Urinary nuclear matrix protein 22 (NMP22) is a nuclear mitotic protein released from apoptotic cells, playing a role in DNA recombination and replication [5]. The NMP22 test presents advantages in terms of sample collection and operational simplicity, with the potential to fundamentally transform approaches to bladder cancer diagnosis and monitoring [6]. The only two protein-based urinary assays that have received FDA approval and CE marking are NMP22, which is recommended in conjunction with cystoscopy for surveillance purposes, but not for initial diagnosis due to the risk of false positive results stemming from hematuria, urolithiasis, infectious diseases, and endoscopic interventions [7]. NMP22 is a nuclear matrix protein that is highly expressed in malignant urothelial cells and is released into urine by necrotic cells [8]. Both quantitative ELISA assays and qualitative tests are available for targeting NMP22. A systematic review assessing the clinical utility of urinary biomarkers indicated that NMP22 assays exhibited a pooled sensitivity of 69% (CI 50 - 85%) and a pooled specificity of 81% (46 - 93%) [9]. More recent studies have highlighted considerable variability among findings, with instances of notably low sensitivity values reported [10]. Research has demonstrated a sensitivity of 79% and specificity of 64% [11] for NMP22 testing in BLCA. BLCA diagnosis has been explored through various studies, revealing a sensitivity range of 11% to 85% and specificity between 69.6% and 100% [12] for NMP22 testing in detecting BLCA recurrence. In the present investigation, the sensitivity of NMP22 testing as a standalone method for identifying BLCA recurrence was determined to be 60.78%, with a specificity of 78.18%. It is clear that the sensitivity and specificity of NMP22 testing can fluctuate considerably across different research efforts, which can be attributed to variations in study populations, designs, and numerous factors that influence NMP22 testing outcomes [13]. Some studies have indicated that the integration of NMP22 testing with exfoliated urothelial cells significantly enhances diagnostic sensitivity without a notable reduction in specificity. In high-grade bladder cancer (e.g., Grade 3), the NMP22 test (such as the BladderChek NMP22) demonstrates a high positive rate. One study reported that in patients with high-grade tumors, the BladderChek NMP22 achieved a positive rate of 68.4%, which was comparable to the NMP22 ELISA and urinary cytology (68.4% and 63.2%, respectively), indicating no significant differences among these methods in high-grade cancer detection [14]. Additionally, the study noted that the NMP22 test is relatively unaffected by contamination with red or white blood cells, enhancing its clinical utility. In contrast, the performance of the NMP22 test is more pronounced in low-grade bladder cancer (e.g., Grade 1). The same study found that the BladderChek NMP22 yielded a positive rate of 58.3% in low-grade tumors, significantly higher than that of the NMP22 ELISA (33.3%) and urinary cytology (8.3%) [14]. This suggests that the NMP22 test exhibits higher sensitivity in low-grade cancers, potentially addressing the limitations of traditional methods like cytology in detecting such tumors. Overall, in a cohort of 51 patients with urothelial carcinoma, the NMP22 test showed an overall sensitivity of 56.8%, further supporting its advantage in low-grade disease [14]. Other investigations have compared NMP22 with novel biomarkers; for instance, in low-grade non-muscle-invasive bladder cancer, the urine laminin-γ2 monomer demonstrated a higher area under the curve (AUC) than NMP22, indicating that NMP22 may underperform relative to certain emerging markers but still surpasses conventional approaches such as BTA or cytology [15]. In summary, the NMP22 test serves as a rapid and convenient screening tool, exhibiting favorable sensitivity in the graded diagnosis of bladder cancer, particularly for low-grade tumors, and can be utilized as an adjunct in clinical practice. Although NMP22 is also present in normal urothelial cells, which can lead to false-positive results and affect diagnostic specificity, the combined detection of exfoliated urothelial cells and NMP22 cannot replace cystoscopy in the postoperative follow-up of bladder cancer, but it can significantly improve the detection rate of positive cases. Consequently, this combined testing method can offer a more thorough and precise evaluation for bladder cancer patients prior to cystoscopy, thus holding substantial clinical relevance.

Schiffer et al. developed a model featuring four urine polypeptides capable of differentiating NMIBC from MIBC and offering timely indications of BLCA progression [16]. Additionally, a broad array of cytokines has been recognized as valuable targets for monitoring recurrence in NMIBC patients treated with Bacillus Calmette-Guerin (BCG). Research by Cai et al. established that the IL-6/IL-10 ratio serves as an independent predictor of recurrence [17]. Furthermore, baseline urine IL-8 concentrations were significantly associated with tumor recurrence following BCG treatment, indicating that higher urine IL-8 levels in NMIBC patients correlate with shorter tumor recurrence intervals [18]. Kamat et al. expanded on this by employing nine cytokines (IL-2, IL-8, IL-6, IL-1ra, IL-10, IL-12, TRAIL, and TNF-α) to formulate a model for tracking tumor recurrence in NMIBC patients undergoing BCG treatment [19], achieving an accuracy of 85.5%.However, these models are still in the research phase.

The activated leukocyte cell adhesion molecule (ALCAM), a cell adhesion molecule involved in tumor cell migration, has been shown to predict overall survival following cystectomy in bladder cancer patients [20]. Snell et al. demonstrated that urine HAI-1 and EpCAM could forecast prognoses in NMIBC patients and potentially inform treatment strategies for those at high risk for NMIBC [21]. Moreover, overexpression of Snail has been identified as an indicator of NMIBC recurrence [22]. Azevedo et al. utilized a dextran affinity glycoproteomic nanoplatform to pinpoint specific glycoproteins in the urine of patients with low-grade (LG) and high-grade (HG) NMIBC, finding that urine CD44 concentrations were significantly elevated in HG MIBC patients and correlated with unfavorable prognoses [23]. Research into serum or plasma proteins is less extensive compared to urine proteins, with most prognostically relevant proteins being cytokines [24]-[28]. For instance, the expression levels of soluble E-cadherin, MMP2, MMP7, endothelial inhibitors, TGF-β, and uPA have been associated with advanced pathological stages or poor prognoses. Elevated baseline levels of circulating IL-8 have been linked to adverse outcomes in patients receiving sunitinib or pazopanib [29][30]. Comprehensive analyses of multiple cytokines may yield cytokine profiles that contribute to the prognostic evaluation of patients. Kumari et al. found that elevated levels of cytokines such as IL-1RA, IL-10, IL-4, IL-6, IP-10, TNF-α, PDGF, and VEGF correlated with poorer recurrence-free survival in bladder cancer patients [31]. The prognostic potential of markers such as EGFR, EpCAM, BTA, MMP2, tenascin-C, and cystatin B has also been examined [32]; however, none of these markers have undergone independent validation. Additionally, while urine EGFR may relate to bladder cancer prognoses, its concentrations are typically too low for practical measurement via ELISA, presenting a significant barrier to its clinical application and thus keeping it still in the clinical research stage.

The Cxbladder assay is a reverse transcription quantitative PCR test designed to detect and quantify urinary mRNA levels of CDK1, MDK, HOXA13, IGFBP5, and CXCR2. There are two versions of this assay: Cxbladder Detect and Cxbladder Monitor. The former exhibits high sensitivity and specificity (82% and 85%, respectively) for detecting bladder cancer in individuals presenting with hematuria [53]. The Cxbladder Monitor version demonstrates significant sensitivity and negative predictive value (NPV) for identifying patients at low risk of recurrence who might not require repeated cystoscopy [54][55]. Koya et al. illustrated that the inclusion of Cxbladder Monitor in clinical guidelines enabled the safe selection of patients who could be monitored through cystoscopy every two years [56]. Studies have indicated that Cxbladder, as a urinary biomarker, demonstrates a sensitivity ranging from approximately 0.57 to 0.82 and a specificity ranging from approximately 0.74 to 0.88 in the overall diagnosis of bladder cancer, though the number of related studies remains limited [57]. With respect to tumor grading, literature also suggests that sensitivity increases with higher tumor grade, implying that Cxbladder exhibits lower sensitivity and reduced accuracy in low-grade tumors, whereas its sensitivity is notably higher in high-grade tumors. Overall, the diagnostic performance of Cxbladder is inferior in low-grade tumors compared to high-grade tumors; however, these findings are constrained by methodological issues and statistical heterogeneity across studies.By reducing the frequency of cystoscopic examinations, this test has the potential to alleviate the economic burden associated with bladder cancer surveillance and enhance patient adherence to follow-up protocols.

The Xpert® Bladder Cancer test is a urinary assay that evaluates the expression levels of five mRNA markers (ABL1, ANXA10, CRH, IGF2, and UPK1B) that may be overexpressed in bladder cancer, utilizing RT-PCR technology [58]. The test is conducted using the Gene Xpert System (Cepheid), which automates the processes of nucleic acid amplification and detection of mRNA targets, providing a rapid and straightforward assay [58]. Two types of assays have been developed: the first for initial diagnosis, and the second for ongoing surveillance [59]. The Xpert Detection assay has demonstrated an NPV of 99% (CI 98 - 100%) for high-grade tumors [59]. Numerous studies have indicated that active surveillance represents a safe and cost-effective alternative therapeutic strategy for patients with recurrent NMIBC [60]. D’Elia et al. conducted a prospective study involving 230 patients with 52 recurrences, revealing that Xpert BC Monitor achieved a sensitivity of 85.7% for high-grade tumors [61][62]. Additionally, Pichler et al. noted a high sensitivity of 77% for low-grade disease [63]. In a recently published study, the Xpert Bladder Monitor exhibited an overall sensitivity of 74% and specificity of 80%, with sensitivities of 83% for high-grade and 63% for low-grade cancers [64]. Notably, Hurle et al. reported that the Xpert Bladder Monitor could prevent unnecessary cystoscopies without overlooking high-grade cancers, based on a cohort of 106 patients undergoing active surveillance when a cutoff value of <0.4 was employed [65].

Exosomes are bilayered vesicles found in various bodily fluids, including blood, urine, and saliva, and their formation is intricately linked to cellular processes [66]. Cells, as the fundamental units of the human body, secrete a variety of cellular factors, growth factors, and extracellular matrix proteins during their growth, thereby creating a conducive environment for tumor cell proliferation [67]. Research has demonstrated that exosomes, as a form of extracellular vesicle, can penetrate tumor cells and facilitate the transport of proteins and information between them, positioning exosomes as significant tumor biomarkers [68]. Recent studies on the content of urinary exosomes in NMIBC have revealed that the biomacromolecules encapsulated in urinary exosomes, particularly lncRNA, play a significant role in the prognostic evaluation of NMIBC [69]. LncRNAs constitute a category of RNA molecules that do not code for proteins, yet they are integral to various cellular processes such as proliferation, differentiation, and tumor progression [70]. Research conducted by Zhang et al. [71] highlighted the essential function of lncRNAs derived from extracellular vesicles in differentiating tumor cells from normal cells, thereby reflecting the pathological alterations and progression of the disease within tumor cells. One notable lncRNA, PCAT-1, initially identified as a transcription factor in prostate cancer, has been found by Guo et al. [72] to exhibit significantly increased expression levels in NMIBC, with its expression correlating closely with cellular proliferation and apoptosis. Furthermore, urinary extracellular vesicle lncRNA PCAT-1 serves as a critical biomarker for predicting recurrence in patients with NMIBC. However, as of now, research on its use as a diagnostic marker remains very limited, and it is still in the research phase.

In the realm of enhanced cytology, fluorescence in situ hybridization (FISH) employs the principle of complementary base pairing, facilitating the specific attachment of labeled DNA probes to exfoliated urinary cells. This method enables the detection of bladder cancer through immunofluorescence staining. A study by Dimashkieh et al. [73] monitored patients diagnosed with bladder cancer, revealing a postoperative recurrence rate of 45.51%. The utilization of FISH for detecting bladder cancer recurrence yielded a positive detection rate of 57.75%. An examination of the medical histories of recurrent patients during follow-up indicated that FISH exhibited heightened sensitivity in identifying recurrence among patients presenting with hematuria compared to those with a prior history of urinary tract tumors, with sensitivity rates of 60% and 62.2%, respectively. The efficacy of FISH detection is contingent upon the preparation of cell smears, with results dependent on the number of detectable cells, which complicates the identification of tumor recurrence. Additionally, the reliance on specialized equipment such as fluorescence microscopes and the need for proficient laboratory personnel limit the widespread application of this technique. FISH is a universal molecular cytogenetic technique. As a methodology, it is not a direct subject of certification by regulatory agencies.

Urovysion, a multitarget FISH assay, is performed on exfoliated cells obtained from voided urine, yielding a dichotomous response based on specific criteria, including chromosomal alterations (3, 7, 9, and 17) and morphological changes in cells [74]. Recent studies have reported a sensitivity range of 67% - 69% and specificity of 72% - 76%, although variability has been noted in other investigations, particularly in cases involving atypical urothelial cells, where sensitivity and specificity were recorded at 44% - 48% and 78% - 81%, respectively [75][76]. Given the unique attributes of Urovysion, it has been employed in various clinical applications. In a follow-up context for assessing the recurrence risk of bladder cancer, Urovysion demonstrated effective performance, reporting recurrence rates of 16.5% (one positive test) and 33.3% (two positive tests) following transurethral resection of bladder tumor (TURBT) [77]. Moreover, Urovysion has proven beneficial in clarifying atypical urothelial cells identified in urinary cytology, detecting high-grade bladder cancer in 17.9% of cases [78]. Despite these promising findings, Urovysion exhibits lower sensitivity for low-grade tumors compared to Immunocyt [79]. Lastly, encouraging results have been observed in the application of Urovysion for detecting upper urinary tract urothelial cancer [57][80]. UroVysion is a multi-target FISH-based assay employed for the diagnosis and surveillance of bladder cancer, which detects genetic alterations by identifying abnormalities in chromosomes 3, 7, and 17, as well as deletions at the 9p21 locus in exfoliated urinary cells [81]. Although widely utilized in clinical practice, its performance varies between low-grade and high-grade bladder tumors. In low-grade tumors, UroVysion exhibits relatively low sensitivity. For instance, one study reported a sensitivity of only 8% for UroVysion, compared to 62% for ImmunoCyt, suggesting inferior detection capability potentially due to fewer or atypical genetic changes in these tumors [79]. However, UroVysion maintains high specificity, approximately 90%, which is comparable to urine cytology [79]. Systematic reviews further indicate that while UroVysion’s sensitivity in low-grade tumors shows improvement over urine cytology, the exact values differ across studies [82]. For high-grade tumors, UroVysion’s sensitivity increases but remains limited, with reported values around 18% in contrast to 91% for ImmunoCyt [79]. Despite this, UroVysion demonstrates utility in predicting recurrence, as positive results in follow-up studies can anticipate relapse, particularly in high-grade cases [83]. Comparative analyses note an overall sensitivity of 67% - 69% and specificity of 72% - 76% for UroVysion, similar to urine cytology but without significant superiority [75]. Limitations in sensitivity may partly arise from instances where high-grade tumors display tetraploid chromosome counts yet fail to meet UroVysion’s positive criteria [75]. In summary, UroVysion offers high specificity and potential value in predicting recurrence for high-grade bladder cancer, but its sensitivity is suboptimal in both low-grade and high-grade tumors, showing no clear advantage over urine cytology [79][82]. Future investigations should focus on optimizing this method to enhance its efficacy, particularly in low-grade neoplasms. The reliance of UroVysion fluorescence in situ hybridization technology on specialized equipment presents a significant practical barrier in clinical detection for bladder cancer. This is primarily reflected in its requirement for commercial UroVysion kits to perform fluorescence in situ hybridization analysis, which necessitates high-resolution fluorescence microscopes and specific DNA probe systems to detect chromosomal aneuploidies [83]. Such equipment-intensive characteristics may lead to substantial initial investment and maintenance costs, limiting its adoption in resource-constrained medical institutions. Additionally, the procedure demands strict adherence to manufacturer protocols and relies on well-trained technical personnel, thereby increasing operational complexity and the risk of variability [84]. Although this technique has demonstrated diagnostic potential in specific studies, its strong dependency on specialized equipment may hinder broader clinical application, particularly in primary care or remote settings. Cost-effectiveness analyses should be integrated to optimize implementation strategies. Unlike many urine biomarkers that are still in the research phase, Urovysion is a mature, commercially available diagnostic product that has been approved by regulatory agencies and is used globally in clinical practice.

The Uromark assay represents a non-invasive urine test that analyzes a panel of 150 epigenetic alterations utilizing next-generation DNA sequencing (NGS) techniques and RainDrop bisulfite sequencing (BS-Seq), which involves microdroplet-based polymerase chain reaction (PCR) amplification. The utilization of bisulfite-converted DNA has shown significant advantages over traditional PCR techniques, particularly in the context of next-generation sequencing (NGS), which allows for the extraction and analysis of DNA from nearly all urine samples due to its minimal input requirements [85]. This assay has been developed and validated across various cohorts, encompassing a total of 274 patients, yielding impressive sensitivity and specificity rates of 95% and 96%, respectively [86]. Presently, two multicenter studies are underway to assess the efficacy of the Uromark assay in both initial diagnoses and recurrent bladder cancer (BC) [87][88]. Uromark, as a promising detection method, has seen its related research published in academic journals, demonstrating its potential value in the diagnosis and monitoring of bladder cancer. However, transforming it into a mature diagnostic product for routine clinical use still requires the completion of large-scale clinical trials and a complex regulatory approval process.

In 1997, Fradet et al. [89] pioneered the application of ImmunoCyt detection for the diagnosis of bladder cancer, which received approval from the U.S. Food and Drug Administration (FDA) for prognostic evaluation in 2000. This immunocytochemical detection technique employs three monoclonal antibodies to identify exfoliated urothelial cells present in urine. A meta-analysis conducted by He et al. [90] systematically reviewed data from seven studies involving 1602 subjects, focusing on the diagnostic accuracy of ImmunoCyt detection for prostate cancer. The findings revealed a sensitivity of 0.725 (95% confidence interval [CI] 0.683 to 0.765) and a specificity of 0.657 (95% CI 0.629 to 0.685). Although immunocytochemical detection exhibits high sensitivity, its relatively low specificity indicates its potential role as a supplementary tool for monitoring bladder cancer recurrence. When combined with other diagnostic methods, it may enhance the overall sensitivity and specificity of postoperative surveillance for bladder cancer. Future advancements could involve the identification of novel biomarkers to optimize ImmunoCyt detection.