Prominence of urinary biomarkers for bladder cancer in the COVID-19 era: From the commercially available to new prospective candidates

Molecular markers detected in urine may improve our understanding of the evolution of bladder cancer (BCa) and its micro- and macroenvironment. Detection of such markers will identify disease earlier, allow stratification of patients according to risk, and improve prognostication and prediction of outcomes, thereby facilitating targeted therapy. However, current guidelines have yet to embrace such markers for routine management of BCa, and most research studies have focused on urine-based tumor markers. In this review, we summarize known urinary biomarkers for BCa and highlight newly identified molecules. We then discuss the challenges that must be overcome to incorporate these markers into clinical care.


INTRODUCTION
Bladder cancer (BCa) is a common disease worldwide, with high morbidity [1,2]; however, developments in medical technology mean that it is no longer fatal in many cases. Despite this, mortality rates for those with muscle-invasive bladder cancer (MIBC) or those who do not receive optimal treatment remain high [3]. One of the hallmarks of BCa is its heterogeneity, which makes it difficult to manage [4]. This heterogeneity is due to genetic, transcriptomic, epigenetic, and/or phenotypic changes, which result in a molecularly heterogeneous tumor comprising cancer cells with diverse molecular signatures [4,5]. Therefore, tumor heterogeneity is the major barrier to successful management of BCa, includ-ing early identification of non-muscle-invasive bladder cancer (NMIBC) or MIBC. Appropriate and early identification will enable suitable treatment planning and assessment of prognosis. Consequently, identification of suitable and reliable tumor biomarkers is essential for diagnosis, prognosis, and treatment planning [6].
Diagnosis and follow-up of BCa are dependent on cystoscopy. This is a highly invasive procedure, which itself can cause complications such as infection or hematuria. Moreover, the high recurrence rate of BCa, along with the frequent requirement for surveillance, place huge economic and quality-of-life burdens on patients [7]. Voided urine cytology (VUC), which has been applied to BCa diagnosis, is a noninvasive option; however, its use is restricted by relatively low Urinary biomarker for bladder cancer sensitivity (particularly for diagnosing low-grade tumors); indeed, a previous study reported sensitivity of only 25% [8]. Thus, efforts have been made to explore cost-effective and noninvasive alternatives to cystoscopy. In particular, delivering cancer care during the present COVID-19 pandemic is challenging given the competing risks of cancer-specific death vs. a potentially lethal coronavirus infection. This highlights an urgent need to develop a guide to pragmatic management of BCa.
Clinical decision-making in a pandemic requires a balance between the probable benefits and risks; patients should attend hospitals only when strictly necessary, but care must be given to those most in need. Given this, the European Association of Urology developed guidelines suggesting a traffic-light surveillance pathway based on primary tumor grade and the presence of hematuria [9,10]. This guideline recommends that patients with low-risk or intermediate-risk tumors, and who are asymptomatic, wait a further 6 months for cystoscopy [11]. Although the guidelines are adaptable to the current situation, some patients are happy to defer this often costly and painful process; however, others would rather undergo the procedure quickly than worry about the ambiguity of their disease status.
This period of uncertainty requires timely action and innovation. Urinary biomarkers would enable early detection of cancer, particularly in patients for whom cystoscopy has been deferred in accordance with the up-to-date guidelines. In this review, we summarize the urinary biomarkers used for BCa diagnosis and discuss research and development of new advanced biomarkers.

Bladder cancer markers: what for?
Tumor markers are molecules secreted directly by tumor cells or indirectly by other cells in response to a tumor [12]. Biomarkers can be used for screening, diagnosis, monitoring/surveillance, and prognosis. However, there are no currently accepted biomarkers for BCa screening; therefore, the gold standard tests (a combination of VUC and cystoscopy) are still used for diagnosis in practice [13]. The main role of surveillance markers is to reduce the need for invasive cystoscopy; however, like diagnostic markers, they are not sufficiently reliable for routine clinical use [13,14]. Prognostic markers can be used to stratify patients according to clinical outcome (e.g., recurrence or progression), thereby helping clinicians decide which treatments are most beneficial in a particular case [15]. Consequently, the main goal for those developing biomarkers is to identify relevant molecules or tests that can improve clinical decision-making in a costeffective way.

Biomarker sources: liquid biopsy vs. tissue biopsy
Tissue biopsy is the traditional approach used for cancer diagnosis. The analysis of biopsy samples detects abnormal tumor cells in tumor-like tissue and surrounding tissue. However, this procedure is highly invasive, painful, expensive, and time-consuming. In addition, it requires the intervention of a skilled clinician owing to difficulties in obtaining the right sample for analysis [16]. In addition, the heterogeneity of BCa is a critical limitation in that a tissue biopsy may not always reflect the entire tumor landscape. The tumors within a bladder vary with respect to morphology, gene expression profile, and mutations. This heterogeneity takes several forms: (1) patient-to-patient (interpatient) heterogeneity; (2) spatial region-to-region variation within a tumor in the same patient (intra-tumoral heterogeneity); and (3) tumor-to-tumor variation, including primary tumor to primary tumor, primary tumor to metastatic site, and metastatic site to metastatic site in the same patient (intertumoral heterogeneity) [4]. Consequently, tissue obtained from different areas of the same tumor, from different sites (primary/metastatic) within the same patient, or from different patients may harbor radically different mutations and gene expression patterns [17]. Thus, the use of minimally invasive procedures such as liquid biopsies is gaining traction [18]. Liquid biopsies, which work by measuring circulating tumor-derived material such as circulating tumor cells (CTCs), circulating cell-free tumor DNA (ctDNA), circulating cell-free tumor RNA (ctRNA), proteins, and extracellular vesicles (EVs) in body fluids, have great potential to overcome the limitations inherent to tissue sampling [18]. Because liquid biopsies are minimally invasive, the risk of complications and pain is reduced. Most importantly, liquid biopsies may better represent tumor heterogeneity and allow monitoring of changes in real time. Although liquid biopsies require minimal medical skills and surgical facilities, they still require specialized laboratory equipment and qualified personnel. Another limitation of liquid-biopsy-based research is that it lacks standard protocols, and the low concentrations of materials may complicate interpretation of the results [19]. Nevertheless, the ease of sample collection and the possibility of time-independent analysis are tremendous assets. Ideally, use of reliable urinary biomarkers for BCa will facilitate patient management and could even provide an at-home service. Tissue and liquid biopsies are depicted and

Urine as a source of biomarkers
The majority of tumor markers are secreted into blood and can be measured in blood; however, they can also be measured in other types of liquid biopsy (e.g., saliva, urine or seminal plasma, and tissues). Some markers are specific to a single type of cancer, whereas others are associated with several types of cancer [12,20]. Unfortunately, unlike the prostate, which secretes prostate-specific antigen into serum, the bladder secretes no organ-specific markers. The urinary bladder is a hollow muscular organ that stores urine (the capacity is about 300-500 mL). It is a small organ, and most BCa tumors are usually less than several centimeters in size; thus, markers secreted by the tumor may not be easy to detect in blood. However, because bladder tumors are in direct contact with urine, many studies focus on identification of urinary biomarkers of BCa. To date, six urine-based methods have been approved by the U.S. Food and Drug Administration (FDA) for clinical use; however, they must still be used in combination with cystoscopy, and their use remains controversial [21,22]. These FDA-approved markers/tests are the Nuclear matrix protein 22 (NMP22) quantitative and qualitative tests, the bladder tumor antigen (BTA) STAT/ TRAK (Polymedco, New York, NY, USA), the ImmunoCyt/ uCyt+ assay (DiagnoCure Inc, Quebec, QC, Canada), and the UroVysion bladder cancer kit (Abbott Molecular Inc, Chicago, IL, USA). Most studies of urine-based biomarkers for BCa are of emerging biomarkers to be applied in the future.

FDA-approved urinary biomarkers for bladder cancer
NMP is a nonchromatin structure responsible for regulating DNA replication, transcription, and RNA processing [23][24][25]. Expression of NMP22 is increased in urothelial tu- Urinary biomarker for bladder cancer mors, and shedding of apoptotic tumor cells into the urine enables detection of this protein in body fluid. However, NMP22 is also present in normal urothelial cells. NMP22 tests include a quantitative enzyme-linked immunosorbent assay (ELISA; the NMP22 test; Abbott) and a qualitative point-of-care (POC) test (NMP22 BladderChek test; Abbott), which are designed to detect the NMP22 antigen in urine, thereby assisting both BCa diagnosis and monitoring of BCa recurrence. The tests are painless and noninvasive assay and provide a positive or negative result within 30 minutes, and the cost is less than half that of cytology. A previous study showed that the sensitivity of the NMP22 ELISA for primary BCa ranges from 44% to 100%, with a specificity of 60% to 95% [26]. Another study reported that the sensitivity in a cancer cohort (comprising patients with primary and recurrent BCa) was 40% (the sensitivity in the primary and recurrent groups alone was 42% and 34%, respectively). These results are not as good as those for the POC assay, which showed a sensitivity of 59%, 63%, and 48% in these same groups. Both assays showed a specificity of 100% in healthy individuals, while the NMP22 ELISA was 99% specific and the POC test was 93% specific in patients with benign disease [27]. A meta-analysis of 19 studies demonstrated that the pooled sensitivity and specificity of the NMP22 POC test was 56% (95% confidence interval [CI], 52%-59%) and 88% (95% CI, 87%-89%), respectively; indeed, the test showed a good ability to detect BCa in both Asian and White populations [28]. Another study compared the NMP22 biomarker with VUC and found that NMP22 was more sensitive for detecting BCa than VUC, especially among patients with microscopic hematuria (60% vs. 35%). However, the specificity of NMP22 was lower than that of VUC (78% vs. 97%, respectively) owing to the presence of NMP22 in normal urothelial cells [29]. Consequently, false-positive results are common in patients with stones, inflammation, and hematuria [30].
The UroVysion bladder cancer kit is a multicolor fluorescent in situ hybridization (FISH) assay designed to estimate aneuploidy of chromosomes 3, 7, and 17, or loss of the 9p21 locus. Its performance with respect to diagnosis and surveillance for BCa has been approved by the FDA. The sensitivity of this test ranges from 69% to 87%, with a specificity between 89% and 96% [38,39]. Similarly, the sensitivity and specificity from a recent meta-analysis (11 studies) was 63% (95% CI, 50%-75%) and 87% (95% CI, 79%-93%) [31]. Another study showed that this kit detected almost twice as many NMIBC tumors as VUC, and identified 88% of invasive tumors (32% of these tumors were missed by VUC) [40]. This test is superior to the NMP22 and BTA tests owing to its high specificity. The assay is not affected by hematuria, inflammation, or other conditions that may result in false-positive readings. Thus, it could be used as an adjunct to VUC, thereby increasing sensitivity while maintaining specificity [41]. Moreover, a preponderance of evidence suggests a role for the UroVysion test for predicting responses to intravesical immunotherapy and BCG treatment [42,43].
In common with other protein-based assays, the Immuno-Cyt fluorescent test is also significantly affected by urinary tract infections, urolithiasis, and benign prostate hyperplasia, which may lead to false-positive results. The detailed characteristics of these assays are described in Table 1.

Non-FDA-approved urinary biomarkers for bladder cancer
Here, we summarize proposed DNA-based, RNA-based, and proteomic/peptidomic markers of BCa.

1) DNA-based urinary biomarkers
DNA tests used for surveillance usually detect loss of heterozygosity, gene methylations, and mutations in tumor cells. DNA methylation and mutations are important for the etiology and pathogenesis of many cancers [54][55][56], including BCa, which is a highly heterogenetic disease [57]. Thus, recent studies have explored molecular classification of both NMIBC and MIBC based on these alterations [58,59]. Alterations in DNA methylation patterns are hallmarks of cancer. Hypomethylation events may result in abnormal activation of genes, which are commonly repressed by DNA methylation. However, hypermethylation of CpG dinucleotides in the promoter regions of tumor suppressor genes can inhibit their transcription in human cells, which gives cancer cells tremendous benefits [59,60]. Thus, methylation status is one of the most studied biomarkers in the follow-up scenario; it is also used to predict treatment responses because it is both chemically stable and quantifiable in liquid biopsies [61].
Recently, Nucleix announced the launch of its BE Safe @Home project, which provides an informatics service for NMIBC patients under surveillance with the Bladder Epi-Check urine test. In consultation with world-leading urologists, the project was implemented in Israel, Spain, and the Netherlands with a view to making surveillance more convenient during the COVID-19 pandemic and beyond.
Other markers related to methylation status are also used to follow-up BCa. Zuiverloon et al. [66] developed a methylation detection assay (based on voided urine) for specific detection of recurrence in patients with NMIBC. A logistic regression model based on methylation of a four-gene panel that combines the APC_a (APC regulator of WNT signaling pathway), TERT_a (telomerase reverse transcriptase), TERT_b, and EDNRB (endothelin receptor type B) genes correlated with BCa recurrence, providing a sensitivity and specificity of 63.3% and 58.3%, respectively, in the test cohort, and of 72.3% and 55.2%, respectively, in the validation cohort [66]. Another study showed a considerably higher sensitivity and specificity (80% and 97%, respectively) using a model based on hypermethylation of SOX1 (SRY-box transcription factor 1) and IRAK3 (interleukin 1 receptor-associated kinase 3) and hypomethylation of a specific LINE1 element in MET (mesenchymal epithelial transition) in urine from BCa patients [67]. Methylation of TWIST1 (twist homolog 1) and NID2 (nidogen 2) is linked to BCa [68][69][70]. One study showed that under adjusted thresholds, methylation of TWIST1 and NID2 has a sensitivity and specificity of 75% and 71%, respectively [69]. More recently, a multi-institutional study reported comparable results using these two methylated genes; this study showed a sensitivity of 58% to 67% and a specificity of 61% to 69%. However, the article noted that prior BCG treatment for NMIBC reduced the accuracy [70]. These findings are promising, despite the limited sensitivity and specificity. However, these markers were identified by research institutions with limited cohorts; thus, large validation tests and methodologic improvements are needed to achieve more accurate results. Gene mutations are related to carcinogenesis of BCa. FGFR3 (fibroblast growth factor receptor 3) is one of the most studied genes; mutations in this gene are found in over 80% of patients with low-grade BCa and are related to a good prognosis [71]. Another well studied gene is TERT (telomerase reverse transcriptase), which has been investigated as a prognostic marker for NMIBC recurrence [72,73]. Allory et al. [74] found that the sensitivity of detecting NMIBC relapse was 19% for FGFR3, 42% for TERT, and 50% for FGFR3 and TERT combined; for comparison, the specificities of mutations in TERT, FGFR3, and a combination of the two, were 73%, 90%, and 71%, respectively.
The Uromonitor urine-based test (Uromonitor, Porto, Portugal) is an ultra-sensitive assay capable of detecting trace amounts of TERT promoter and FGFR3 mutations in tumor cells shed into urine [75]. Screening of targeted alterations is based on a highly sensitive multiplex competitive allelespecific discrimination PCR that allows clear interpretation of results. Compared with Sanger sequencing, this test can detect a very small number of altered cells in a large pool of unaltered cells. In addition, it is superior to next generation sequencing (NGS)-based assays in terms of cost and time. A multicenter validation study revealed that the Uromonitor urine-based test has a sensitivity of 73.5% and a specificity of 73.2% for detection of NMIBC recurrence [76]. Higher sensitivity and specificity were reported in a study based on a cohort of 72 patients first diagnosed with BCa and under surveillance for NMIBC; the data suggest that the sensitivity, specificity, and NPV of the Uromonitor urine-based test are 100%, 96.3%, and 88.9%, respectively [75]. Concerning lowgrade and high-grade recurrence-positive patients, the test showed a detection rate of 62.5% and 75%, respectively. Also, the presence of inflammation or other benign lesions in the urinary tract did not affect results. Thus, routine use of this urine-based test plus cystoscopy could be very cost-effective. Indeed, the sensitivity and specificity are similar to those of cystoscopy (which were 79.4% and 73.2%, respectively) in one study, which suggests that the Uromonitor urine-based test is an appropriate option when cystoscopy cannot be performed or is not available routinely [76].
The UroSEEK urine-based molecular assay is another noninvasive commercially available test (although not approved by the FDA or the European organization). The assay is designed to detect alterations in 11 genes, including 10 typical mutations associated with BCa (FGFR3 [78]. Springer et al. [78] found that this test could detect recurrence with a sensitivity of 68% and a specificity of 80%. Another study revealed that in the setting of early detection, the sensitivity and specificity were 96% and 88%, respectively, with an NPV of 99%; however, the results from a surveillance cohort were less robust (sensitivity of 74%, specificity of 72%, and NPV of 53%) [79]. Nevertheless, the UroSEEK molecular assay was more sensitive than cytology both in the surveillance cohort (71% vs. 25%, respectively) and in the primary detection cohort (95% vs. 43%, respectively); however, the specificity of cytology was superior in the detection cohort (100% vs. 93%) [78]. Accordingly, this test does not show excellent performance for the follow-up of patients with a prior diagnosis of BCa.
All these findings strongly suggest that mutations or methylation status of several genes are promising biomarkers for BCa; thus, a combination of genetic and epigenetic markers for BCa diagnosis and surveillance is both logical and appealing.
The combination of FGFR3 mutations and methylation biomarkers has been tested, with promising results. Beukers et al. [80] investigated the performance of FGFR3 and TERT mutations combined with OTX1 (orthodenticle homeobox 1) methylation in 977 patients with NMIBC. They reported that the sensitivity for detecting NMIBC recurrence was 57%, with a specificity of 59% [80]. Similarly, a 3-plex methylation (combination of OTX1, ONECUT2 [one cut homeobox 2], and OSR1 [odd-skipped related transcription factor 1] methylation) assay combined with the FGFR3 mutation assay detects recurrent NMIBC in voided urine with a sen-Urinary biomarker for bladder cancer sitivity of 79% and a specificity of 77% [81]. Another study combined FGFR3 mutation with methylation of a set of DNA markers (HS3ST2 [heparan sulfate-glucosamine 3-sulfotransferase 2], SEPTIN9, and SLIT2 [slit guidance ligand 2]) and reported a sensitivity of 94.5% for discriminating recurrent tumors. The specificity and NPV for this assay were 75.9% and 98.5%, respectively [82]. Recently, MDxHealth SA (Euronext: MDXH.BR; Herstal, Belgium) announced the commercial launch of its AssureMDx laboratory-based test in the United States, which combines methylation (OTX1, ONECUT2, and TWIST1) and mutation (FGFR3, TERT, and HRAS) biomarkers to identify BCa in patients with hematuria [83]. A multicenter study verified a sensitivity of 93% and a specificity of 86% for BCa diagnosis [84].
Thus, combined analyses of DNA mutations and DNA methylation markers could be used for risk stratification of patients with BCa and for surveillance, forming the foundation for a promising noninvasive urine test.

2) RNA-based urinary biomarkers
RNA-based urinary biomarkers are less well studied than DNA markers. One commercially available RNA test is the Cxbladder Monitor (Pacific Edge Diagnostics, Dunedin, New Zealand). This test evaluates the expression of five urinary mRNAs (insulin-like growth factor binding protein 5 [IGFBP5], homeobox protein Hox-A13 [HOXA13], midkine [MDK], cyclin-dependent kinase 1 [CDK1], and chemokine receptor type 2 [CXCR2]) and incorporates them into a mathematical algorithm that also includes clinical variables, such as primary vs. recurrent BCa and time since tumor occurrence, to generate a score to give a positive or negative result [85]. Lotan et al. [85] compared the Cxbladder Monitor with current FDA-approved urine tests; they examined 1,036 urine samples from 803 patients undergoing surveillance for recurrent BCa and found that the Cxbladder Monitor test significantly outperformed the other tests. The Cxbladder Monitor showed a sensitivity of 91% (95% CI, 86%-95%) when monitoring a BCa population. This is obviously higher than that of other tests: 22% for cytology, 26% for the NMP22 ELISA test, and 11% for NMP22 BladderChek. The NPV of the Cxbladder Monitor was also superior (96%) to that of cytology (87%), the NMP22 ELISA (87%), and NMP22 BladderChek (86%) [85]. In addition, the Cxbladder Monitor showed a sensitivity of 93% (95% CI, 85%-97%) and an NPV of 94% (95% CI, 88%-97%) in patients undergoing routine surveillance for recurrent BCa [85,86]. By contrast, cytology, the NMP22 ELISA, and NMP22 BladderChek showed sensitivities of 22%, 29%, and 8%, and NPVs of 83%, 83%, and 81%, respectively. After seeing the evidence, New Zealand's public healthcare providers have integrated the Cxbladder Monitor into their routine clinical surveillance of BCa patients. A recent study demonstrated that the Cxbladder Monitor accurately detected about 77.8% of recurrence-free patients per year who could avoid unnecessary cystoscopy [87]. During the COVID-19 lockdown, two of New Zealand's public healthcare providers started using the Cxbladder Monitor for in-home testing as an out-patient solution for BCa monitoring; this is especially useful for older patients at high risk for COVID-19.
Another  [88] developed this urine-based test using 450 urine specimens collected from 18 multinational sites and obtained an overall sensitivity of 73%, with specificities of 90% and 77% in hematuria and surveillance patient populations, respectively. In another study, Pichler et al. [89] examined 140 patients with a history of NMIBC who were undergoing routine surveillance and reported for the first time that the Xpert Bladder Cancer Monitor outperforms VUC in terms of sensitivity (84% vs. 33%, respectively) and NPV (93% vs. 76%, respectively), even in those with lowgrade and Ta tumors; however, the specificity of the two tests was similar (91% vs. 94%, respectively). Another multicenter study compared the Xpert Bladder Cancer Monitor, VUC, and the UroVysion bladder cancer kit to determine their follow-up performance in patients previously diagnosed with NMIBC. The Xpert Bladder Cancer Monitor showed a higher sensitivity and NPV (74% [95% CI, 94%-99%] and 93% [95% CI, 89%-96%]), respectively, than did VUC or the UroVysion bladder cancer kit. Moreover, the sensitivity and NPV for high-grade tumors were 83% (95% CI, 64%-93%) and 98% (95% CI, 94%-99%), respectively [90]. The improved NPV of this test in patients under follow-up for BCa suggests that the Xpert Bladder Cancer Monitor is a promising tool for excluding BCa and reducing the need for cystoscopy. However, D´Elia et al. [91] indicated a lower overall sensitivity: 46.2% for detecting NMIBC recurrence. In addition, results from the Bladder Cancer Italian Active Surveillance project, which enrolled 106 patients with low-grade NMIBC who developed recurrence during follow-up and underwent active surveillance, suggest the need to optimize the cutoff value [92]. Thus, further research on larger populations is mandatory before this test can be used routinely in clinical https://doi.org/10.4111/icu.20210194 practice.
In addition to these mRNA-based biomarkers, a number of urinary microRNA (miRNA) biomarkers are emerging. miRNAs interact with their target mRNAs to modulate their expression, thereby controlling many physiologic processes, including carcinogenesis [93]. Most miRNA-based studies have focused on the diagnostic performance of the miRNAs (one special miRNA or miRNA panels) that are differentially expressed in BCa urine [8,94,95]. As a prognostic marker for predicting NMIBC recurrence, Kim et al. [96] found that urinary miR-214 was down-regulated in NMIBC patients who experienced recurrence during surveillance, with a hazard ratio of 2.011 (95% CI, 1.027-3.937), when compared with those without recurrence. Sapre et al. [97] examined the potential of a urinary miRNA panel for predicting the presence of BCa in NMIBC patients undergoing surveillance. They found that a combination of six miRNAs (miR16, miR200c, miR205, miR21, miR221, and miR34a ) yielded an area under the curve (AUC) of 0.85 for distinguishing NMIBC patients with recurrence from those without in the discovery cohort, and they showed high sensitivity (88%) and adequate specificity (48%) (AUC=0.74) in the validation cohort; these data suggest that cystoscopy rates in the validation cohort would have been reduced by 30%.
Lately, another group of noncoding RNAs, named long noncoding RNAs (lncRNAs), which are longer than miRNAs, have emerged as an informative tool for the management of BCa [98]. The advent of high-throughput technology, such as RNA-seq, has identified more than 10,000 unique lncRNAs and clarified their biological functions. lncRNAs play a crucial role in BCa tumorigenesis by modulating cellular pathways involved in cell transformation [99][100][101]. Zhang et al. [102] investigated the potential application of an lncRNA called urothelial cancer associated 1 (UCA1), which is found in BCa patients' urine. They identified that UCA1 showed high sensitivity and specificity (84.4% and 92.4%, respectively) for BCa (AUC=0.898). Moreover, the study highlighted the role of UCA1 as a prognostic biomarker for NMIBC patients who may progress to MIBC (sensitivity, 86.4%; specificity, 92.3%) [102]. Similarly, Eissa et al. [103] showed that UCA1 has great sensitivity (91.5%) and specificity (96.5%) for detecting BCa. Indeed, urinary UCA1 was more accurate than VUC in NMIBC patients [103]. Therefore, accumulation of UCA1 in urine may be a prospective marker for BCa diagnosis and surveillance. HOX antisense intergenic RNA (HOTAIR) is a recently discovered lncRNA that plays an important role in BCa. A study revealed that HOTAIR expression has prognostic value for BCa progression, recurrence, and survival [104]; recurrent NMIBC tumors showed significantly higher HOTAIR expression than did nonrecurrent tumors. Kaplan-Meier analysis revealed that patients with higher HOTAIR expression exhibited significantly earlier recurrence and earlier progression after recurrence. Another pilot study detected lncRNA H19 in urine sediment from 90.5% of BCa patients and 25.9% of healthy controls, making it a supplemental tool for BCa diagnosis [105]. Although expression of other lncRNAs correlates with BCa, most studies were based on tissues or cell lines; therefore, the results require validation in urine samples to confirm their practical applicability as noninvasive BCa biomarkers [106][107][108]. In addition, urinebased research mostly relies on urine sediment, and different lncRNA targets are detected in urine cells. Thus, results may vary because of the paucity of genitourinary-derived cells in urine [109,110], the presence of urinary crystals [111], and the concentration of inhibitors in urine sediment [112,113]. A recent study reported a 16 cell-free urinary lncRNAbased panel, which showed differential expression between NMIBC and urocystitis patients [114]. Among them, the AUCs for four biomarkers (UCA1-201, HOTAIR, HYMA1, and metastasis associated lung adenocarcinoma transcript 1 [MALAT1]) were higher than 0.80, suggesting superior diagnostic performance in differentiating NMIBC from urocystitis. Next, machine learning was used to train the four lncRNA panel as a predictive model; the panel made good predictions in the validation phase, showing a sensitivity of 93.3% and a specificity of 96.7% for discriminating NMIBC from urocystitis.

3) Proteomic/peptidomic urinary biomarkers
The UBC urinary bladder cancer test (IDL Biotech, Borläbger, Sweden) is a commercially available test that measures soluble fragments of cytokeratin 8 and 18 (CK8 and CK18) in urine samples for the purpose of diagnosis and monitoring of BCa. The UBC test is available in ELISA or POC formats. The sensitivity and specificity vary from 12% to 88% and from 77% to 92%, respectively [115]. Pichler et al. [116] found a sensitivity of 61.3% and 64.5%, and a specificity of 77.3% and 81.8%, for the qualitative and quantitative UBC tests, respectively; thus, the assay is more sensitive than VUC (sensitivity, 25.8%) and NMP22 (sensitivity, 12.9%). A multicenter study demonstrated another interesting outcome: the sensitivity for high-grade NMIBC was greater than that for low-grade NMIBC (75.0% vs. 38.8%, respectively), suggesting the potential of UBC as a clinically valuable urinary protein biomarker for detection of high-grade NMIBC [117]. Babjuk et al. [118] reported that the sensitivity and specificity of the UBC ELISA test were 12.1% and 97.2%, respectively, for detecting NMIBC in patients under surveillance. However, when setting a new cutoff to reach a sensitivity of 90%, the specificity declined to 20.4%, indicating that individually installed cutoffs are of no benefit [118]. Concerning the issue of an appropriate cutoff for the UBC test, a study compared the uncorrected and corrected cutoff values of the UBC test for distinguishing BCa patients with and without a recurrence. The overall sensitivity, specificity, and NPV of the uncorrected UBC test were 20.7%, 84.7%, and 72.6%, respectively; those for the corrected UBC test were 20.7%, 79.2%, and 71.3%, respectively. Nonetheless, the receiver operating characteristic analyses showed no statistical significance (both p>0.05), indicating that the UBC test has no diagnostic value [119]. In contrast to other markers, the UBC test is rapid, with results available within 10 minutes; however, the clinical utility of the UBC test for followup of BCa patients remains unconvincing.
The ADXBLADDER in vitro diagnostic test (Arquer Diagnostics, Sunderland, UK) is an up-to-date commercial urine test that detects mini-chromosome maintenance complex component 5 (MCM5), which is a marker of cells that are replicating (or that still have the capability to replicate). In July 2020, the UK National Health Service approved the use of the ADXBLADDER test to help with diagnosis and surveillance of BCa [11]. The test was superior to VUC for detecting BCa recurrence [120]. The ADXBLADDER MCM5 test has sensitivities of 44.1% and 58.8% for low-and highgrade recurrence, respectively, which is more accurate than VUC (sensitivity of 17.6% for both low-grade and high-grade recurrence); thus, this test could be a reliable alternative to VUC for follow-up monitoring. Similar results were reported in a multicenter prospective, blinded study carried out from August 2017 to July 2019 at 21 European Union centers. The study demonstrated that the ADXBLADDER test excluded the presence of high-grade recurrence in 97.8% of cases (compared with 97. 1% with VUC). Meanwhile, the sensitivity of the test was 51.9%, which was much higher than that of VUC (16.7%) [121]. The test demonstrated an impressive NPV of 92% to 99%, using a standard ELISA and with a rapid 2-hour turnaround time. Despite having advantages over VUC, the overall performance of the ADXBLADDER test remains relatively low. The clinical implementation of these biomarkers for the follow-up of BCa must be investigated further in prospective randomized trials in patients with low-grade as well as high-grade tumors.
Cytokeratin fragment 21.1 (CYFRA 21.1) is an ELISAbased assay that measures the concentration of a soluble fragment of cytokeratin 19, the levels of which in urine samples differ between healthy persons and those with BCa (sensitivity, 82%; specificity, 80%) [122]. An extensive meta-analysis study reported not only that the CYFRA 21. 1 level has diagnostic value for BCa, but also that CYFRA 21. 1 levels are higher in those with metastatic BCa than in those with locally invasive disease, inferring a role for detecting metastases [123]. Moreover, Nisman et al. [124] showed that CYFRA 21. 1 detected 100% of carcinoma in situ cases, 92.8% of MIBC cases, and 91.9% of grade 3 tumors; also, the assay detected 65% of recurrent tumors and 71% of primary tumors missed by VUC. Unfortunately, like other protein markers in urine, CYFRA 21. 1 has a high false-positive rate in patients with urinary tract infections, stones, or a history of pelvic radiotherapy, urethral catheterization, or BCG intravesical instillation within the 3 previous months.
The characteristics of non-FDA-approved but commercially available biomarkers are shown in Table 2.

Limitations of current urinary biomarkers
Although newly developed molecular biomarkers for BCa management show superior sensitivity to VUC, few are included in clinical guidelines. Most studies concur that the currently FDA-approved biomarkers do not perform well in real clinical practice as they lack sensitivity or specificity for detecting BCa, or the studies suffered from high false-positive rates. Indeed, a systemic meta-analysis of 57 studies concluded that the false-positive rates of FDA-approved markers range from 44% to 78% [31]. This review also showed that these urinary biomarkers missed 18% to 43% of patients with BCa. These poor performances suggest that limited sensitivity, specificity, and high false-positive rates are the greatest challenge to application of urinary biomarkers in clinical practice.
Most studies of these markers reported "promising" results and initially "positive" observations, which may contribute to early clinical application. However, these preliminary findings lose their glory when tested in real clinical practice; such differences in performance may be related to study design (i.e., selection of suitable cohorts, sufficient number to achieve statistical significance, inadequate followup, and poor validation of results). Moreover, the definition of an ideal marker contains the words easier, faster, better, and cheaper [125]. Easier allows performance in a clinical environment; faster means a rapid turnaround; better means that it must at least be equal to currently clinically available alternatives and provide information that is helpful to clinicians with respect to management of the disease; and cheaper means a reduced economic burden on patients and health services. Nevertheless, many urine tests require highly skilled personnel and specialized laboratory equipment, which increases both time and cost. Initially, the cost of https://doi.org/10.4111/icu.20210194 Owing to the lack of disease-specific symptoms, diagnosis and follow-up of BCa are challenging. Noninvasive urine tests are designed to diagnose BCa, but it remains unclear how they can be integrated effectively into clinical decisionmaking, particularly with respect to discrimination of BCa from other diseases presenting with similar signs and symptoms. This pertains largely to patients with gross hematuria, non-urinary tract infection-related irritative voiding symptoms, and those with microscopic hematuria found on routine urinalysis. These variables should be taken into account during study design, although they are often neglected. The majority of studies are case-control trials comparing artificially composed study cohorts, in which the prevalence of the cancer frequently exceeds 50%. This high disease prevalence is not common in urological practice, and such evaluations lead to an overly optimistic calculation of positive predictive value. Finally, poor study design with respect to selection of patient cohorts and endpoints, and statistical considerations, is one of the reasons for the limited incorporation of novel BCa urinary markers into clinical decision-making. This may be connected to the lack of coherent and comprehensive processes (pipelines) for biomarker development. Therefore, to improve and standardize BCa marker development, Goebell et al. [126] proposed a stepwise procedure comprising four phases (analogous to therapeutic trials). Shariat et al. [127] placed emphasis on this four-phase process and stressed the importance of statistical considerations when conducting research into clinical biomarkers for BCa. It is crucial that researchers are aware of the complexity and poor success rate of biomarkers trying to enter the clinical arena. Fig. 2 describes the limitations of the current urine tests.

THE MICROBIOME AS A SOURCE OF BIOMARKERS
Mounting evidence indicates that the microbiota plays an important role in carcinogenesis and response to treatment. The dogma that urine is sterile has been discredited, and dysbiosis of the urinary microbiota is linked to urological disorders [128][129][130][131][132]. Developed techniques, including highthroughput sequencing, single-cell transcriptomics, and mass spectrometry, offer precise characterization of single enteric, neoplastic, and immune cells, and culture assays enable detection of microbes throughout the urinary system [131,[133][134][135]. A recent study demonstrated an innovative mechanism underlying intravesical immunotherapy with BCG, a live attenuated strain of Mycobacterium bovis. The study suggests that the effects of BCG on BCa may be related to the action of certain microbiomes. The local microbiota may inactivate BCG directly in the bladder or may modulate urothelial sensitivity to BCG through competitive binding to fibronectin [136]. All these advances have focused BCa research on an attempt to understand the relationship between the commensal urinary microbiome and BCa development, as well as its impact on treatment efficacy through modulation of the anti-cancer immune response. The exact nature and role of the most applicable microbes remain unclear, but their potential involvement in BCa is apparent. A study by Xu et al. [137] undertook 16S sequencing analysis of voided urine from six healthy adults and eight BCa patients. They reported that the genus Streptococcus was enriched in the BCa patients [137]. Similarly, another study compared voided urine from 12 BCa patients with that from 11 healthy adults using 16S sequencing. They identified a known colorectal cancer-related genus, Fusobacterium, and showed that it was enriched in the BCa group; however, they found no significant differences in microbial diversity or overall microbiome composition between the groups [138]. Wu et al. [139] compared 31 male BCa patients with 18 healthy control subjects using 16S sequencing of midstream voided urine; they found that enrichment of the genera Acinetobacter, Anaerococcus, and Sphingobacterium was associated with BCa. In addition, greater bacterial richness was also present in urine from NMIBC patients with a high risk for recurrence or progression, indicating that higher bacterial richness may be a potential indicator of NMIBC prognosis. More recently, Chipollini et al. [140] performed 16S ribosomal RNA sequencing in voided urine samples from 10 noncancerous controls, 12 NMIBC patients, and 15 MIBC patients and discovered that the noncancerous group had less species variation and phylogenetic diversity than both the NMIBC and MIBC groups (all p<0.05). The authors also found that Bacteroides, Lachnoclostridium, and Burkholderiaceae species were significantly enriched in noncancerous samples, whereas Bacteroides and Faecalibacterium species were enriched in MIBC samples [140]. Unfortunately, these studies have a critical limitation in that they all analyzed the microbiome in voided urine. Voided urine is not representative of the bladder microbiome because the bacterial DNA detected in midstream voided urine differ substantially from the DNA detected in transurethral catheterized urine, which may be free from external contamination [141,142]. Moreover, although 16S sequencing is a powerful and sensitive tool for microbiome research, there are limitations. First, the results of 16S rRNA gene sequencing are relative rather than absolute; this is because the actual quantity of a particular bacteria is uncertain. Second, the outcomes can be biased due to fluctuating PCR amplification frequency and incomplete reference databases used for sequence analysis. Third, it does not determine cause-andeffect relationships. Fourth, it cannot determine whether bacteria were alive or dead, thereby requiring further urine culture for confirmation [143]. Connections between the urinary microbiome and BCa seemed real, and the possibility of using it as a noninvasive biomarker is highly intriguing. Further investigations should be conducted to improve the studies in this intriguing area.

CONCLUSIONS
Urinary biomarkers have been disregarded because of a perceived lack of sensitivity, a high rate of false positivity, and a paucity of independent validation studies, even though they have great potential as biomarkers of BCa owing to their noninvasive and easy sampling methods. For this reason, substantial improvements in this area have been made in the past few years. In addition, the current COVID-19 pandemic, which has caused inevitable delays in diagnosis, has highlighted the value of quick, efficient, easy, and contactless methods that can be used in clinical practice. Thus, much effort should be devoted to translating potential urinary biomarkers into clinical practice to alleviate the backlog of patients awaiting diagnostic procedures. However, current screening and surveillance urine tests are hampered by a low disease prevalence, which results in a high cost-tobenefit ratio. Although preliminary reports suggest that urinary biomarkers are feasible, there is insufficient detailed information from large study populations to make a final judgment. We urge the scientific and clinical communities to join hands to tackle these problems by modifying protocols and conducting prospective trials to provide a basis for integrating molecular markers into clinical practice. Guidelines on the use of biomarkers would also be welcome. Also, there should be direct head-to-head comparisons of urinary biomarkers to determine the best combination that provides the greatest sensitivity and specificity. The limitations of urinary biomarkers are solvable, albeit time-consuming; however, such markers will be of great benefit to BCa patients around the world. Accordingly, we can use them to improve care for patients with BCa during the COVID-19 pandemic and beyond.

CONFLICTS OF INTEREST
The authors have nothing to disclose.