For many years, researchers have attempted to identify and utilize urinary markers for bladder cancer detection. Voided urine cytology has been the mainstay of urine-based diagnosis of bladder cancer since the original description by Papanicolaou and Marshall.110 Urine cytology, however, has several drawbacks, including a poor sensitivity for low-grade/stage tumors, a lack of interobserver consistency, a range of readings (e.g., atypical, atypical-suspicious, non-diagnostic), a need to send the specimen to an external laboratory, and a delay in obtaining results.111 These shortcomings have inspired the search for a more sensitive urinary bladder cancer marker. Many markers have been investigated and developed over the past three decades, with multiple of which have been approved by the U.S. Food and Drug Administration (FDA) and/or are commercially available in the U.S.112, 113 The NMP22® and BTA® tests are protein-based, while UroVysion® fluorescence in-situ hybridization (FISH), ImmunoCyt™ and Cxbladder™ are cell-based. The pooled sensitivity, specificity, and positive and negative likelihood ratios are shown in Table 6.114 The NMP22® test is available as a point-of-care test (NMP22® BladderChek®) or in a more quantitative format. This test identifies a nuclear matrix protein that is involved in the mitotic apparatus. The BTA® test identifies a basement membrane antigen that is related to complement factor H and is present within urine at higher levels in patients with bladder cancer. Like NMP22®, the BTA® test is also available in qualitative and quantitative formats. Both tests are FDA-approved for initial evaluation and surveillance of bladder cancer; however, these protein-based urine markers have a tendency to be falsely positive in the presence of inflammation, resulting in lower specificity than urine cytology. This can result in subjecting patients to unnecessary diagnostic evaluations. The UroVysion® FISH test identifies altered copy numbers of four specific chromosomes or loss of regions of chromosome 9p using fluorescent probes. The ImmunoCyt™ test identifies three cell surface glycoproteins that are present on the membrane of cancer cells and can be used in conjunction with cytology to enhance the sensitivity of cytology. The Cxbladder™ test identifies the presence of five messenger ribonucleic acid (mRNA) fragments in the urine that are expressed at high levels in patients with bladder cancer.113 One such fragment, CXCR2, is an inflammatory marker that helps discriminate false positive cases. This test appears to be able to distinguish between low- and high-grade tumors and may perform better than protein-based markers, such as NMP22® and BTA®. Although not a complete listing, given the lower specificity of all of the other currently available urinary markers to urine cytology as well as other concerns, the use of these markers has not been widely adopted. Direct comparisons of protein markers, such as NMP22® and BTA®, suggest that there is little difference in sensitivity or specificity between them.115 Comparing ImmunoCyt™ to UroVysion® FISH suggests that ImmunoCyt™ has a higher sensitivity but a lower specificity than UroVysion® FISH. Most of the studies evaluating these markers utilized cystoscopy as the gold standard for detecting tumors, while some utilized a pathologic evaluation of the biopsy specimen as the final reference. Direct comparisons between markers are difficult, and given the uncertainty in sensitivity, these tests may not be used to replace cystoscopy. Recently, an RCT (UroFollow) evaluated a biomarker-based surveillance strategy compared to standard cystoscopic surveillance in patients with low- and intermediate-risk NMIBC.116 In this study, biomarker-driven surveillance demonstrated a lower sensitivity for the detection of recurrence compared to standard surveillance (73.7-81.5% versus 95.8-96.5%), although these differences were not statistically significant (81.5% versus 96.5% overall, p=0.1; 73.7% versus 95.8% after 3 months, p=0.07). The marker-based strategy substantially reduced cystoscopy use (148 versus 589 WLC procedures). This reduction was accompanied by more missed low-grade Ta recurrences, with no missed tumors progressing in stage or grade. These findings support continued evaluation of marker-based surveillance in selected patients, but prospective validation is needed before it can replace cystoscopy-based surveillance. The update review identified six new observational studies (in seven publications) including 1,604 participants and one systematic review relevant to this Guideline statement. Since the Guideline’s initial publication in 2016, several new urinary biomarkers have been developed to detect recurrent bladder cancer in NMIBC patients on surveillance.117-119 One such marker is CxBladder Monitor™. It was designed as a high sensitivity rule-out test such that a negative result can be used to defer cystoscopy or confirm negative cystoscopy. In a cohort of 763 patients with NMIBC on surveillance, CxBladder Monitor™ had a 93% sensitivity and 97% negative predictive value for recurrent NMIBC; however, the test specificity was not reported. Approximately one-third of patients had a negative test and could potentially avoid cystoscopy. The test performed well for both low- and high-grade recurrences117 and outperformed urine cytology, NMP22®, and UroVysion® FISH.120 In a pragmatic single-arm surveillance study of a mixed-risk cohort (low-risk n=9, intermediate-risk n=26, high-risk n=52), Cxbladder Monitor™ was also evaluated in a surveillance pathway in which patients with negative tests deferred cystoscopy, and no recurrences were reported among those with negative tests during the limited deferred-surveillance interval (median follow-up: 4 months; interquartile range: 3-6 months).121 Other more novel urinary biomarkers have also been evaluated for surveillance. A cytokeratin re-expressed in urothelial carcinoma cells, URO17® which detects keratin 17, was evaluated in a small surveillance study that demonstrated higher sensitivity but lower specificity than urine cytology for recurrence detection.122 In another study of high - and very high-risk NMIBC patients on surveillance, Xpert® Monitor BC demonstrated high sensitivity with moderate specificity for recurrence detection.123 Although early data on these biomarkers are encouraging, further validation studies are needed to determine if a negative test is sufficient to defer or delay surveillance cystoscopy. The role of markers as adjuncts to cystoscopy or their potential to reduce or defer cystoscopy in select instances, continues to be evaluated in surveillance and related clinical contexts. More recently, Bladder EpiCheck® (urinary methylation biomarker) has been studied for prediction of residual disease prior to second TURBT. In a single-arm study of patients with primary Ta/T1 NMIBC scheduled for repeat resection, Bladder EpiCheck® demonstrated 63.6% sensitivity and 78.6% specificity for detection of residual disease.124 Comprehensive literature analysis showed that urinary markers have an increased sensitivity and specificity as tumor grade and stage increase. Urinary marker sensitivity is improved in patients with larger tumors. Several studies have examined markers for bladder cancer screening in high-risk populations.125, 126 Utilization of urine markers could potentially reduce the frequency of cystoscopy in screening.127 Moreover, the AUA/SUFU Microhematuria Guideline now recognizes a selective role for urine cytology and validated urine-based tumor markers in appropriately counseled intermediate-risk patients seeking to avoid or delay cystoscopy.23 In the STRATA RCT, CxBladder™ Triage showed a 99% negative predictive value and reduced cystoscopy use by 59% in lower-risk patients.128 While this is an intriguing idea, the prevalence of bladder cancer even in high-risk individuals is not high enough to justify routine screening at this time. Further, the point-of-care protein markers used in screening do not appear helpful in identifying the screen-detectable cancers;127, 129 therefore, this approach cannot be recommended.