While patients with non-muscle invasive bladder cancer (NMIBC) have historically had favorable survival outcomes, patients with high-grade (HG) NMIBC have the potential to progress to muscle-invasive disease that is typically not amenable to the bladder-sparing treatments often utilized for NMIBC.1 Contemporary evidence has found that as many as 70% to 80% of NMIBC tumors will recur after initial treatment, and nearly 20% will progress to muscle-invasive disease within 5 years.2-6 As a result, the risk of disease recurrence and progression to muscle-invasive bladder cancer (MIBC) necessitate timely and extended surveillance strategies.
Surveillance for NMIBC has historically relied on the diagnostic combination of cystoscopy and urinary cytology, which has been reported to have high specificity (>90%) for HG lesions, including carcinoma in situ (CIS).3,7 More recently, novel urinary markers also have been evaluated to augment these conventional methods. In this article, we evaluate current and emerging urinary markers used in the surveillance for NIMBC.
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Urine cytology
The use of urine cytology involves the microscopic evaluation of voided urine or bladder-washes for exfoliated cancer cells. Although it has historically been reported to have a high sensitivity for detecting HG lesions (84%), contemporary series do not support this notion. Cytology has been reported to have a sensitivity ranging from 34%-60% for grade 3 or HG lesions in recent studies.8-13 Conversely, urine cytology has a high specificity (>90%) for both low- and high-grade tumors, including CIS.3,7 As a result, a positive cytology reading, regardless of cystoscopic or radiographic findings, suggests the existence of malignancy in the vast majority of patients (Table 1).12 One study found that even in the setting of negative diagnostic evaluation (cystoscopic and upper tract studies), 41% of patients with persistently positive cytology were found to have a genitourinary cancer within 24 months, with a mean time to diagnosis of 5.6 months.14 A positive voided urinary cytology can therefore indicate the presence of a malignant lesion in the urinary tract; a negative cytology does not, however, rule out the presence of a tumor.3,15
Urine cytology, however, also has several known drawbacks. Unlike tumor markers, urine cytology is not a laboratory test: It is an interpretation of the morphologic features of exfoliated urothelial cells. As a result, cytology is often associated with a lack of inter-observer consistency and a wide range of readings (e.g., atypical, atypical-suspicious, non-diagnostic).4 Consequently, the accuracy of cytology can vary among pathologists.7
To address these concerns, The Paris System for Reporting Urine Cytology (PSRUC) recently has emerged as a significant international and multi-institutional measure to help standardize urine cytology reporting and to further focus the application of cytology towards the detection primarily of HG urothelial carcinoma. (Table 1).16 The requirement of additional cytological features such as hyperchromasia, irregular nuclear membranes, or clumpy chromatin pattern to define a cell as atypical, for example, has resulted in fewer cases being assigned the anomalous interpretation. By excluding atypia related to low-grade urothelial neoplasms and reactive conditions such as polyomavirus infection and urolithiasis from the atypical category, urologists can use cytology more effectively to separate patients with concerning high-risk lesions from those with low-grade urothelial carcinomas, most of which have a low risk of progression. Thus, the PSRUC takes important steps towards improving the clinical utility of urine cytology.
Urinary biomarkers
Several novel urinary biomarkers have been developed and investigated over the last 3 decades to complement or replace urine cytology. Current urinary markers have been developed to detect tumor-associated antigens, blood group antigens, growth factors, cell cycle/apoptosis, and extracellular matrix proteins. Some of these markers have been approved by the FDA and are commercially available in the US.17 The NMP22® and BTA® tests are protein-based, while UroVysion® FISH and ImmunoCyt™ are cell-based. While most biomarkers have demonstrated adequate sensitivity, they are associated with poor specificity that can result in substantial false-positive readings and thus create the need for further diagnostic testing.
Protein-based urinary biomarkers. The NMP22BladderChek® test (Matritech, Inc., Newton, MA) identifies nuclear matrix protein 22, part of the mitotic apparatus released from urothelial nuclei upon cellular apoptosis.3 The protein is elevated in urothelial carcinoma and is released in dying urothelial cells. In a recent multi-institutional trial with 1,331 patients, NMP22 was found to be more sensitive than cytology, but less specific.8 NMP22 demonstrated sensitivities of 50% and 90% for noninvasive and invasive cancer, respectively, with an overall sensitivity of 55.7%. Cytology, however, demonstrated a specificity of 99.2% compared with 85.7% for NMP22.
Similar to NMP22, the BTA® test (Polymedco, Inc.,Cortlandt Manor, NY) is a protein-based marker that identifies a basement membrane antigen that is related to complement factor H and is present at high levels in urine in patients with bladder cancer. Like NMP22, the BTA test is available in both qualitative and quantitative formats. A direct comparison between the NMP22 and BTA tests demonstrated similar sensitivities and specificities.18 Both protein-based markers are FDA-approved for the diagnosis and surveillance of bladder cancer. Some benign conditions, however, such as infection, inflammation, hematuria, and cystoscopy can cause false-positives, resulting in lower specificity than urine cytology in both tests.4
Cell-based urinary biomarkers. UroVysion® (Abbott Molecular, Des Plaines, IL) is a cytology-based test that uses fluorescence in situ hybridization (FISH) of DNA probes to identify aneuploidy in chromosomes 3, 7, and 17 and alterations to the chromosome 9p21 locus. Cumulative data from comparative studies demonstrated sensitivity for cytology compared with FISH of 35% vs 64% for Ta, 66% vs 83% for T1, and 76% vs 94% for muscle-invasive carcinoma.19 Notably, cytology detected only 67% of cases with CIS vs 100% detection by FISH. UroVysion has the highest specificity of the available tumor markers and is not affected by hematuria, inflammation, or other factors that can cause false-positive readings with some tumor markers, making it useful as a marker of bacillus Calmette-Guérin (BCG) response.3,20
ImmunoCyt™ (DiagnoCure, Inc., Sainte- Foy, Canada) is a hybrid of cytology and an immunofluorescent assay. The test identifies 3 cell surface glycoproteins present on cancer cell membranes and can be used to enhance the sensitivity of cytology.4 In one study, the sensitivity of cytology for stages Ta, T1, T2 or greater, and Tis tumors was 12%, 67%, 47% and 50%, respectively, whereas it reached 78%, 83%, 79%, and 100% when combined with ImmunoCyt.21 It has not been shown to be affected by benign conditions, but interpretation is complex and operator-dependent.3
The Cxbladder™ (Pacific Edge Ltd., Dunedin, New Zealand) test is a laboratory-developed test, so it has not been approved for use by the FDA. It identifies 4 mRNA fragments in the urine that demonstrated high levels of expression in urothelial carcinoma.22 A fifth RNA marker, CXCR2, is typically highly expressed in non-malignant inflammatory conditions; its inclusion helps discriminate against false-positive cases in patients with acutely or chronically inflamed urothelium. Initial studies show that this test can readily distinguish between low- and high-grade tumors, and may perform better than urinary markers such as NMP22 and BTA4. In one study, Cxbladder™ was found to have a sensitivity of 82%, including 97% of high grade tumors and 100% of tumors stage 1 or greater.22
Other urinary biomarkers. Several recent publications have evaluated the utility of telomerase reverse transcriptase (TERT) promoter mutations as a biomarker for bladder cancer. Telomerase is essential in proliferating cells, such as stem cells, and increases telomere length at chromosome ends. Telomerase is subsequently downregulated in differentiated cells in somatic tissues. It can, however, become reactivated in tumors. Kinde et al noted that TERT mutations are detectable in urine, occur in papillary and flat lesions, and are also strongly associated with bladder cancer recurrence.23 Allory et al subsequently found that 70%-79% of urothelial tumors harbored TERT mutations. TERT mutations were also found more frequently among fibroblast growth factor receptor 3 (FGFR3) mutant tumors (p=0.0002), which is the most commonly mutated gene in urothelial carcinoma. TERT Mutations, however, were not associated with clinical outcomes or pathologic parameters.24 As the TERT mutation has been found to be highly prevalent in both noninvasive and invasive bladder tumors, however, this may represent a useful biomarker for future urine-based disease diagnosis and monitoring.23,25
A number of other biomarkers—including those based on detection of FGFR3, cytokeratin fragments (e.g., CYFRA 21-1, TPA, TPS), surviving, vascular endothelial growth factor (VEGF), urine cell-free DNA, aurora kinase, or metalloproteinases (MMP-2 and MMP-9)—also have been developed, but they are not FDA-approved.
The large number of available and emerging biomarkers, each with potential trade-offs in diagnostic accuracy, risks, and patient preferences, pose significant challenges in determining optimal testing and surveillance strategies. Biomarkers with high false-positive rates could lead to unnecessary invasive procedures for further evaluation, whereas biomarkers with high false-negative rates could lead to missed diagnoses.26 Therefore, further prospective studies are needed to identify the optimal biomarkers and surveillance strategies prior to clinical application. The most significant issue limiting widespread adoption of tumor markers at this time appears to be the lack of prospective data to support their impact on prognosis or disease management.7
Currently, while many urine markers exhibit promising sensitivity, particularly for lower-grade tumors, their specificity is still lower than that of urine cytology.3,4 Furthermore, lack of prospective data to support the impact of urinary biomarkers on patient prognosis has limited their widespread adoption at this time. There are also limited data on the efficacy of urinary biomarkers in predicting BCG response and treatment outcomes. As result, and given the uncertainty in their sensitivity and specificity, urinary biomarkers cannot be used to replace cystoscopy and cytology at this time. As a result, until a more effective urinary marker is developed for detecting potentially lethal urothelial carcinoma, urine cytology will continue to serve as a cornerstone for surveillance for NMIBC. Continued improvements in the accuracy and reporting of urinary cytology, through efforts such as the PSRUC, will also be necessary. Nevertheless, future investigations of urinary biomarkers are indicated for the surveillance of patients with NMIBC, and especially in the post-BCG therapy setting, where the identification of patients at risk of disease recurrence and progression is paramount.
1Department of Urology, James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA
Financial Disclosures/Funding Source: No financial disclosures.
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Table 1: Risk of Malignancy of Diagnostic Categories Outlined in the Paris Reporting System |
|
|
Category |
Risk of Malignancy, % |
|
Unsatisfactory/non-diagnostic |
<5–10 |
|
Negative for high-grade urothelial carcinoma |
0–10 |
|
Atypical urothelial cells |
8–35 |
|
Suspicious for high-grade urothelial carcinoma |
50–90 |
|
Low-grade urothelial neoplasm |
~10 |
|
High-grade urothelial carcinoma |
>90 |
Adapted from Barkan GA, Wojcik EM, Nayar R, et al. The Paris System for Reporting Urinary Cytology: The Quest to Develop a Standardized Terminology. Acta Cytol. 2016;60(3):185-197.
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