carcinoma in situ (CIS) and therefore it has insufficient diagnostic value for detecting superficial bladder cancer [46, 122]. Compared to other bladder tumor markers and cytology, UBC tests have generally lower sensitivity.

The expression of cytokeratin 20 is restricted to the superficial and occasionally the intermediate cells of the normal urothelium, but not the basal cells. Aberrant cytokeratin 20 (CK 20) expression is seen in bladder cancer cells [112]. Reverse transcription (RT)-PCR assays have been used to evaluate CK 20 expression in urine samples. Several studies have shown that CK 20 RT-PCR has a 78-87% sensitivity for detecting bladder cancer in urine. The specificity of CK 20 RT-PCR ranges from 55.7% to 98% [111, 123-129]. CK 20 RT-PCR on blood specimens has also been studied for early detection of systemic bladder cancer progression [126]. Overall, 17-29% of bladder cancer patients were positive for CK 20 RT-PCR [130, 131]. The high sensitivity intrinsic to the RT-PCR methodology may also be associated with low specificity [125].

Cytokeratin 20 immunocytochemistry has also been evaluated as an adjunctive marker for atypical cytology. Klein etal reported a sensitivity of 91% and a specificity of 67% in a study of 87 patients [132]. Specimens with false-positive results had cytology consistent with premalignant conditions such as atypia, hyperplasia, or metaplasia [111, 132]. All completely healthy patients had negative CK 20 levels. Lin et al showed that overall sensitivity and specificity of CK20 immunocytochemistry for the detection of urothelial carcinoma were 94.4% and 80.5% respectively [133]. This study demonstrated that CK20 is a useful adjunct marker for urine cytology, that is, analysis of CK20 can be conveniently performed on the same slide after routine morphological evaluation and be used to triage atypical urine cytology into low and high risk categories for clinical follow-up. Golijanin et al also reported a high sensitivity (82%) for CK 20 immunocytochemistry in patients with microhematuria and those with bladder cancer. The specificity in this study was 76% [134]. Although overall sensitivity was high, the Golijanin study also showed that the sensitivity varied depending on tumor grade, with only 56.5% sensitivity for grade 1 tumors. CK 20 staining had a much higher sensitivity with grade 2 and 3 bladder tumors (93% and 92% respectively) [134]. More recent studies have supported these findings, demonstrating sensitivities ranging from 65-86%, specificities from 86-100%, and advantages over urine cytology in the detection of primary, recurrent, stage pT1 and grade 2/3 tumors [135-137]. One of the pitfalls of the CK20 immunocytochemical staining is that often benign umbrella cells are positive. Thus, in our practice, we only use CK20 in samples containing few small basaloid cells and to distinguish whether these cells represent normal basal cells (negative CK20) versus dysplastic or malignant cells (positive CK20).

Cytokeratin 19 (CK 19) is expressed in normal urothelium. CYFRA 21-1 is a soluble fragment of CK 19 that can be measured in the urine when urothelial cells are exfoliated and lysed. There are two commercially available tests that can measure CYFRA 21-1; one is a solid phase sandwich immunoradiometric assay (Cis Bio International, Gif-sur-Yvette, France) and the other is an electrochemiluminescent immunoas-say with the Elecsys 2010 system (Roche Diagnostics). CK 19 levels are measured after urinary creatinine is normalized [138]. A retrospective cohort study showed that CYFRA 21-1 levels are increased in bladder cancer patients when compared to patients with other urologic conditions and normal controls. The level of CYFRA 21-1 in patients with bladder cancer, patients with other urologic conditions, and normal controls were 154.4 ng/mL, 22.3 ng/mL, and 2.4 ng/mL respectively [139]. When a cutoff level of 4 ng/mL was applied, the sensitivity and specificity of CYFRA 21-1 for detection of bladder cancer were 96.9% and 67.2% respectively. The low specificity was attributed to high CK 19 levels in patients with urolithiasis and urinary tract infection. Another study reported the sensitivity and specificity of CYFRA 21-1 to be 75.5% and 71% respectively when using the electro-chemiluminescent assay [140]. This study showed the sensitivity of detecting bladder cancer increased with higher grade tumors (sensitivities to detect grade 1, 2, and 3 tumors were 54.5%, 66.7%, and 88.2% respectively). However, the study also reported a false positive rate of approximately 33% in patients with various urologic conditions including urolithi-asis, stenosis, BPH and urinary tract infections. Subsequent later studies have shown sensitivities ranging from 43-79% and specificities ranging from 68-88% with a 4 ng/mL cut-off [118, 141-143]. Some have reported improved sensitivity over cytology in detecting Grade 1 tumors [141].

In summary, cytokeratin 20 detected by RT-PCR or immunocytochemistry appears to be a useful and simple marker. However the often positive findings of CK20 in normal umbrella cells preclude widespread application of the test as a primary screening tool. Rather, it is better used as a test in specific settings; for example, in samples that contain scant small basaloid cells. The UBC tests appear to have lower sensitivity compared to other tumor markers and currently do not have sufficient diagnostic value for the detection of bladder cancer. Overall, the use of CYFRA 21-1 is promising with some conflicting studies of its benefit over urine cytology.

Hyaluronic acid/Hyaluronidase

Hyaluronic acid (HA) is a glycosaminoglycan that promotes tumor cell adhesion and angiogenesis [13, 144]. Hyaluronidase (HAase) is an enzyme which cleaves HA into fragments; these cleaved fragments then aid tumor growth and propagation by promoting angiogenesis [145, 146]. Initial case control studies measured both HA levels and HAase activity in the urine. The results showed that there was a 2.5 to 6.5-fold increase in HA levels (83% sensitivity and 90.1% specificity) in patients with bladder cancer, regardless of tumor grade [146]. HAase activity levels were also increased 3 to 7-fold in patients with high grade bladder cancers (81.5% sensitivity and 83.8% specificity) [147]. The combination of tests increases the overall sensitivity to 92%.

A prospective study to monitor bladder cancer recurrence showed that the HA-HAase was more sensitive and more accurate than BTA stat (sensitivities of 94% and 61% respectively). The BTA stat test, however, had better specificity than the HA-HAase test (74% and 63% respectively) [49]. Subsequent comparative studies showed that HA-HAase testing had the highest sensitivity in detecting both low grade/low-stage, and high grade/high stage tumors [46, 67]. Passerotti et al compared accuracy of the HA test to UroVysion™, BTA stat, and cytology in a prospective study involving bladder cancer patients with either primary or recurrent tumors. The specificity of the test was determined in patients with a history of bladder cancer not evident at the time of testing and in patients with BPH. HA testing had the highest sensitivity among all the tests (83%) and a slightly higher specificity [148]. Eissa et al examined HAase RNA in urine detected by RT-PCR and found superior sensitivity (90.8%) over cytology (68.9%) and CK 20 (78.1%) with specificities of 93.4%, 98.1% and 80.2%, respectively [149].

In summary, HA-HAase testing is a promising marker for the detection of primary and recurrent bladder tumors. The test has high sensitivity with the ability to detect low grade/low stage and high grade/high stage tumors. The test may be most useful in screening bladder cancer patients for recurrence.

Survivin

Survivin is an inhibitor of apoptosis that extends cell viability in bladder tumors [150, 151]. Survivin is undetectable in most normal adult tissue and correlates with unfavorable disease and shortened overall survival in neuroblastoma, colorectal cancers, and non-small cell lung cancers [152-155]. Gazzaniga etal demonstrated using RT-PCR that Survivin mRNA is expressed in 30% of bladder tumors [156]. Schultz et al found Survivin mRNA expression in 100% of bladder tumors [157].

Smith et al analyzed urine specimens with a polyclonal antibody for Survivin and then validated findings with both western blot and RT-PCR. Survivin was detected in 31 of 31 patients with new onset or recurrent bladder cancer using the polyclonal antibody system and 15 of 15 patients with RT-PCR, giving a sensitivity of 100%. Only 3 of 35 patients with treated bladder cancers and negative cystoscopies tested positive, suggesting Survivin could be used for surveillance. Additional data showed Survivin was negative in 17 healthy volunteers and 30 patients with non-urothelial genitourinary cancers. There were only 4 false positive results amongst the 30 patients with non-neoplastic urinary tract disease, including 3 with bladder abnormalities on cystoscopy and one patient with an elevated PSA. The overall specificity for Survivin was 95% [151]. Other studies have shown that urinary Survivin levels are higher in patients with recurrence of carcinoma compared to those who achieved remission after treatment with BCG or mitomycin C. The sensitivity and specificity for detecting recurrence were 100% and 78% respectively [158]. Finally, urinary assays detecting Survivin mRNA by RT-PCR have shown sensitivities ranging from 53-94% and specificities from 88-100% [159-162].

In short, Survivin could potentially be a valuable marker for both detection and monitoring of bladder cancer, but its validation awaits further testing.

DNA ploidy and S-phase fraction

DNA ploidy and S-phase fractions can be evaluated from urine samples by either flow cytometry, image cytometry (ICM), laser scanning cytometry (LSC), or fluorescence in situ hybridization (FISH) [3]. While an FDA-approved FISH methodology has already been discussed in this article (UroVysion™), other methodologies such as flow cytometry can identify neoplastic cells with increased nuclear size and increased nuclear chromatin ratios and further determine their DNA ploidy (e.g., diploid, tetraploid, or ane-uploid). High grade tumors may be detected by the presence of aneuploidy and a higher percentage of cells in the S phase [3]. Sensitivity for high grade urothelial tumors or carcinoma in situ may reach 90% [3, 163-165]. Because this technique is expensive and requires a large number of cells as well as highly trained personnel, flow cytometry has not gained widespread acceptance.

Image cytometry (ICM), especially a fluorescence-based system, allows the measurement of DNA content in each individual cell, making this an attractive alternative to flow cytometry, which requires a large cell population. Hemstreet's group, using a specific platform called Quantitative Fluorescence Image Analysis (QFIA), demonstrated that single cell-based DNA content analysis (by detecting cells over 5c DNA) is more sensitive than cytology or flow cytometry in detecting low grade tumors [166]. Moreover, such a system allows the analysis of multiple other protein (or potentially DNA)-based markers at the same time. Subsequent studies have shown similar findings [92, 167]. Laser scanning cytometry combines the advantages of flow cytometry and ICM by laser-scanning individual cells to quantify fluorescence [168]. While numerous studies have generally shown that imaging-based DNA content analysis is a useful marker for detecting bladder cancer, the need for expensive instrumentation and careful quality control measures for fluorescence quantification precludes the widespread application of this useful technology.

Microsatellite instability assays

Similar to other malignancies, bladder cancer DNA repair mechanisms may be defective, leading to persistent errors in replication, i.e., genomic instability. Microsatellites are inherited tandem repeat DNA sequences that can be analyzed to detect replication errors [169, 170], also known as microsatellite instability. PCR amplification of these tandem repeat sequences can detect microsatellite instability and loss of heterozygosity (LOH) of tumor suppressor genes. While microsatellite instability tends to be found more frequently in advanced bladder cancers, it can be demonstrated in low grade tumors when more microsatellite markers are used. The methodology requires a substantial number of microsatellite markers to achieve high sensitivity [171-174].

Microsatellite analysis has been used to confirm that low grade papillary urothelial carcinoma has instability and/or loss of chromosome 9 and p16 (MTS1) tumor suppressor gene [169, 175]. Regardless of tumor grade and stage, bladder tumors typically have LOH in the 9p region on microsatellite analysis [169, 176]. Using microsatellite analysis and PCR, Mao et al were able to identify 19 of 20 patients (95% sensitivity) with genetic alterations; however, 2 of 4 samples with inflammatory atypia were also positive [13, 177]. Recent studies by van Rhijn et al showed that activating FGFR3 mutations are detectable in low grade superficial bladder cancers, and, when used in conjunction with microsatellite analysis, the sensitivity for bladder cancer detection increases to 89% (compared to 71% for negative FGFR3 mutations) [178]. Other studies have shown that microsatellite instability may be used to predict recurrence of urothelial carcinoma. The sensitivity of selected studies ranged from 58% to 95% with specificity ranging from 73-100% [169, 177, 179-185] with some studies showing a prediction of recurrence months before positive cystoscopy. However, large-scale analysis will be needed to determine specificity, especially in symptomatic populations, to understand the true clinical utility of the test.

DNA chips (HuSNP chip) can detect alleles differing by a single nucleotide polymorphism. With this technology, LOH can be detected at 1500 different loci at once. Preliminary results in thirty-one patients show LOH at 24 or more loci, demonstrating the ability of chip technology to detect bladder tumors with 100% sensitivity. Nine control subjects and 4 of 5 patients with hematuria had negative chip findings [186].

Although studies to date show that microsatellite analysis has excellent sensitivity and specificity regardless of tumor grade and stage, tumor multiplicity, or previous history of bladder cancer, the studies are based on relatively small sample sizes. Disadvantages include a potential contamination of non-urothelial cells that may cause either false negative or positive findings, long turnaround time, high equipment cost, and need for trained personnel, rendering this test impractical for routine clinical use. Currently, the testing of urine for microsatellite instability is not recommended for monitoring or for detection of primary tumors.

DD23

A monoclonal antibody called DD23 resulted from the immunization of a BALB/c mouse with fresh bladder cancer. The antigen recognized by DD23 is identified in 81% of bladder tumors. Testing for this antigen utilizing Quantitative Fluorescence Image Analysis (QFIA) has an 85% sensitivity and a 95% specificity [13, 187]. When used in combination with cytology, the sensitivity is 94%, and the specificity is 85% [3]. UroCor, Inc., (now part of LabCorp Inc.) licensed the DD23 monoclonal antibody, and the analytic method was converted to an alkaline phosphatase immunohistochemical assay. A prospective study evaluating the utility of DD23 immunohistochemistry showed that the overall sensitivity and specificity of DD23 were 81% and 60%, respectively compared to a sensitivity and specificity of 66% and 85%, respectively with cytology alone. The combination of cytology and DD23 had a sensitivity of 85% and specificity of 55% [188]. Another study showed a sensitivity of 70% and specificity of 60% with improved sensitivity in patients with a prior history of intravesical treatment [189]. Overall, DD23 is a promising monoclonal antibody that can be used in the detection of bladder cancer. Fluorescent assays seem to have better overall sensitivity and specificity when compared to immu-nocytochemistry. When both the fluorescent and the immunocytochemical assays are used in combination with cytology, the sensitivity is increased with a slight decrease in specificity. Further studies are necessary, however, to validate the utility of both methods in larger prospective trials.

Quanticyt nuclear karyometry

Quanticyt is an automated quantitative karyometric cytology system that objectively interprets nuclear features (nuclear shape and DNA content) based on microscopic images. Cytospin preparations of ethanol-polyethylene glycolfixed bladder wash specimens are made. Light microscopy nuclear images are transferred to a computerized image analysis system. Using an internal lymphocyte standard, mean nuclear shape (MPASS) and DNA content (2c deviation index, or 2cDI) are measured. The samples are then stratified into low, intermediate, or high-risk groups [39, 190-194].

Van der Poel et a l reported that Quanticyt test had a 59% sensitivity and a 70% specificity for detecting bladder cancer. Wiener et al reported a sensitivity of 69%. The sensitivity increased for higher grade tumors, with a sensitivity of 85% for grade 3 tumors [39, 195]. Additionally, a 2cDI of ≥ 2.00 was a significant predictor of carcinoma in situ, invasive bladder cancer, and progression [190].

The utility of Quanticyt is somewhat limited by the low sensitivity. In one study by van der Poel et al the rate of finding invasive disease was 10% among individuals classified as high-risk by Quanticyt but only after five consecutive samples were collected from the patient [190]. Other studies suggest that Quanticyt also overestimates the risk for bladder abnormalities, and therefore has a lower specificity than bladder wash cytology and voided urine cytology [87, 195].

In summary, Quanticyt is a potential adjunctive test for risk stratifying patients for bladder cancer. However, the test is limited by its low sensitivity, its need for sophisticated instrumentation and technical expertise, and its potential to overestimate the risk of bladder cancer. At the present time, general applicability of this methodology is restricted.

Prostate Stem Cell Antigen

Recent studies have shown expression of prostate stem cell antigen (PSCA), a glycosylphos-phatidylinosital (GPI)-anchored cell surface antigen, to be increased in human urothelial carcinoma. PSCA expression is detected in more than 80% of local tumors and in 60 to 100% of metastatic tumors [196-198]. In 2003, a study utilizing PSCA immunocytochemistry as an adjunctive marker for urothelial carcinoma in voided urine samples showed that positive stainingwith PSCA had increased sensitivity and specificity for detection of urothelial carcinoma when compared to cytology alone. Cheng et al showed that the sensitivity and specificity of PSCA staining alone were 80% and 85.7% respectively compared to cytology (46.7% sensitivity). Of the false positive cases, one had a history of interstitial cystitis and the other had a history of hematuria. The sensitivity and specificity increased when cytology and PSCA immunocytochemical staining were combined in an either/or situation to 83.3% and 85.7% respectively [199]. Wu et al recently conducted a genome-wide association study on 969 bladder cancer cases and 957 controls and identified a missense variant in the PSCA gene consistently associated with bladder cancer in US and European populations [200].

In summary, it appears that PSCA may be a useful adjunctive marker to urine cytology in the detection of urothelial carcinoma in voided urine samples. Initial studies, however, need to be validated with a larger number of samples and in a wider variety of clinical settings before any definite conclusions can be made.

DNA methylation

CpG dinucleotides are present in the promoters of many genes and may become methylated which in turn inhibits gene expression. Alteration in methylation status is a frequent occurrence in cancer where, for example, methylation inactivates a tumor suppressor gene and results in cancer development. Methylation of the p16/CDKN2A gene in bladder cancer was first described by Gonzalez-Zulueta et al [201] and since then many studies have examined various loci and combination methylation marker panels in urine specimens for the detection of bladder cancer.

Dulaimi et al [202] examined the methylation status of APC, RASSF1A, and p14(ARF) tumor suppressor genes and demonstrated an 87% sensitivity in the urine of 45 tumor patients obtained prior to bladder cancer surgery. Others have examined a combination of panels ranging from 2 to 15 separate gene panels and have reported sensitivities and specificities ranging from 69-92% and 60-100%, respectively [203-207]. Renard et a l [207] recently identified two genes, TWIST1 and NID2, frequently methylated in bladder cancer including early-stage and low grade tumor. The two-gene panel detected bladder cancer in three separate sets of urine specimens (selection, training, and validation sets) for a total of 496 patients with a sensitivity of 90% and specificity of 93%.

Studies demonstrating the use of DNA methylation for screening and surveillance are promising. Further multi-institutional studies to examine the various proposed methylation panels and validate this methodology are required to better understand its applicability to the management of bladder cancer.

Perspective

Cystoscopy in combination with Papanicolaou [15] cytology remains the most effective means of detecting bladder cancer. However, cystoscopy is an invasive procedure, and while cytology remains a useful method for detecting high grade tumors, its utility in detecting low grade tumors remains limited due to the lack of distinguishing cytologic features between low grade disease and reactive processes. The selection of the ideal biomarker depends on whether the goal is detection/screening, monitoring/surveillance, or predicting progression to invasion or metastatic disease. This article has focused on markers that are currently used or are being investigated for detection purposes, keeping in mind that many of the markers can also be used for other objectives.

Most of the current markers in use have higher sensitivities than cytology, especially when used to identify low grade disease. Most of these markers also have lower specificities when compared to cytology. Furthermore, all of these tests must still be utilized in conjunction with cystoscopy findings. Complete elimination of cystoscopy or cytology to detect bladder cancers does not appear feasible, at least in the near future. One or more of these tests may eventually prove to be a useful adjunct for cytology and cystoscopy, but each and every one of the markers awaits further validation. Currently, with all information on hand, the best approach still-seems to be using cytomorphologic analysis as the initial screen test, using uCyt+™ as a reflex test for atypical cytology, and using UroVysion™ as a confirmatory test for either positive cytology or uCyt+™. Whether such an approach would withstand time remains to be seen.

References

1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59:225–249. [PubMed]
2. Lokeshwar VB, Habuchi T, Grossman HB, Murphy WM, Hautmann SH, Hemstreet GP, 3rd, Bono AV, Getzenberg RH, Goebell P, Schmitz-Drager BJ, Schalken JA, Fradet Y, Marberger M, Messing E, Droller MJ. Bladder tumor markers beyond cytology: International Consensus Panel on bladder tumor markers. Urology. 2005;66:35–63. [PubMed]
3. Burchardt M, Burchardt T, Shabsigh A, De La Taille A, Benson MC, Sawczuk I. Current concepts in biomarker technology for bladder cancers. Clin Chem. 2000;46:595–605. [PubMed]
4. Chao D, Freedland SJ, Pantuck AJ, Zisman A, Belldegrun AS. Bladder cancer 2000: molecular markers for the diagnosis of transitional cell carcinoma. Rev Urol. 2001;3:85–93. [PubMed]
5. Badalament RA, Kimmel M, Gay H, Cibas ES, Whitmore WF, Jr, Herr HW, Fair WR, Melamed MR. The sensitivity of flow cytometry compared with conventional cytology in the detection of superficial bladder carcinoma. Cancer. 1987;59:2078–2085. [PubMed]
6. Gregoire M, Fradet Y, Meyer F, Tetu B, Bois R, Bedard G, Charrois R, Naud A. Diagnostic accuracy of urinary cytology, and deoxyribonucleic acid flow cytometry and cytology on bladder washings during followup for bladder tumors. J Urol. 1997;157:1660–1664. [PubMed]
7. Brown FM. Urine cytology. It is still the gold standard for screening? Urol Clin North Am. 2000;27:25–37. [PubMed]
8. Rao JY. Molecular Pathology of Early Cancer. Amsterdam, Netherlands: IOS Press; 1999. Molecular Pathology of Early Bladder Cancer.
9. Marsh GM, Leviton LC, Talbott EO, Callahan C, Pavlock D, Hemstreet G, Logue JN, Fox J, Schulte P. Drake Chemical Workers' Health Registry Study: I. Notification and medical surveillance of a group of workers at high risk of developing bladder cancer. Am J Ind Med. 1991;19:291–301. [PubMed]
10. Iczkowski KA, Katz G, Cascione CJ. Postoperative bladder washing cytology after transure-thral resection. Can it predict the recurrence of urothelial carcinoma? Acta Cytol. 2004;48:380–384. [PubMed]
11. Braun WE. BK polyomavirus: a newly recognized threat to transplanted kidneys. Cleve Clin J Med. 2003;70:1056. 1059–1060, passim 1062. [PubMed]
12. Koss LG, Deitch D, Ramanathan R, Sherman AB. Diagnostic value of cytology of voided urine. Acta Cytol. 1985;29:810–816. [PubMed]
13. Konety BR, Getzenberg RH. Urine based markers of urological malignancy. J Urol. 2001;165:600–611. [PubMed]
14. Pagano F, Bassi P, Galetti TP, Meneghini A, Milani C, Artibani W, Garbeglio A. Results of contemporary radical cystectomy for invasive bladder cancer: a clinicopathological study with an emphasis on the inadequacy of the tumor, nodes and metastases classification. J Urol. 1991;145:45–50. [PubMed]
15. Giannopoulos A, Manousakas T, Mitropoulos D, Botsoli-Stergiou E, Constantinides C, Giannopoulou M, Choremi-Papadopoulou H. Comparative evaluation of the BTAstat test, NMP22, and voided urine cytology in the detection of primary and recurrent bladder tumors. Urology. 2000;55:871–875. [PubMed]
16. Keesee SK, Briggman JV, Thill G, Wu YJ. Utilization of nuclear matrix proteins for cancer diagnosis. Crit Rev Eukaryot Gene Expr. 1996;6:189–214. [PubMed]
17. Soloway MS, Briggman V, Carpinito GA, Chodak GW, Church PA, Lamm DL, Lange P, Messing E, Pasciak RM, Reservitz GB, Rukstalis DB, Sarosdy MF, Stadler WM, Thiel RP, Hayden CL. Use of a new tumor marker, urinary NMP22, in the detection of occult or rapidly recurring transitional cell carcinoma of the urinary tract following surgical treatment. J Urol. 1996;156:363–367. [PubMed]
18. Miyanaga N, Akaza H, Tsukamoto S, Shimazui T, Ohtani M, Ishikawa S, Noguchi R, Manabe F, Nishijima Y, Kikuchi K, Sato K, Hayashi H, Kondo F, Shiraiwa H, Aoyama O. Usefulness of urinary NMP22 to detect tumor recurrence of superficial bladder cancer after transurethral resection. Int J Clin Oncol. 2003;8:369–373. [PubMed]
19. Shariat SF, Marberger MJ, Lotan Y, Sanchez-Carbayo M, Zippe C, Ludecke G, Boman H, Sawczuk I, Friedrich MG, Casella R, Mian C, Eissa S, Akaza H, Serretta V, Huland H, Hedelin H, Raina R, Miyanaga N, Sagalowsky AI, Roehrborn CG, Karakiewicz PI. Variability in the performance of nuclear matrix protein 22 for the detection of bladder cancer. J Urol. 2006;176:919–926. discussion 926. [PubMed]
20. Landman J, Chang Y, Kavaler E, Droller MJ, Liu BC. Sensitivity and specificity of NMP-22, telomerase, and BTA in the detection of human bladder cancer. Urology. 1998;52:398–402. [PubMed]
21. Stampfer DS, Carpinito GA, Rodriguez-Villanueva J, Willsey LW, Dinney CP, Grossman HB, Fritsche HA, McDougal WS. Evaluation of NMP22 in the detection of transitional cell carcinoma of the bladder. J Urol. 1998;159:394–398. [PubMed]
22. Zippe C, Pandrangi L, Agarwal A. NMP22 is a sensitive, cost-effective test in patients at risk for bladder cancer. J Urol. 1999;161:62–65. [PubMed]
23. Nguyen CT, Jones JS. Defining the role of NMP22 in bladder cancer surveillance. World J Urol. 2008;26:51–58. [PubMed]
24. Lotan Y, Roehrborn CG. Sensitivity and specificity of commonly available bladder tumor markers versus cytology: results of a comprehensive literature review and meta-analyses. Urology. 2003;61:109–118. discussion 118. [PubMed]
25. Sharma S, Zippe CD, Pandrangi L, Nelson D, Agarwal A. Exclusion criteria enhance the specificity and positive predictive value of NMP22 and BTA stat. J Urol. 1999;162:53–57. [PubMed]
26. Messing EM, Young TB, Hunt VB, Roecker EB, Vaillancourt AM, Hisgen WJ, Greenberg EB, Kuglitsch ME, Wegenke JD. Home screening for hematuria: results of a multiclinic study. J Urol. 1992;148:289–292. [PubMed]
27. Boman H, Hedelin H, Holmang S. Four bladder tumor markers have a disappointingly low sensitivity for small size and low grade recurrence. J Urol. 2002;167:80–83. [PubMed]
28. Grossman HB, Soloway M, Messing E, Katz G, Stein B, Kassabian V, Shen Y. Surveillance for recurrent bladder cancer using a point-of-care proteomic assay. Jama. 2006;295:299–305. [PubMed]
29. Grossman HB, Messing E, Soloway M, Tomera K, Katz G, Berger Y, Shen Y. Detection of bladder cancer using a point-of-care proteomic assay. Jama. 2005;293:810–816. [PubMed]
30. Tritschler S, Scharf S, Karl A, Tilki D, Knuechel R, Hartmann A, Stief CG, Zaak D. Validation of the diagnostic value of NMP22 BladderChek test as a marker for bladder cancer by photody-namic diagnosis. Eur Urol. 2007;51:403–407. discussion; 407-408. [PubMed]
31. Moonen PM, Kiemeney LA, Witjes JA. Urinary NMP22 BladderChek test in the diagnosis of superficial bladder cancer. Eur Urol. 2005;48:951–956. discussion 956. [PubMed]
32. Kumar A, Kumar R, Gupta NP. Comparison of NMP22 BladderChek test and urine cytology for the detection of recurrent bladder cancer. Jpn J Clin Oncol. 2006;36:172–175. [PubMed]
33. Ponsky LE, Sharma S, Pandrangi L, Kedia S, Nelson D, Agarwal A, Zippe CD. Screening and monitoring for bladder cancer: refining the use of NMP22. J Urol. 2001;166:75–78. [PubMed]
34. Schamhart DH, de Reijke TM, van der Poel HG, Witjes JA, de Boer EC, Kurth K, Schalken JA. The Bard BTA test: its mode of action, sensitivity and specificity, compared to cytology of voided urine, in the diagnosis of superficial bladder cancer. Eur Urol. 1998;34:99–106. [PubMed]
35. Pirtskalaishvili G, Konety BR, Getzenberg RH. Update on urine-based markers for bladder cancer. How sensitive and specific are the new noninvasive tests? Postgrad Med. 1999;106:85–86. 91–84. [PubMed]
36. Sarosdy MF, deVere White RW, Soloway MS, Sheinfeld J, Hudson MA, Schellhammer PF, Jarowenko MV, Adams G, Blumenstein BA. Results of a multicenter trial using the BTA test to monitor for and diagnose recurrent bladder cancer. J Urol. 1995;154:379–383. discussion 383-374. [PubMed]
37. Ianari A, Sternberg CN, Rossetti A, Van Rijn A, Deidda A, Giannarelli D, Pansadoro V. Results of Bard BTA test in monitoring patients with a history of transitional cell cancer of the bladder. Urology. 1997;49:786–789. [PubMed]
38. Johnston B, Morales A, Emerson L, Lundie M. Rapid detection of bladder cancer: a comparative study of point of care tests. J Urol. 1997;158:2098–2101. [PubMed]
39. Van der Poel HG, Van Balken MR, Schamhart DH, Peelen P, de Reijke T, Debruyne FM, Schalken JA, Witjes JA. Bladder wash cytology, quantitative cytology, and the qualitative BTA test in patients with superficial bladder cancer. Urology. 1998;51:44–50. [PubMed]
40. Zimmerman RL, Bagley D, Hawthorne C, Bibbo M. Utility of the Bard BTA test in detecting upper urinary tract transitional cell carcinoma. Urology. 1998;51:956–958. [PubMed]
41. Nasuti JF, Gomella LG, Ismial M, Bibbo M. Utility of the BTA stat test kit for bladder cancer screening. Diagn Cytopathol. 1999;21:27–29. [PubMed]
42. Ramakumar S, Bhuiyan J, Besse JA, Roberts SG, Wollan PC, Blute ML, O'Kane DJ. Comparison of screening methods in the detection of bladder cancer. J Urol. 1999;161:388–394. [PubMed]
43. Gutierrez Banos JL, del Henar Rebollo Rodrigo M, Antolin Juarez FM, Garcia BM. Usefulness of the BTA STAT Test for the diagnosis of bladder cancer. Urology. 2001;57:685–689. [PubMed]
44. Raitanen MP, Marttila T, Kaasinen E, Rintala E, Aine R, Tammela TL. Sensitivity of human complement factor H related protein (BTA stat) test and voided urine cytology in the diagnosis of bladder cancer. J Urol. 2000;163:1689–1692. [PubMed]
45. Raitanen MP, Marttila T, Nurmi M, Ala-Opas M, Nieminen P, Aine R, Tammela TL. Human complement factor H related protein test for monitoring bladder cancer. J Urol. 2001;165:374–377. [PubMed]
46. Schroeder GL, Lorenzo-Gomez MF, Hautmann SH, Friedrich MG, Ekici S, Huland H, Lokeshwar V. A side by side comparison of cytology and biomarkers for bladder cancer detection. J Urol. 2004;172:1123–1126. [PubMed]
47. Glas AS, Roos D, Deutekom M, Zwinderman AH, Bossuyt PM, Kurth KH. Tumor markers in the diagnosis of primary bladder cancer. A systematic review. J Urol. 2003;169:1975–1982. [PubMed]
48. Halling KC, King W, Sokolova IA, Karnes RJ, Meyer RG, Powell EL, Sebo TJ, Cheville JC, Clayton AC, Krajnik KL, Ebert TA, Nelson RE, Burkhardt HM, Ramakumar S, Stewart CS, Pankratz VS, Lieber MM, Blute ML, Zincke H, Seelig SA, Jenkins RB, O'Kane DJ. A comparison of BTA stat, hemoglobin dipstick, telomerase and Vysis UroVysion assays for the detection of urothelial carcinoma in urine. J Urol. 2002;167:2001–2006. [PubMed]
49. Lokeshwar VB, Schroeder GL, Selzer MG, Hautmann SH, Posey JT, Duncan RC, Watson R, Rose L, Markowitz S, Soloway MS. Bladder tumor markers for monitoring recurrence and screening comparison of hyaluronic acid-hyaluronidase and BTA-Stat tests. Cancer. 2002;95:61–72. [PubMed]
50. Raitanen MP. The role of BTA stat Test in followup of patients with bladder cancer: results from FinnBladder studies. World J Urol. 2008;26:45–50. [PubMed]
51. Wald M, Halachmi S, Amiel G, Madjar S, Mullerad M, Miselevitz I, Moskovitz B, Nativ O. Bladder tumor antigen stat test in non-urothelial malignant urologic conditions. Isr Med Assoc J. 2002;4:174–175. [PubMed]
52. Heicappell R, Muller M, Fimmers R, Miller K. Qualitative determination of urinary human complement factor H-related protein (hcfHrp) in patients with bladder cancer, healthy controls, and patients with benign urologic disease. Urol Int. 2000;65:181–184. [PubMed]
53. Serretta V, Pomara G, Rizzo I, Esposito E. Urinary BTA-stat, BTA-trak and NMP22 in surveillance after TUR of recurrent superficial transitional cell carcinoma of the bladder. Eur Urol. 2000;38:419–425. [PubMed]
54. Oge O, Kozaci D, Gemalmaz H. The BTA stat test is nonspecific for hematuria: an experimental hematuria model. J Urol. 2002;167:1318–1319. discussion 1319-1320. [PubMed]
55. Heicappell R, Wettig IC, Schostak M, Muller M, Steiner U, Sauter T, Miller K. Quantitative detection of human complement factor H-related protein in transitional cell carcinoma of the urinary bladder. Eur Urol. 1999;35:81–87. [PubMed]
56. Ellis WJ, Blumenstein BA, Ishak LM, Enfield DL. Clinical evaluation of the BTA TRAK assay and comparison to voided urine cytology and the Bard BTA test in patients with recurrent bladder tumors. The Multi Center Study Group. Urology. 1997;50:882–887. [PubMed]
57. Babjuk M, Soukup V, Pesl M, Kostirova M, Drncova E, Smolova H, Szakacsova M, Getzenberg R, Pavlik I, Dvoracek J. Urinary cytology and quantitative BTA and UBC tests in surveillance of patients with pTapT1 bladder urothelial carcinoma. Urology. 2008;71:718–722. [PubMed]
58. Heicappell R, Schostak M, Muller M, Miller K. Evaluation of urinary bladder cancer antigen as a marker for diagnosis of transitional cell carcinoma of the urinary bladder. Scand J Clin Lab Invest. 2000;60:275–282. [PubMed]
59. Mahnert B, Tauber S, Kriegmair M, Schmitt UM, Hasholzner U, Reiter W, Hofmann K, Schmeller N, Stieber P. BTA-TRAK–a useful diagnostic tool in urinary bladder cancer? Anticancer Res. 1999;19:2615–2619. [PubMed]
60. Chautard D, Daver A, Bocquillon V, Verriele V, Colls P, Bertrand G, Soret JY. Comparison of the Bard Trak test with voided urine cytology in the diagnosis and follow-up of bladder tumors. Eur Urol. 2000;38:686–690. [PubMed]
61. Thomas L, Leyh H, Marberger M, Bombardieri E, Bassi P, Pagano F, Pansadoro V, Sternberg CN, Boccon-Gibod L, Ravery V, Le Guludec D, Meulemans A, Conort P, Ishak L. Multicenter trial of the quantitative BTA TRAK assay in the detection of bladder cancer. Clin Chem. 1999;45:472–477. [PubMed]
62. Khaled HM, Abdel-Salam I, Abdel-Gawad M, Metwally A, El-Demerdash S, El-Didi M, Morsi A, Ishak L. Evaluation of the BTA tests for the detection of bilharzial related bladder cancer: the Cairo experience. Eur Urol. 2001;39:91–94. [PubMed]
63. Tsihlias J, Grossman HB. The utility of fibrin/fibrinogen degradation products in superficial bladder cancer. Urol Clin North Am. 2000;27:39–46. [PubMed]
64. Schmetter BS, Habicht KK, Lamm DL, Morales A, Bander NH, Grossman HB, Hanna MG, Jr, Silberman SR, Butman BT. A multicenter trial evaluation of thefibrin/fibrinogen degradation products test for detection and monitoring of bladder cancer. J Urol. 1997;158:801–805. [PubMed]
65. McCabe RP, Lamm DL, Haspel MV, Pomato N, Smith KO, Thompson E, Hanna MG., Jr A diagnostic-prognostic test for bladder cancer using a monoclonal antibody-based enzyme-linked immunoassay for detection of urinary fibrin(ogen) degradation products. Cancer Res. 1984;44:5886–5893. [PubMed]
66. Topsakal M, Karadeniz T, Anac M, Donmezer S, Besisik A. Assessment of fibrin-fibrinogen degradation products (Accu-Dx) test in bladder cancer patients. Eur Urol. 2001;39:287–291. [PubMed]
67. Hautmann S, Toma M, Lorenzo Gomez MF, Friedrich MG, Jaekel T, Michl U, Schroeder GL, Huland H, Juenemann KP, Lokeshwar VB. Immunocyt and the HA-HAase urine tests for the detection of bladder cancer: a side-by-side comparison. Eur Urol. 2004;46:466–471. [PubMed]
68. Mian C, Pycha A, Wiener H, Haitel A, Lodde M, Marberger M. Immunocyt: a new tool for detecting transitional cell cancer of the urinary tract. J Urol. 1999;161:1486–1489. [PubMed]
69. Olsson H, Zackrisson B. ImmunoCyt a useful method in the follow-up protocol for patients with urinary bladder carcinoma. Scand J Urol Nephrol. 2001;35:280–282. [PubMed]
70. Lodde M, Mian C, Negri G, Berner L, Maffei N, Lusuardi L, Palermo S, Marberger M, Brssner C, Pycha A. Role of uCyt+ in the detection and surveillance of urothelial carcinoma. Urology. 2003;61:243–247. [PubMed]
71. Feil G, Zumbragel A, Paulgen-Nelde HJ, Hennenlotter J, Maurer S, Krause S, Bichler KH, Stenzl A. Accuracy of the ImmunoCyt assay in the diagnosis of transitional cell carcinoma of the urinary bladder. Anticancer Res. 2003;23:963–967. [PubMed]
72. Pfister C, Chautard D, Devonec M, Perrin P, Chopin D, Rischmann P, Bouchot O, Beurton D, Coulange C, Rambeaud JJ. Immunocyt test improves the diagnostic accuracy of urinary cytology: results of a French multicenter study. J Urol. 2003;169:921–924. [PubMed]
73. Tetu B, Tiguert R, Harel F, Fradet Y. ImmunoCyt/uCyt+ improves the sensitivity of urine cytology in patients followed for urothelial carcinoma. Mod Pathol. 2005;18:83–89. [PubMed]
74. Lodde M, Mian C, Comploj E, Palermo S, Longhi E, Marberger M, Pycha A. uCyt+ test: alternative to cystoscopy for less-invasive follow-up of patients with low risk of urothelial carcinoma. Urology. 2006;67:950–954. [PubMed]
75. Mian C, Maier K, Comploj E, Lodde M, Berner L, Lusuardi L, Palermo S, Vittadello F, Pycha A. uCyt+/ImmunoCyt in the detection of recurrent urothelial carcinoma: an update on 1991 analyses. Cancer. 2006;108:60–65. [PubMed]
76. Messing EM, Teot L, Korman H, Underhill E, Barker E, Stork B, Qian J, Bostwick DG. Performance of urine test in patients monitored for recurrence of bladder cancer: a multicenter study in the United States. J Urol. 2005;174:1238–1241. [PubMed]
77. Sullivan PS, Nooraie F, Sanchez H, Hirschowitz S, Levin M, Rao PN, Rao J. Comparison of ImmunoCyt, UroVysion, and urine cytology in detection of recurrent urothelial carcinoma: a “split-sample” study. Cancer Cytopathol. 2009;117:167–173. [PubMed]
78. Piaton E, Daniel L, Verriele V, Dalifard I, Zimmermann U, Renaudin K, Gobet F, Caratero A, Desvaux D, Pouille Y, Seigneurin D. Improved detection of urothelial carcinomas with fluorescence immunocytochemistry (uCyt+assay) and urinary cytology: results of a French Prospective Multicenter Study. Lab Invest. 2003;83:845–852. [PubMed]
79. Junker K, Boerner D, Schulze W, Utting M, Schubert J, Werner W. Analysis of genetic alterations in normal bladder urothelium. Urology. 2003;62:1134–1138. [PubMed]
80. Knowles MA. What we could do now: molecular pathology of bladder cancer. Mol Pathol. 2001;54:215–221. [PMC free article] [PubMed]
81. Cordon-Cardo C, Cote RJ, Sauter G. Genetic and molecular markers of urothelial premalig-nancy and malignancy. Scand J Urol Nephrol Suppl. 2000:82–93. [PubMed]
82. Placer J, Espinet B, Salido M, Sole F, Gelabert-Mas A. Clinical utility of a multiprobe FISH assay in voided urine specimens for the detection of bladder cancer and its recurrences, compared with urinary cytology. Eur Urol. 2002;42:547–552. [PubMed]
83. Halling KC, King W, Sokolova IA, Meyer RG, Burkhardt HM, Halling AC, Cheville JC, Sebo TJ, Ramakumar S, Stewart CS, Pankratz S, O'Kane DJ, Seelig SA, Lieber MM, Jenkins RB. A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma. J Urol. 2000;164:1768–1775. [PubMed]
84. Sokolova IA, Halling KC, Jenkins RB, Burkhardt HM, Meyer RG, Seelig SA, King W. The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine. J Mol Diagn. 2000;2:116–123. [PubMed]
85. Varella-Garcia M, Akduman B, Sunpaweravong P, Di Maria MV, Crawford ED. The UroVysion fluorescence in situ hybridization assay is an effective tool for monitoring recurrence of bladder cancer. Urol Oncol. 2004;22:16–19. [PubMed]
86. Gudjonsson S, Isfoss BL, Hansson K, Domanski AM, Warenholt J, Soller W, Lundberg LM, Liedberg F, Grabe M, Mansson W. The value of the UroVysion assay for surveillance of non-muscle-invasive bladder cancer. Eur Urol. 2008;54:402–408. [PubMed]
87. Moonen PM, Merkx GF, Peelen P, Karthaus HF, Smeets DF, Witjes JA. UroVysion compared with cytology and quantitative cytology in the surveillance of non-muscle-invasive bladder cancer. Eur Urol. 2007;51:1275–1280. discussion 1280. [PubMed]
88. Junker K, Fritsch T, Hartmann A, Schulze W, Schubert J. Multicolor fluorescence in situ hybridization (M-FISH) on cells from urine for the detection of bladder cancer. Cytogenet Genome Res. 2006;114:279–283. [PubMed]
89. Toma MI, Friedrich MG, Hautmann SH, Jakel KT, Erbersdobler A, Hellstern A, Huland H. Comparison of the ImmunoCyt test and urinary cytology with other urine tests in the detection and surveillance of bladder cancer. World J Urol. 2004;22:145–149. [PubMed]
90. Friedrich MG, Toma MI, Hellstern A, Pantel K, Weisenberger DJ, Noldus J, Huland H. Comparison of multitarget fluorescence in situ hybridization in urine with other noninvasive tests for detecting bladder cancer. BJU Int. 2003;92:911–914. [PubMed]
91. Sarosdy MF, Schellhammer P, Bokinsky G, Kahn P, Chao R, Yore L, Zadra J, Burzon D, Osher G, Bridge JA, Anderson S, Johansson SL, Lieber M, Soloway M, Flom K. Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer. J Urol. 2002;168:1950–1954. [PubMed]
92. Dalquen P, Kleiber B, Grilli B, Herzog M, Bubendorf L, Oberholzer M. DNA image cytometry and fluorescence in situ hybridization for noninvasive detection of urothelial tumors in voided urine. Cancer. 2002;96:374–379. [PubMed]
93. Laudadio J, Keane TE, Reeves HM, Savage SJ, Hoda RS, Lage JM, Wolff DJ. Fluorescence in situ hybridization for detecting transitional cell carcinoma: implications for clinical practice. BJU Int. 2005;96:1280–1285. [PubMed]
94. Bergman J, Reznichek RC, Rajfer J. Surveillance of patients with bladder carcinoma using fluorescent in-situ hybridization on bladder washings. BJU Int. 2008;101:26–29. [PubMed]
95. Skacel M, Fahmy M, Brainard JA, Pettay JD, Biscotti CV, Liou LS, Procop GW, Jones JS, Ulchaker J, Zippe CD, Tubbs RR. Multitarget fluorescence in situ hybridization assay detects transitional cell carcinoma in the majority of patients with bladder cancer and atypical or negative urine cytology. J Urol. 2003;169:2101–2105. [PubMed]
96. Bubendorf L, Grilli B, Sauter G, Mihatsch MJ, Gasser TC, Dalquen P. Multiprobe FISH for enhanced detection of bladder cancer in voided urine specimens and bladder washings. Am J Clin Pathol. 2001;116:79–86. [PubMed]
97. Getzenberg RH, Konety BR, Oeler TA, Quigley MM, Hakam A, Becich MJ, Bahnson RR. Bladder cancer-associated nuclear matrix proteins. Cancer Res. 1996;56:1690–1694. [PubMed]
98. Myers-Irvin JM, Van Le TS, Getzenberg RH. Mechanistic analysis of the role of BLCA-4 in bladder cancer pathobiology. Cancer Res. 2005;65:7145–7150. [PubMed]
99. Konety BR, Nguyen TS, Dhir R, Day RS, Becich MJ, Stadler WM, Getzenberg RH. Detection of bladder cancer using a novel nuclear matrix protein, BLCA-4. Clin Cancer Res. 2000;6:2618–2625. [PubMed]
100. Van Le TS, Miller R, Barder T, Babjuk M, Potter DM, Getzenberg RH. Highly specific urine-based marker of bladder cancer. Urology. 2005;66:1256–1260. [PubMed]
101. Myers-Irvin JM, Landsittel D, Getzenberg RH. Use of the novel marker BLCA-1 for the detection of bladder cancer. J Urol. 2005;174:64–68. [PubMed]
102. Orlando C, Gelmini S, Selli C, Pazzagli M. Telomerase in urological malignancy. J Urol. 2001;166:666–673. [PubMed]
103. Kinoshita H, Ogawa O, Kakehi Y, Mishina M, Mitsumori K, Itoh N, Yamada H, Terachi T, Yoshida O. Detection of telomerase activity in exfoliated cells in urine from patients with bladder cancer. J Natl Cancer Inst. 1997;89:724–730. [PubMed]
104. Yoshida K, Sugino T, Tahara H, Woodman A, Bolodeoku J, Nargund V, Fellows G, Goodison S, Tahara E, Tarin D. Telomerase activity in bladder carcinoma and its implication for non-invasive diagnosis by detection of exfoliated cancer cells in urine. Cancer. 1997;79:362–369. [PubMed]
105. Casadio V, Bravaccini S, Gunelli R, Nanni O, Zoli W, Amadori D, Calistri D, Silvestrini R. Accuracy of urine telomerase activity to detect bladder cancer in symptomatic patients. Int J Biol Markers. 2009;24:253–257. [PubMed]
106. Bravaccini S, Sanchini MA, Granato AM, Gunelli R, Nanni O, Amadori D, Calistri D, Silvestrini R. Urine telomerase activity for the detection of bladder cancer in females. J Urol. 2007;178:57–61. [PubMed]
107. Sanchini MA, Gunelli R, Nanni O, Bravaccini S, Fabbri C, Sermasi A, Bercovich E, Ravaioli A, Amadori D, Calistri D. Relevance of urine telomerase in the diagnosis of bladder cancer. JAMA. 2005;294:2052–2056. [PubMed]
108. Dalbagni G, Han W, Zhang ZF, Cordon-Cardo C, Saigo P, Fair WR, Herr H, Kim N, Moore MA. Evaluation of the telomeric repeat amplification protocol (TRAP) assay for telomerase as a diagnostic modality in recurrent bladder cancer. Clin Cancer Res. 1997;3:1593–1598. [PubMed]
109. Kavaler E, Landman J, Chang Y, Droller MJ, Liu BC. Detecting human bladder carcinoma cells in voided urine samples by assaying for the presence of telomerase activity. Cancer. 1998;82:708–714. [PubMed]
110. Liu BC, Loughlin KR. Telomerase in human bladder cancer. Urol Clin North Am. 2000;27:115–123. x. [PubMed]
111. Buchumensky V, Klein A, Zemer R, Kessler OJ, Zimlichman S, Nissenkorn I. Cytokeratin 20: a new marker for early detection of bladder cell carcinoma? J Urol. 1998;160:1971–1974. [PubMed]
112. Southgate J, Harnden P, Trejdosiewicz LK. Cytokeratin expression patterns in normal and malignant urothelium: a review of the biological and diagnostic implications. Histol Histopathol. 1999;14:657–664. [PubMed]
113. Eissa S, Swellam M, el-Mosallamy H, Mourad MS, Hamdy N, Kamel K, Zaglol AS, Khafagy MM, el-Ahmady O. Diagnostic value of urinary molecular markers in bladder cancer. Anticancer Res. 2003;23:4347–4355. [PubMed]
114. Boman H, Hedelin H, Jacobsson S, Holmang S. Newly diagnosed bladder cancer: the relationship of initial symptoms, degree of microhematuria and tumor marker status. J Urol. 2002;168:1955–1959. [PubMed]
115. Babjuk M, Kostirova M, Mudra K, Pecher S, Smolova H, Pecen L, Ibrahim Z, Dvoracek J, Jarolim L, Novak J, Zima T. Qualitative and quantitative detection of urinary human complement factor H-related protein (BTA stat and BTA TRAK) and fragments of cytokeratins 8, 18 (UBC rapid and UBC IRMA) as markers for transitional cell carcinoma of the bladder. Eur Urol. 2002;41:34–39. [PubMed]
116. Sanchez-Carbayo M, Herrero E, Megias J, Mira A, Soria F. Comparative sensitivity of urinary CYFRA 21-1, urinary bladder cancer antigen, tissue polypeptide antigen, tissue polypeptide antigen and NMP22 to detect bladder cancer. J Urol. 1999;162:1951–1956. [PubMed]
117. Mian C, Lodde M, Haitel A, Vigl EE, Marberger M, Pycha A. Comparison of the monoclonal UBC-ELISA test and the NMP22 ELISA test for the detection of urothelial cell carcinoma of the bladder. Urology. 2000;55:223–226. [PubMed]
118. Gkialas I, Papadopoulos G, Iordanidou L, Stathouros G, Tzavara C, Gregorakis A, Lykourinas M. Evaluation of urine tumor-associated trypsin inhibitor, CYFRA 21-1, and urinary bladder cancer antigen for detection of high-grade bladder carcinoma. Urology. 2008;72:1159–1163. [PubMed]
119. May M, Hakenberg OW, Gunia S, Pohling P, Helke C, Lubbe L, Nowack R, Siegsmund M, Hoschke B. Comparative diagnostic value of urine cytology, UBC-ELISA, and fluorescence in situ hybridization for detection of transitional cell carcinoma of urinary bladder in routine clinical practice. Urology. 2007;70:449–453. [PubMed]
120. Shoshtari MA, Soleimani M, Moslemi M. Comparative evaluation of urinary bladder cancer antigen and urine cytology in the diagnosis of bladder cancer. Urol J. 2005;2:137–140. [PubMed]
121. Hakenberg OW, Fuessel S, Richter K, Froehner M, Oehlschlaeger S, Rathert P, Meye A, Wirth MP. Qualitative and quantitative assessment of urinary cytokeratin 8 and 18 fragments compared with voided urine cytology in diagnosis of bladder carcinoma. Urology. 2004;64:1121–1126. [PubMed]
122. Mungan NA, Vriesema JL, Thomas CM, Kiemeney LA, Witjes JA. Urinary bladder cancer test: a new urinary tumor marker in the followup of superficial bladder cancer. Urology. 2000;56:787–792. [PubMed]
123. Retz M, Lehmann J, Amann E, Wullich B, Roder C, Stockle M. Mucin 7 and cytokeratin 20 as new diagnostic urinary markers for bladder tumor. J Urol. 2003;169:86–89. [PubMed]
124. Rotem D, Cassel A, Lindenfeld N, Mecz Y, Sova Y, Resnick M, Stein A. Urinary cytokeratin 20 as a marker for transitional cell carcinoma. Eur Urol. 2000;37:601–604. [PubMed]
125. Cassel A, Rahat MA, Lahat N, Lindenfeld N, Mecz Y, Stein A. Telomerase activity and cytokeratin 20 as markers for the detection and followup of transitional cell carcinoma: an unfulfilled promise. J Urol. 2001;166:841–844. [PubMed]
126. Retz M, Lehmann J, Roder C, Weichert-Jacobsen K, Loch T, Romahn E, Luhl C, Kalthoff H, Stockle M. Cytokeratin-20 reverse-transcriptase polymerase chain reaction as a new tool for the detection of circulating tumor cells in peripheral blood and bone marrow of bladder cancer patients. Eur Urol. 2001;39:507–515. discussion 516-507. [PubMed]
127. Guo B, Luo C, Xun C, Xie J, Wu X, Pu J. Quantitative detection of cytokeratin 20 mRNA in urine samples as diagnostic tools for bladder cancer by real-time PCR. Exp Oncol. 2009;31:43–47. [PubMed]
128. Eissa S, Zohny SF, Swellam M, Mahmoud MH, El-Zayat TM, Salem AM. Comparison of CD44 and cytokeratin 20 mRNA in voided urine samples as diagnostic tools for bladder cancer. Clin Biochem. 2008;41:1335–1341. [PubMed]
129. Pu XY, Wang ZP, Chen YR, Wu YL, Wang HP, Wang XH. [Clinical value of combined detection with urinary bladder cancer antigen, hyaluronic acid and cytokeratin 20 in diagnosis of bladder cancer] Ai Zheng. 2008;27:970–973. [PubMed]
130. Gazzaniga P, Gandini O, Giuliani L, Magnanti M, Gradilone A, Silvestri I, Gianni W, Gallucci M, Frati L, Agliano AM. Detection of epidermal growth factor receptor mRNA in peripheral blood: a new marker of circulating neoplastic cells in bladder cancer patients. Clin Cancer Res. 2001;7:577–583. [PubMed]
131. Okegawa T, Kinjo M, Nutahara K, Higashihara E. Value of reverse transcription polymerase chain assay in peripheral blood of patients with urothelial cancer. J Urol. 2004;171:1461–1466. [PubMed]
132. Klein A, Zemer R, Buchumensky V, Klaper R, Nissenkorn I. Expression of cytokeratin 20 in urinary cytology of patients with bladder carcinoma. Cancer. 1998;82:349–354. [PubMed]
133. Lin S, Hirschowitz SL, Williams C, Shintako P, Said J, Rao JY. Cytokeratin 20 as an immu-nocytochemical marker for detection of urothelial carcinoma in atypical cytology: preliminary retrospective study on archived urine slides. Cancer Detect Prev. 2001;25:202–209. [PubMed]
134. Golijanin D, Shapiro A, Pode D. Immunostaining of cytokeratin 20 in cells from voided urine for detection of bladder cancer. J Urol. 2000;164:1922–1925. [PubMed]
135. Soyuer I, Sofikerim M, Tokat F, Soyuer S, Ozturk F. Which urine marker test provides more diagnostic value in conjunction with standard cytology- ImmunoCyt/uCyt+ or Cytokeratin 20 expression. Diagn Pathol. 2009;4:20. [PMC free article] [PubMed]
136. Bhatia A, Dey P, Kumar Y, Gautam U, Kakkar N, Srinivasan R, Nijhawan R. Expression of cytokeratin 20 in urine cytology smears: a potential marker for the detection of urothelial carcinoma. Cytopathology. 2007;18:84–86. [PubMed]
137. Melissourgos ND, Kastrinakis NG, Skolarikos A, Pappa M, Vassilakis G, Gorgoulis VG, Salla C. Cytokeratin-20 immunocytology in voided urine exhibits greater sensitivity and reliability than standard cytology in the diagnosis of transitional cell carcinoma of the bladder. Urology. 2005;66:536–541. [PubMed]
138. Sanchez-Carbayo M, Ciudad J, Urrutia M, Navajo JA, Orfao A. Diagnostic performance of the urinary bladder carcinoma antigen ELISA test and multiparametric DNA/cytokeratin flow cytometry in urine voided samples from patients with bladder carcinoma. Cancer. 2001;92:2811–2819. [PubMed]
139. Pariente JL, Bordenave L, Jacob F, Gobinet A, Leger F, Ferriere JM, Le Guillou M. Analytical and prospective evaluation of urinary cytokeratin 19 fragment in bladder cancer. J Urol. 2000;163:1116–1119. [PubMed]
140. Sanchez-Carbayo M, Urrutia M, Silva JM, Romani R, De Buitrago JM, Navajo JA. Comparative predictive values of urinary cytology, urinary bladder cancer antigen, CYFRA 21-1 and NMP22 for evaluating symptomatic patients at risk for bladder cancer. J Urol. 2001;165:1462–1467. [PubMed]
141. Nisman B, Barak V, Shapiro A, Golijanin D, Peretz T, Pode D. Evaluation of urine CYFRA 21-1 for the detection of primary and recurrent bladder carcinoma. Cancer. 2002;94:2914–2922. [PubMed]
142. Bian W, Xu Z. Combined assay of CYFRA 21-1, telomerase and vascular endothelial growth factor in the detection of bladder transitional cell carcinoma. Int J Urol. 2007;14:108–111. [PubMed]
143. Fernandez-Gomez J, Rodriguez-Martinez JJ, Barmadah SE, Garcia Rodriguez J, Allende DM, Jalon A, Gonzalez R, Alvarez-Mugica M. Urinary CYFRA 21.1 is not a useful marker for the detection of recurrences in the follow-up of superficial bladder cancer. Eur Urol. 2007;51:1267–1274. [PubMed]
144. Hautmann SH, Schroeder GL, Civantos F, Duncan RC, Gnann R, Friedrich MG, Hellstern A, Huland H, Soloway MS, Lokeshwar VB. [Hyaluronic acid and hyaluronidase. 2 new bladder carcinoma markers] Urologe A. 2001;40:121–126. [PubMed]
145. Hautmann SH, Lokeshwar VB, Schroeder GL, Civantos F, Duncan RC, Gnann R, Friedrich MG, Soloway MS. Elevated tissue expression of hyaluronic acid and hyaluronidase validates the HA-HAase urine test for bladder cancer. J Urol. 2001;165:2068–2074. [PubMed]
146. Lokeshwar VB, Block NL. HA-HAase urine test. A sensitive and specific method for detecting bladder cancer and evaluating its grade. Urol Clin North Am. 2000;27:53–61. [PubMed]
147. Pham HT, Block NL, Lokeshwar VB. Tumor-derived hyaluronidase: a diagnostic urine marker for high-grade bladder cancer. Cancer Res. 1997;57:778–783. [PubMed]
148. Passerotti CC, Bonfim A, Martins JR, Dall'Oglio MF, Sampaio LO, Mendes A, Ortiz V, Srougi M, Dietrich CP, Nader HB. Urinary hyaluronan as a marker for the presence of residual transitional cell carcinoma of the urinary bladder. Eur Urol. 2006;49:71–75. [PubMed]
149. Eissa S, Kassim SK, Labib RA, El-Khouly IM, Ghaffer TM, Sadek M, Razek OA, El-Ahmady O. Detection of bladder carcinoma by combined testing of urine for hyaluronidase and cytokeratin 20 RNAs. Cancer. 2005;103:1356–1362. [PubMed]
150. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997;3:917–921. [PubMed]
151. Smith SD, Wheeler MA, Plescia J, Colberg JW, Weiss RM, Altieri DC. Urine detection of survivin and diagnosis of bladder cancer. Jama. 2001;285:324–328. [PubMed]
152. Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res. 2000;6:127–134. [PubMed]
153. Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998;58:5071–5074. [PubMed]
154. Monzo M, Rosell R, Felip E, Astudillo J, Sanchez JJ, Maestre J, Martin C, Font A, Barnadas A, Abad A. A novel anti-apoptosis gene: Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol. 1999;17:2100–2104. [PubMed]
155. Adida C, Berrebi D, Peuchmaur M, Reyes-Mugica M, Altieri DC. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet. 1998;351:882–883. [PubMed]
156. Gazzaniga P, Gradilone A, Giuliani L, Gandini O, Silvestri I, Nofroni I, Saccani G, Frati L, Agliano AM. Expression and prognostic significance of LIVIN, SURVIVIN and other apoptosis-related genes in the progression of superficial bladder cancer. Ann Oncol. 2003;14:85–90. [PubMed]
157. Schultz IJ, Kiemeney LA, Karthaus HF, Witjes JA, Willems JL, Swinkels DW, Gunnewiek JM, de Kok JB. Survivin mRNA copy number in bladder washings predicts tumor recurrence in patients with superficial urothelial cell carcinomas. Clin Chem. 2004;50:1425–1428. [PubMed]
158. Hausladen DA, Wheeler MA, Altieri DC, Colberg JW, Weiss RM. Effect of intravesical treatment of transitional cell carcinoma with bacillus Calmette-Guerin and mitomycin C on urinary survivin levels and outcome. J Urol. 2003;170:230–234. [PubMed]
159. Eissa S, Swellam M, Shehata H, El-Khouly IM, El-Zayat T, El-Ahmady O. Expression of HYAL1 and survivin RNA as diagnostic molecular markers for bladder cancer. J Urol. 183:493–498. [PubMed]
160. Kenney DM, Geschwindt RD, Kary MR, Linic JM, Sardesai NY, Li ZQ. Detection of newly diagnosed bladder cancer, bladder cancer recurrence and bladder cancer in patients with he-maturia using quantitative rt-PCR of urinary survivin. Tumour Biol. 2007;28:57–62. [PubMed]
161. Horstmann M, Bontrup H, Hennenlotter J, Taeger D, Weber A, Pesch B, Feil G, Patschan O, Johnen G, Stenzl A, Bruning T. Clinical experience with survivin as a biomarker for urothelial bladder cancer. World J Urol. 28:399–404. [PubMed]
162. Moussa O, Abol-Enein H, Bissada NK, Keane T, Ghoneim MA, Watson DK. Evaluation of survivin reverse transcriptase-polymerase chain reaction for noninvasive detection of bladder cancer. J Urol. 2006;175:2312–2316. [PubMed]
163. Melamed MR. Flow cytometry of the urinary bladder. Urol Clin North Am. 1984;11:599–608. [PubMed]
164. Mora LB, Nicosia SV, Pow-Sang JM, Ku NK, Diaz JI, Lockhart J, Einstein A. Ancillary techniques in the followup of transitional cell carcinoma: a comparison of cytology, histology and deoxyribonucleic acid image analysis cytometry in 91 patients. J Urol. 1996;156:49–54. discussion 54-45. [PubMed]
165. Barlandas-Rendon E, Muller MM, Garcia-Latorre E, Heinschink A. Comparison of urine cell characteristics by flow cytometry and cytology in patients suspected of having bladder cancer. Clin Chem Lab Med. 2002;40:817–823. [PubMed]
166. Parry WL, Hemstreet GP., 3rd Cancer detection by quantitative fluorescence image analysis. J Urol. 1988;139:270–274. [PubMed]
167. Caraway NP, Khanna A, Payne L, Kamat AM, Katz RL. Combination of cytologic evaluation and quantitative digital cytometry is reliable in detecting recurrent disease in patients with urinary diversions. Cancer. 2007;111:323–329. [PubMed]
168. Kamentsky LA, Kamentsky LD. Microscope-based multiparameter laser scanning cytome-ter yielding data comparable to flow cytometry data. Cytometry. 1991;12:381–387. [PubMed]
169. Ross JS, Cohen MB. Ancillary methods for the detection of recurrent urothelial neoplasia. Cancer. 2000;90:75–86. [PubMed]
170. Brentnall TA. Microsatellite instability. Shifting concepts in tumorigenesis. Am J Pathol. 1995;147:561–563. [PubMed]
171. Cairns P, Tokino K, Eby Y, Sidransky D. Homozygous deletions of 9p21 in primary human bladder tumors detected by comparative multiplex polymerase chain reaction. Cancer Res. 1994;54:1422–1424. [PubMed]
172. Mao L, Lee DJ, Tockman MS, Erozan YS, Askin F, Sidransky D. Microsatellite alterations as clonal markers for the detection of human cancer. Proc Natl Acad Sci U S A. 1994;91:9871–9875. [PubMed]
173. Ruppert JM, Tokino K, Sidransky D. Evidence for two bladder cancer suppressor loci on human chromosome 9. Cancer Res. 1993;53:5093–5095. [PubMed]
174. van der Riet P, Nawroz H, Hruban RH, Corio R, Tokino K, Koch W, Sidransky D. Frequent loss of chromosome 9p21-22 early in head and neck cancer progression. Cancer Res. 1994;54:1156–1158. [PubMed]
175. Orntoft TF, Wolf H. Molecular alterations in bladder cancer. Urol Res. 1998;26:223–233. [PubMed]
176. Christensen M, Jensen MA, Wolf H, Orntoft TF. Pronounced microsatellite instability in transitional cell carcinomas from young patients with bladder cancer. Int J Cancer. 1998;79:396–401. [PubMed]
177. Mao L, Schoenberg MP, Scicchitano M, Erozan YS, Merlo A, Schwab D, Sidransky D. Molecular detection of primary bladder cancer by microsatellite analysis. Science. 1996;271:659–662. [PubMed]
178. van Rhijn BW, Lurkin I, Chopin DK, Kirkels WJ, Thiery JP, van der Kwast TH, Radvanyi F, Zwarthoff EC. Combined microsatellite and FGFR3 mutation analysis enables a highly sensitive detection of urothelial cell carcinoma in voided urine. Clin Cancer Res. 2003;9:257–263. [PubMed]
179. Mourah S, Cussenot O, Vimont V, Desgrandchamps F, Teillac P, Cochant-Priollet B, Le Duc A, Fiet J, Soliman H. Assessment of microsatellite instability in urine in the detection of transitional-cell carcinoma of the bladder. Int J Cancer. 1998;79:629–633. [PubMed]
180. Linn JF, Lango M, Halachmi S, Schoenberg MP, Sidransky D. Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients. Int J Cancer. 1997;74:625–629. [PubMed]
181. Bartoletti R, Dal Canto M, Cai T, Piazzini M, Travaglini F, Gavazzi A, Rizzo M. Early diagnosis and monitoring of superficial transitional cell carcinoma by microsatellite analysis on urine sediment. Oncol Rep. 2005;13:531–537. [PubMed]
182. Bartoletti R, Cai T, Dal Canto M, Boddi V, Nesi G, Piazzini M. Multiplex polymerase chain reaction for microsatellite analysis of urine sediment cells: a rapid and inexpensive method for diagnosing and monitoring superficial transitional bladder cell carcinoma. J Urol. 2006;175:2032–2037. discussion 2037. [PubMed]
183. Wild PJ, Fuchs T, Stoehr R, Zimmermann D, Frigerio S, Padberg B, Steiner I, Zwarthoff EC, Burger M, Denzinger S, Hofstaedter F, Kristiansen G, Hermanns T, Seifert HH, Provenzano M, Sulser T, Roth V, Buhmann JM, Moch H, Hartmann A. Detection of urothelial bladder cancer cells in voided urine can be improved by a combination of cytology and standardized microsatellite analysis. Cancer Epidemiol Biomarkers Prev. 2009;18:1798–1806. [PubMed]
184. Frigerio S, Padberg BC, Strebel RT, Lenggenhager DM, Messthaler A, Abdou MT, Moch H, Zimmermann DR. Improved detection of bladder carcinoma cells in voided urine by standardized microsatellite analysis. Int J Cancer. 2007;121:329–338. [PubMed]
185. van der Aa MN, Zwarthoff EC, Steyerberg EW, Boogaard MW, Nijsen Y, van der Keur KA, van Exsel AJ, Kirkels WJ, Bangma C, van der Kwast TH. Microsatellite analysis of voided-urine samples for surveillance of low-grade non-muscle-invasive urothelial carcinoma: feasibility and clinical utility in a prospective multicenter study (Cost-Effectiveness of Follow-Up of Urinary Bladder Cancer trial [CEFUB]) Eur Urol. 2009;55:659–667. [PubMed]
186. Hoque MO, Lee J, Begum S, Yamashita K, Engles JM, Schoenberg M, Westra WH, Sidransky D. High-throughput molecular analysis of urine sediment for the detection of bladder cancer by high-density single-nucleotide polymorphism array. Cancer Res. 2003;63:5723–5726. [PubMed]
187. Grossman HB, Washington RW, Jr, Carey TE, Liebert M. Alterations in antigen expression in superficial bladder cancer. J Cell Biochem Suppl. 1992;16I:63–68. [PubMed]
188. Sawczuk IS, Pickens CL, Vasa UR, Ralph DA, Norris KA, Miller MC, Ng AY, Grossman HB, Veltri RW. DD23 Biomarker: a prospective clinical assessment in routine urinary cytology specimens from patients being monitored for TCC. Urol Oncol. 2002;7:185–190. [PubMed]
189. Gilbert SM, Veltri RW, Sawczuk A, Shabsigh A, Knowles DR, Bright S, O'Dowd GJ, Olsson CA, Benson MC, Sawczuk IS. Evaluation of DD23 as a marker for detection of recurrent transitional cell carcinoma of the bladder in patients with a history of bladder cancer. Urology. 2003;61:539–543. [PubMed]
190. van der Poel HG, van Rhijn BW, Peelen P, Debruyne FM, Boon ME, Schalken JA. Consecutive quantitative cytology in bladder cancer. Urology. 2000;56:584–588. [PubMed]
191. van der Poel HG, Debruyne FM. Can biological markers replace cystoscopy? An update. Curr Opin Urol. 2001;11:503–509. [PubMed]
192. Witjes JA, van der Poel HG, van Balken MR, Debruyne FM, Schalken JA. Urinary NMP22 and karyometry in the diagnosis and follow-up of patients with superficial bladder cancer. Eur Urol. 1998;33:387–391. [PubMed]
193. Vriesema JL, van der Poel HG, Debruyne FM, Schalken JA, Kok LP, Boon ME. Neural network-based digitized cell image diagnosis of bladder wash cytology. Diagn Cytopathol. 2000;23:171–179. [PubMed]
194. van Rhijn BW, van der Poel HG, Boon ME, Debruyne FM, Schalken JA, Witjes JA. Presence of carcinoma in situ and high 2C-deviation index are the best predictors of invasive transitional cell carcinoma of the bladder in patients with high-risk Quanticyt. Urology. 2000;55:363–367. [PubMed]
195. Wiener HG, Mian C, Haitel A, Pycha A, Schatzl G, Marberger M. Can urine bound diagnostic tests replace cystoscopy in the management of bladder cancer? J Urol. 1998;159:1876–1880. [PubMed]
196. Ross S, Spencer SD, Lasky LA, Koeppen H. Selective expression of murine prostate stem cell antigen in fetal and adult tissues and the transgenic adenocarcinoma of the mouse prostate model of prostate carcinogenesis. Am J Pathol. 2001;158:809–816. [PubMed]
197. Saffran DC, Raitano AB, Hubert RS, Witte ON, Reiter RE, Jakobovits A. Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts. Proc Natl Acad Sci USA. 2001;98:2658–2663. [PubMed]
198. Gu Z, Thomas G, Yamashiro J, Shintaku IP, Dorey F, Raitano A, Witte ON, Said JW, Loda M, Reiter RE. Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer. Oncogene. 2000;19:1288–1296. [PubMed]
199. Cheng L, Reiter RE, Jin Y, Sharon H, Wieder J, Lane TF, Rao J. Immunocytochemical analysis of prostate stem cell antigen as adjunct marker for detection of urothelial transitional cell carcinoma in voided urine specimens. J Urol. 2003;169:2094–2100. [PubMed]
200. Wu X, Ye Y, Kiemeney LA, Sulem P, Rafnar T, Matullo G, Seminara D, Yoshida T, Saeki N, Andrew AS, Dinney CP, Czerniak B, Zhang ZF, Kiltie AE, Bishop DT, Vineis P, Porru S, Buntinx F, Kellen E, Zeegers MP, Kumar R, Rudnai P, Gurzau E, Koppova K, Mayordomo JI, Sanchez M, Saez B, Lindblom A, de Verdier P, Steineck G, Mills GB, Schned A, Guarrera S, Polidoro S, Chang SC, Lin J, Chang DW, Hale KS, Majewski T, Grossman HB, Thorlacius S, Thorsteinsdottir U, Aben KK, Witjes JA, Stefansson K, Amos CI, Karagas MR, Gu J. Genetic variation in the prostate stem cell antigen gene PSCA confers susceptibility to urinary bladder cancer. Nat Genet. 2009;41:991–995. [PMC free article] [PubMed]
201. Gonzalez-Zulueta M, Bender CM, Yang AS, Nguyen T, Beart RW, Van Tornout JM, Jones PA. Methylation of the 5′ CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res. 1995;55:4531–4535. [PubMed]
202. Dulaimi E, Uzzo RG, Greenberg RE, Al-Saleem T, Cairns P. Detection of bladder cancer in urine by a tumor suppressor gene hypermethy-lation panel. Clin Cancer Res. 2004;10:1887–1893. [PubMed]
203. Yu J, Zhu T, Wang Z, Zhang H, Qian Z, Xu H, Gao B, Wang W, Gu L, Meng J, Wang J, Feng X, Li Y, Yao X, Zhu J. A novel set of DNA methylation markers in urine sediments for sensitive/specific detection of bladder cancer. Clin Cancer Res. 2007;13:7296–7304. [PubMed]
204. Hoque MO, Begum S, Topaloglu O, Chatterjee A, Rosenbaum E, Van Criekinge W, Westra WH, Schoenberg M, Zahurak M, Goodman SN, Sidransky D. Quantitation of promoter methylation of multiple genes in urine DNA and bladder cancer detection. J Natl Cancer Inst. 2006;98:996–1004. [PubMed]
205. Yates DR, Rehman I, Meuth M, Cross SS, Hamdy FC, Catto JW. Methylational urinaly-sis: a prospective study of bladder cancer patients and age stratified benign controls. Oncogene. 2006;25:1984–1988. [PubMed]
206. Lin HH, Ke HL, Huang SP, Wu WJ, Chen YK, Chang LL. Increase sensitivity in detecting superficial, low grade bladder cancer by combination analysis of hypermethylation of E-cadherin, p16, p14, RASSF1A genes in urine. Urol Oncol. 2009 [PubMed]
207. Renard I, Joniau S, van Cleynenbreugel B, Collette C, Naome C, Vlassenbroeck I, Nicolas H, de Leval J, Straub J, Van Criekinge W, Hamida W, Hellel M, Thomas A, de Leval L, Bierau K, Waltregny D. Identification and Validation of the Methylated TWIST1 and NID2 Genes through Real-Time Methylation-Specific Polymerase Chain Reaction Assays for the Noninvasive Detection of Primary Bladder Cancer in Urine Samples. Eur Urol. 2009 [PubMed]

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