Clinical Decision Making in Surveillance of Non–Muscle-Invasive Bladder Cancer: The Evolving Roles of Urinary Cytology and Molecular Markers

Cystoscopy and urine cytology are the gold-standard tests for detection of recurrent disease during follow-up in patients with a history of non–muscle-invasive bladder cancer (NMIBC). High associated costs, as well as side effects, have driven the desire for inexpensive, noninvasive, accurate, and easy-to-use urine markers to detect bladder cancer recurrence. While many urine markers have been developed, very few have been clinically implemented. In this article, we discuss the requirements for development and validation of urine markers and the factors that hamper their clinical implementation. We also review current surveillance guidelines for NMIBC and provide an overview of approved urine markers for the detection and surveillance of NMIBC.

Introduction

At first diagnosis, approximately 75% of bladder cancer patients present with non–muscle-invasive bladder cancer (NMIBC), with 70% having Ta disease, 20% having T1, and 10% having Tis.[1] All patients initially undergo transurethral resection of the bladder tumor (TURBT) for diagnostic and staging purposes. This allows patients with NMIBC to be stratified into low-, intermediate-, and high-risk groups, based on the probability of recurrence and progression.[2] Whereas the high rate of recurrence (70%) is the key clinical concern in low- and intermediate-risk disease, the fairly high rate of progression to muscle-invasive disease (30%) is the main problem in patients with high-risk NMIBC.[1] Therefore, patients are monitored frequently by urine cytology and cystoscopy, the “gold standard” for detection of bladder cancer recurrence.[3,4]

Side effects and costs associated with surveillance, cytology, and cystoscopy in the setting of bladder cancer have driven the discovery of urine markers to detect recurrences. The ideal marker would be noninvasive and urine-based, would yield results at least as accurate as those obtained with cytology and cystoscopy in patients with low- and intermediate-risk disease, and would make possible earlier detection of recurrence or progression in patients with high-risk disease. The net result would be a reduction in costs, improved compliance and comfort, and reduced rates of advanced disease-which would follow from earlier detection of recurrences in patients with high-risk disease.[5,6] Multiple urine-based tests have been approved by the US Food and Drug Administration (FDA) and are commercially available, yet none have been incorporated as routine into American Urological Association and European Association of Urology clinical guidelines for treatment of bladder cancer.[7-9] In this article, we discuss requirements for development and validation of urine markers, discuss which factors hamper clinical implementation of these markers, and provide an overview of approved urine markers.

The Standard of Care: Current Follow-up Guidelines for NMIBC

Depending on the risk of recurrence and progression determined by the initial workup and pathology, patients are monitored every 3 to 6 months for the first 2 years, with longer follow-up intervals thereafter.[3,4] Table 1 summarizes the published guidelines from major oncology organizations for follow-up in NMIBC. Flexible cystoscopy is invasive, relatively expensive, and associated with some discomfort, triggering reduced compliance with established follow-up regimens.[10,11] In addition, approximately 10% of patients develop a urinary tract infection, and 10% to 20% of tumors are missed by conventional white-light cystoscopy (WLC).[12,13] Recently, in a meta-analysis of 1,345 patients, blue-light (fluorescence) cystoscopy (BLC), which uses a photosensitizer (5-aminolevulinic acid or hexaminolevulinate) instilled in the bladder 1 to 3 hours prior to cystoscopy, showed improved detection of bladder tumors independent of tumor stage (Ta, T1, and Tis), risk category (low, intermediate, and high), and primary or recurrent disease compared with WLC.[14,15] The improved detection rate was most pronounced in Tis tumors (40.8%; odds ratio, 12.4; 95% CI, 6.3–24.1; P < .001), and improved detection and subsequent treatment led to reduction of recurrences at 12 months.[14]

In contrast, BLC has a lower specificity than WLC (63% vs 81%), with false-positive biopsies caused by recent TURBT, the presence of inflammation, or intravesical instillations of bacillus Calmette-Guérin (BCG).[15,16] Most guidelines recommend urine cytology as an adjunct to cystoscopy for follow-up of high-risk patients. Urine cytology lacks sufficient sensitivity for detection of low-grade tumors (27% for G1 tumors, 54% for G2 tumors, and 78% for G3 tumors), and its specificity ranges from 83% to 88%. The utility of both urine cytology and cystoscopy in bladder cancer diagnosis and decision making is dependent upon the type of technique used for urine collection and preparation; the presence of stones, urinary tract infections, and intravesical instillations of chemotherapy or immunotherapy; and whether the sample is reviewed by a general pathologist or specialized genitourinary pathologist.[17-21]

Considerations in Urine Marker Development and Validation

Many urine-based tests have been developed for detection and monitoring of bladder cancer. While some of these have been approved by the FDA, none have been mandated in current guidelines; this is because their value-either as a replacement for cystoscopy and cytology or as providing additional relevant diagnostic information-is unclear.[8,9,18] However, in specific cases clinicians can use fluorescence in situ hybridization (FISH) and/or ImmunoCyt to assess response to immunotherapy with intravesical BCG, to determine which patients should be monitored closely (eg, those with a positive FISH test but negative cystoscopy), or to differentiate between subtypes in patients with high-grade bladder cancer of atypical cytology. We will discuss the characteristics of an ideal urine marker and factors that currently hamper the clinical utility of urine markers.

Characteristics of an ideal urine marker

Use of a particular urine marker in clinical practice will occur if it satisfies the need for “easier, better, faster, and cheaper” means of diagnosing and monitoring bladder cancer.[22] The concept of an “easier” marker relates to its analytical performance and robustness in different clinical settings (ie, both community and academic cancer centers). While academic medical centers generally have the ability to snap-freeze and store human samples, many community hospitals cannot do this. “Better” refers to performance of the new marker that is equal or superior to the current standard of care. “Faster” means that the new biomarker readout should be available in a timely manner.[12] “Cheaper” means that the new marker is more cost-effective than the standard of care, in terms of both direct and downstream costs (ie, costs of false-positive results that lead to unnecessary additional diagnostic testing).

Assessing the performance of urine markers

Clinical context. A urine test should be developed and validated in the specific group of patients in whom it will be used in clinical practice, such as for screening (where the prevalence of bladder cancer is low), diagnosis (in which the prevalence of bladder cancer is higher than that of the screening population), or follow-up (high disease prevalence). The importance of this concept is highlighted by studies demonstrating that test sensitivities were higher in bladder cancer patients with a primary tumor compared with those under surveillance, due to the presence in the former group of larger tumor sizes and malignant tissues of higher stages and grades.[9,23-26] Hence, test sensitivity of a follow-up marker will be overestimated if evaluated in primary tumors, leading to “validation failure” in the surveillance setting.[27] This can also occur when validating a urine marker for follow-up of low/intermediate-grade disease in high-risk patients (larger tumors, more genetic changes).[26]

Cross-sectional vs longitudinal study design. Most studies compare the performance of a new marker with that of the standard of care at a single time point (cross-sectional sensitivity). This is not ideal because many such markers can yield a positive result before the tumor is visible on cystoscopy.[28,29] This “anticipatory” positive test emphasizes the need for a longitudinal analysis that incorporates future recurrences. For example, a large multicenter study to detect recurrences by FGFR3 mutations in patients under surveillance showed a cross-sectional sensitivity of 58% and a high number of false-positive results.[28] Since FGFR3 mutations are tumor-specific, these false positives indicate that tumor cells are present in the urinary tract, and findings must be anticipatory or reflect the presence of an upper tract tumor. A subsequent longitudinal analysis showed testing for FGFR3 mutations to have a sensitivity of 81% when future recurrences were included. It also showed that in the presence of a tumor, urine does not always contain tumor cells, highlighting the need to evaluate multiple samples.[28] A follow-up investigation demonstrated that the sensitivity for detection of recurrence increased with sampled urine volume (pooled urine over 24 hours), suggesting that analysis of multiple urine samples would improve test performance.[25]

Clinically Available Urine-Based Markers

Many urine-based markers have been developed for the detection and surveillance of bladder cancer. Only five (NMP22, BTA stat, BTA TRAK, ImmunoCyt/uCyt+, and UroVysion) are FDA approved. CxBladder is available through dedicated Clinical Laboratory Improvement Amendments–certified laboratories. Table 2 provides an overview of the reviewed markers.

NMP22 and NMP22 BladderChek

The NMP22 test is a protein-based assay using nuclear matrix protein 22. The test is based on the principle that during cell death, bladder cancer cells release more NMP22 into urine compared with normal bladder urothelium.[30] It can be performed on voided urine using a quantitative test, the enzyme-linked immunosorbent assay (ELISA). A laboratory environment is required in order to perform NMP22 testing, which costs approximately $25.[31] NMP22 sensitivity for bladder cancer ranges from 26% to 100%, while specificity ranges from 49% to 98% (Table 2).[23,24,32-47] A meta-analysis of eight studies that included Ta–T4, G1–G3, and Tis tumors concluded that NMP22 testing had a pooled sensitivity of 61% and a specificity of 71%.[7] Both sensitivity and specificity were dependent on tumor grade and stage and influenced by differences in cutoff points. Many studies suggested a higher cutoff value than indicated by the manufacturer to reduce false-positive findings caused by infections, urolithiasis, benign prostatic hyperplasia, presence of foreign bodies (eg, catheters) in the bladder, or cell death in the bladder due to intravesical chemotherapy with mitomycin C or immunotherapy with BCG.[33,34,41-43,48] Because of the aforementioned uncertainties about its value as a diagnostic tool in bladder cancer, the ELISA-based NMP22 test has not gained much traction in clinical practice.

In 2003, NMP22 BladderChek (NMP22 BC) was launched as a point-of-care immunoassay based on the same principle as the NMP22 ELISA test, but this test can be completed in less than 30 minutes. The cost is approximately $25 per test, and only four drops of urine are required.[11] The reported sensitivity of NMP22 BC ranges from 11% to 86%, and its specificity ranges from 77% to 98% (Table 2).[21,36,49-51] A large study (N = 668) showed the test to be more sensitive in detecting tumors of higher grade and stage, with a sensitivity of 75% for high-grade lesions and 91% for lesions of stage T2 or higher, with only one T2 lesion missed.[49] In the same study, cystoscopy visualized 75% of high-grade tumors; when combined with NMP22 BC, 97% of high-grade recurrences were detected (P = .008). Cystoscopy combined with NMP22 BC detected 99% of recurrences, compared with 91.3% for cystoscopy alone (P = .005). Unfortunately, other studies could not validate these findings in a smaller cohort (N = 145), but did show that a positive test was associated with 9.5 times higher risk of recurrence.[50] More recently, two studies compared NMP22 BC with CxBladder. NMP22 BC showed a sensitivity of 4% for low-risk NMIBC and an equally poor sensitivity of 16% for high-risk NMIBC, calling into question the test characteristics of NMP22 BC.[36] In summary, NMP22 BC is a fast and inexpensive test, yet its usefulness is unclear due to conflicting study findings. On the other hand, in view of its higher sensitivity compared with cytology, its use in combination with cystoscopy could be considered for future research.

BTA stat & BTA TRAK

Bladder tumor antigen (BTA) has been extensively investigated and is available in two variants. The BTA stat test is a point-of-care rapid immunochromatographic assay, costing $25 and giving results in 5 minutes, while BTA TRAK is a multistep quantitative immunoassay, which requires a laboratory setting.[52] The BTA stat test is based on the use of monoclonal antibodies to detect complement factor H–related protein, which is found more frequently in bladder cancer cells and inhibits essential activation of the complement system.[53] Overall sensitivity of the BTA stat test is 29% to 91%, somewhat higher than sensitivity rates of cytology and comparable to rates seen with NMP22. Specificity ranges from 54% to 86%, which is also comparable to the specificity of NMP22 (Table 2).[24,34,37,38,42,44,46,54-65] A meta-analysis of 11 studies reported an overall sensitivity of 60% and specificity of 76%.[7] In addition, the sensitivity was 82% for stage T2 and higher tumors, 70% for T1 tumors, and 75% for all high-grade tumors.[7,34,46,57]

BTA TRAK has been studied less than the BTA stat test and is rarely used in clinical practice. The sensitivity of BTA TRAK ranges from 52% to 99%, and its specificity ranges from 12% to 95% (Table 2).[32,42,66-69] Both the BTA stat and BTA TRAK tests frequently yield false-positive results because factor H can also be detected in benign conditions and following intravesical therapy for bladder cancer.[42,68] The sensitivity of BTA TRAK for high-grade tumors ranges from approximately 75% to 80%, higher than sensitivity rates reported with BTA stat.[32,66,68] In one investigation, BTA TRAK showed a lower sensitivity than cytology (57% vs 73%).[32] Another study adjusted the cutoff values to reach a sensitivity of 99%, but as a result the specificity dropped to 12%.[66] BTA TRAK and BTA stat frequently yield false-negative results as well, not only for low-grade tumors but also for muscle-invasive tumors, the latter of which can have serious consequences.[32,34,66,69] Given that both tests generate a notable degree of false-positives and false-negatives, their use in follow-up monitoring of NMIBC is not advised.[9,70]

ImmunoCyt/uCyt+

ImmunoCyt/uCyt+ is an immunocytology test that is performed in a laboratory.[71] A single test costs approximately $80 and uses three monoclonal fluorescent antibodies.[72] Two antibodies are directed against tumor mucins, which are often present in bladder cancer and absent in normal urothelial cells, and the third antibody detects carcinoembryonic antigens.[73] While initial data from two studies indicated sensitivities of 86% for detection of low-grade tumor recurrence and 89% to 100% for detection of high-grade tumor recurrence, neither investigation was carried out in a surveillance setting.[74,75] Surveillance studies showed an overall sensitivity of 50%, specificity of 73%, and a high interobserver variability.[76] Several prospective trials demonstrated that sensitivity ranges from 52% to 100% and specificity from 62% to 82% (Table 2).[35,74-87] A French multicenter trial (N = 458) combining ImmunoCyt/uCyt+ and cytology showed an overall increase in sensitivity and specificity compared with cytology alone.[85] However, since only 75% of high-grade tumors were detected, the test does not have sufficient discriminatory power to enable patients with high-risk NMIBC to delay or forgo cystoscopy. Two trials showed excellent specificities for detection of bladder cancer at stages Tis (100%) and T2 or higher (100%), yet only a few patients with muscle-invasive bladder cancer were included in this cohort.[80,84] Interestingly, both studies also showed a high sensitivity of 75% to 79% for the detection of small pTaG1 tumors. One study of 314 patients monitored for NMIBC reported a sensitivity of 71% for recurrences of tumors less than 1 cm, 83% for stage Ta tumors, and 79% for low-grade tumors.[81] Results suggest a potential role for ImmunoCyt/uCyt+ as a follow-up tool in patients with stage Ta low-grade tumors.

A cost-effectiveness analysis of 216 patients (84 of whom had been diagnosed with low-risk bladder cancer) showed that costs for replacement of cystoscopy by a combination of ImmunoCyt/uCyt+ and cytology were $17,600, while regular cystoscopy costs were $40,300 during 26 months of follow-up. In the same study, no tumor stage progression was detected in the low-risk patients.[80] Therefore, previous studies may indicate the use of ImmunoCyt/uCyt+ as an (intermittent) replacement for cystoscopies while at the same time lowering costs of surveillance for patients with low-risk disease. However, this is not the case for high-grade tumors, since the proportion of false-negatives has been too high.[79] One study showed a sensitivity of 75% and a specificity of 49% in patients with atypical cytology.[83] The sensitivity of this test for stage T2 and higher tumors varies; some reports show excellent sensitivity of 100% and a negative predictive value of 100%, yet other studies show 90% sensitivity for T2 tumors when use of ImmunoCyt/uCyt+ is combined with cytology.[81,84] A more recent meta-analysis (N = 7,422) showed that ImmunoCyt/uCyt+ had a sensitivity of 75% for high-grade tumors and 68% for tumors of stage T2 and higher.[77] In summary, ImmunoCyt/uCyt+ has potential as a marker during follow-up of patients with low-grade bladder tumors and can be used as an adjunct evaluative test in low-risk patients with atypical cytology, but its clinical value in the setting of high-risk disease is unclear.

UroVysion

UroVysion is a FISH test that uses multiple fluorescently labelled DNA probes to detect common genetic alterations in urothelial cells in urine samples.[88] The total cost of the test is approximately $800. A prospective cost-effectiveness analysis reported that the total cost per tumor detected by cystoscopy was $7,692 compared with $26,462 for cystoscopy combined with UroVysion FISH, which makes UroVysion the most expensive FDA-approved test.[11] The sensitivity ranges from 13% to 93% and specificity from 65% to 95% (Table 2).[35,36,64,86,89-95] A meta-analysis of 14 studies using 2,477 tests showed a pooled sensitivity of 72% (range, 69% to 75%) and a specificity of 83% (range, 82% to 85%); however, only 9 of the 14 studies included patients under surveillance.[96] Results of another meta-analysis showed a pooled sensitivity of 55% and specificity of 80%. Tumor stage and grade had an impact on test performance; sensitivity rates of 94% for detection of high-grade recurrences and 89% for detection of stage T2 and higher tumors were reported, yet more than half of all low-risk tumors were missed.[7] In contrast, other reports on UroVysion demonstrated a sensitivity of 18% for high-grade NMIBC and 50% for stages T2 to T4.[97,98] Interestingly, within 2.5 years of testing positive by the UroVysion FISH test, 65% of patients experienced tumor relapse, suggesting that UroVysion is able to detect genetic abnormalities before tumors are visible by cystoscopy (ie, the test has an anticipatory effect).[29] Given this significant anticipatory effect, FISH-positive patients should be monitored closely, as indicated by the American Urological Association guidelines.[99]

Since chromosomal integrity remains intact in patients who have the UroVysion test, outcomes are not hampered during treatment with BCG.[97] Results from two studies showed that patients who have a FISH-positive urine test immediately after TURBT are more likely to experience recurrences, although the investigators also found contradictory evidence of a higher risk of progression in these patients.[97,100] Furthermore, BCG therapy is more likely to fail in patients who are FISH-positive after intravesical instillations of BCG.[101] In summary, UroVysion is of questionable value in routine follow-up, since overall sensitivity varies. However, because results in high-risk patients seem acceptable and the anticipatory effect to predict future recurrences is strong, UroVysion may be useful as an adjunct for predicting response to BCG, and/or for stratifying whether patients with high-grade bladder cancer who have a positive UroVysion urine test and negative cystoscopy need to be monitored closely.[99]

CxBladder

This urine-based laboratory test consists of a multiplex evaluation of an RNA gene expression signature and costs approximately $320.[102,103] In a study of initial detection of bladder cancer in patients with hematuria, CxBladder had a higher sensitivity (62.1%) than both NMP22 ELISA (50.0%) and NMP22 BC (37.9%). Its specificity was 85%, with false-positive results seen mainly in patients with urinary stone disease. Recently, results from a large (N = 1,036) multicenter surveillance trial showed that CxBladder had a sensitivity of 86% for low-risk NMIBC and 95% for high-risk NMIBC, detecting 97% of recurrences of high-grade tumors and 100% of tumors of stage T1 and higher.[104] When CxBladder was compared with NMP22 ELISA, NMP22 BC, and FISH, overall sensitivity was 91% and the negative predictive value was 96%, outperforming other tests.[36] Taken together, results of multiple trials are promising, given the high sensitivity of CxBladder for both low-risk and high-risk patients. A prospective randomized comparative study is needed to further validate these findings and to determine whether CxBladder could effectively replace cystoscopy and/or urine cytology.

Conclusions

The goal for newly developed urine markers is to be easier, better, faster, and cheaper.[22] Two studies evaluating patients’ perspectives demonstrated that a urine marker would need a sensitivity of 95% or greater to replace cystoscopy.[105,106] While most markers aim for a sensitivity and specificity of 100%, the “perfect” marker does not exist; even the standard of care does not achieve this level of performance. Although multiple markers have been approved, current guidelines do not recommend their regular use in clinical practice. However, promising and robust experimental assays have been developed for detection of recurrence, and the results of large randomized studies of these markers are eagerly anticipated.

Financial Disclosure:The authors have no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.

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