Urothelial carcinomas of the bladder and upper urothelial tract are widespread diseases related to exposure to several external environmental factors (such as tobacco and various chemicals) and chronic inflammation (for example, caused by persistent infections), and are characterized by a higher prevalence among Caucasian males. They carry a consistent risk of recurrence, thus implying a follow-up period after initial treatment. The use of urinary cytology, cystoscopy, and radiological evaluation of the upper urinary tract is recommended during follow-up by several international urological guidelines ^[1][2][1,2].

Cystoscopy is the standard examination for the first assessment and follow-up of non-muscle invasive bladder carcinoma (NMIBC) [3]. However, repeated cystoscopic examinations cause discomfort to patients and represent the principal factor in the high costs for the follow-up of NMIBC [4]. Therefore, urinary cytology has been employed as a complementary and more manageable tool for NMIBC follow-up, showing high sensitivity in high-grade urothelial carcinoma (HGUC) [5] and carcinoma in situ (CIS) but low sensitivity in low-grade urothelial carcinoma (LGUC). Moreover, the results of this test are observer-dependent [6]. Given the limited reliability of urinary cytology, many diagnostic urinary biomarkers have been studied over the last few years to identify recurrences while at the same time avoiding cystoscopic examinations. These are based on a deeper understanding of the molecular background of urothelial carcinomas. Such neoplasms are characterized by a variety of genetic alterations, and different molecular subtypes of urothelial carcinoma have been identified based on gene expression and genomic profiling [7].

Specifically, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP22), ImmunoCyt/uCyt+, and UroVysion as urinary biomarkers for the detection and surveillance of urothelial carcinomas [8]. The BTA test is an antibody-based diagnostic tool that measures the urinary levels of the complement factor H-related protein (CFHrp), a molecule that is secreted by healthy cells to protect against autoimmunity, and its release has also been demonstrated in tumor cells as a potential aid in evading host immune defenses ^[9][10][11][9,10,11]. When compared to cytology, the BTA test has higher sensitivity but lower specificity because it can be influenced by several factors, such as inflammatory conditions, administration of Bacillus Calmette–Guerin (BCG), calculi, foreign bodies, previous instrumentation, bowel interposition, or concomitant genitourinary neoplasms, which can provide false-positive results ^[9][12][9,12]. Similarly, NMP22 is more common in neoplastic urothelial cells than in their non-malignant counterparts, and the process of apoptosis is associated with urine excretion of this protein, which is up to 25 times higher in bladder cancer ^[9][10][11][9,10,11]. Specifically, NMP22 belongs to the nuclear matrix protein (NMP) group, which is an essential component of the nuclear architecture that acts as a scaffold to provide structural integrity and shape to the nucleus. These proteins are involved in DNA replication, ribonucleic acid transcription, and gene expression regulation. However, NMP22 excretion can also occur due to urothelial cell apoptosis in non-malignant pathologies. Elevated NMP22 levels have been observed in individuals with urinary infections, concomitant urolithiasis, a history of bladder interposition, other neoplasms, intravesical therapies, and even cystoscopic examinations, similar to the BTA test. These factors may lead to false-positive results ^[9][13][9,13]. While the BTA and NMP22 tests are based on the detection of urinary molecules, the other tests detect cellular changes. The ImmunoCyt/uCyt+ test is an immunocytological fluorescence test that adopts a combination of three monoclonal antibodies to identify antigens exclusively derived from transitional epithelial cell tumors ^[9][10][11][9,10,11]. Two of these antibodies are conjugated with fluorescein (a green fluorescent dye) and bind to a mucin-like antigen found in the urine of exfoliated neoplastic cells, while the other is conjugated with Texas Red (a red fluorescent dye) and binds to a high-molecular-weight glycosylated variant of carcinoembryonic antigen. ImmunoCyt/uCyt+ is not a diagnostic tool in itself, but rather a monitoring test used in conjunction with cytology. Some authors have shown that ImmunoCyt/uCyt+ has higher sensitivity than cytology for pathological stage Ta-T2 and grade 1–2 tumors and comparable or increased sensitivity for grade 3 tumors and CIS ^[14][15][14,15]. However, other studies have reported a lower sensitivity [16]. In contrast to molecular diagnostic assays, ImmunoCyt/uCyt+ test results are unaffected by inflammatory conditions or instillation therapy. However, the observed variability in reported sensitivity may be attributed to factors such as observer expertise, and specimen stability, handling, and size [9]. In contrast, the UroVysion assay is a fluorescence-based cellular test that employs fluorescence in situ hybridization (FISH) to allow the direct visualization of chromosome copy numbers and specific DNA sequences within the cellular nucleus ^[9][10][11][9,10,11]. Chromosomal aberrations are common in the pathogenesis of bladder cancer (particularly on chromosomes 1, 3, 5, 7, 9, 11, and 17), with the loss of the 9p21 locus of chromosome 9, which houses the p16 tumor suppressor gene, being the most common and earliest genetic alteration observed. The UroVysion assay employs a multitargeted group of probes that bind to the centromeres of chromosomes 3, 7, and 17, as well as to the 9p21 locus. This assay has been shown to have a higher sensitivity than urinary cytology while maintaining comparable or marginally lower specificity ^[9][17][9,17]. Moreover, sensitivity was positively correlated with cancer grade across all studies. Similar to the ImmunoCyt/uCyt+ test, the results are unaffected by concurrent non-malignant conditions since the FISH technique analyzes nuclear alterations. The observed variability in the performance of the UroVysion assay can be attributed to several factors, including variations in scoring criteria, the use of voided urine versus bladder-wash urine, observer expertise, and specimen stability and handling. Furthermore, this method is burdened by non-negligible costs and necessitates the use of skilled technical personnel [9].

Globally, when compared to urine cytology, most of the above-mentioned urinary biomarkers show increased sensitivity with comparable or lower specificity. However, the heterogeneity of outcomes among studies is high [17], and some authors have highlighted the limitations of such screening tests over time [18]. Therefore, cystoscopy cannot currently be replaced by any of these biomarkers, and the need to search for new biomarkers is becoming increasingly important. For example, recent advancements in mRNA (e.g., “Xpert^® BC” [19]) and protein-based enzyme-linked immunosorbent assay (ELISA) analytical biochemical technique (e.g., “UBC^®” [20]) have led to the development of several commercially accessible assays with enhanced sensitivity and specificity; however, their efficacy still needs to be verified in further independent research.

In this context, the Bladder EpiCheck Test, a novel urine assay that analyzes the degree of methylation of a group of genes implicated in bladder carcinogenesis, could represent an interesting alternative [21].

DNA methylation is an epigenetic mechanism that alters gene expression without changing the underlying DNA sequence, and represents a well-known oncogenic process in several cancers, such as urothelial carcinomas [22]. In general, this process involves both tumor suppressor genes and oncogenes that when have a promoter hypermethylation or a hypomethylation, usually shows a low or iper expression. It has been postulated that this mechanism could be at the base of oncogenic processes and could represent a target for tailored therapies in an era in which tumor molecular signatures may represent crucial points in personalized medicine ^[23][24][23,24]. For example, in colorectal cancer, abnormal DNA hypermethylation of potential tumor suppressor genes has been identified as a promising biomarker for cancer screening, and it has recently been clinically applied [25]. Moreover, DNA methylation has been correlated with disease progression in NMIBC ^[21][26][21,26].

The Bladder EpiCheck test (Nucleix Ltd., Pekeris 3, Rehovot 7670203, Israel) analyzes 15 methylation biomarkers and detects the presence of urothelial neoplastic cells based on their methylation profiles. This test is performed on urine samples processed by centrifugation (twice at 1000× g for 10 min at room temperature) and DNA extraction from the cell pellet (using the Bladder EpiCheck DNA extraction kit). Then, the DNA is digested using a methylation-sensitive restriction enzyme, which cleaves the DNA at its recognition sequence if it is unmethylated. Subsequently, quantitative real-time polymerase chain reaction (qPCR) amplification is performed using a real-time platform and the results are analyzed using Bladder EpiCheck software version 1.9. The outcome of this assay is defined as the EpiScore; it consists of a number between 0 and 100, and a value ≥ 60 indicates a positive result [high risk for HGUC], and a score < 60 indicates a high probability of no bladder cancer or that the cancer is still in remission [negative or low risk for HGUC] [26]. In particular, an EpiScore of ≥90 indicates a diagnosis of HGUC. This test showed a higher sensitivity than urine cytology during the follow-up of NMIBC patients but a significantly lower specificity [27].

Databases, including MEDLINE, Embase, and the Cochrane Library, were searched. Combinations of the following keywords were used to search for results up to March 2023: “non-muscle-invasive carcinoma”, “urinary biomarkers”, “urinary cytology”, “cystoscopy”, “Bladder EpiCheck test”, and “DNA methylation”. No language restrictions were applied. Moreover, reviews and articles on Bladder EpiCheck and upper urinary tract carcinoma were included. All studies, abstracts, and non-full text articles concerning the use of Bladder EpiCheck in patients with bladder carcinoma were included without exclusion criteria.

All cohort patients had a histologically proven diagnosis of NMIBC and were undergoing surveillance and therapy for NMIBC. Any stage (CIS, non-invasive papillary carcinoma (Ta), carcinoma with invasion of connective tissue (T1), without clinically identified regional lymph node metastasis (cN0), or clinically demonstrated metastasis (cM0)) according to the tumor, node, and metastasis (TNM) staging system (all versions) was included, and no gender limit was considered. In all studies considered, data from patients with a first diagnosis and patients who underwent intravesical therapy and surveillance cystoscopies at 3–6-month intervals were also included; data from patients with muscle-invasive bladder carcinoma (T2) and patients with non-localized disease [with neoplastic involvement of lymph nodes and clinically demonstrated distant metastases (cN1-3 and cM1)] were excluded.

Moreover, researchers included all studies on neoplasms of the upper urothelial tract with the same pathological and clinical criteria adopted for bladder carcinoma.

The bladder EpiCheck test was performed on patient urinary samples (voided, bladder washing, and ureteral washing) collected before standard cystoscopy. The sensitivity and specificity of Bladder EpiCheck were compared to those of cystoscopy, cytology, and histology results.

Information on study design, inclusion and exclusion criteria, patient data, and outcome measures were analyzed in all consulted studies.

Eleven studies that recruited 2516 patients met the inclusion criteria. All the studies were prospective, blinded, and single-cohort studies, with five regarding multicentric tumors ^{[21][27][28][29][30]}[21,27,28,29,30] and six regarding monocentric tumors ^{[26][31][32][33][34][35]}[26,31,32,33,34,35].

Two studies performed secondary external independent analyses with and without additional patients ^[27][30][27,30], and one study compared the sensitivity of two different urinary markers using qPCR in a population analyzed in a previous study [29].

The sensitivity and specificity of Bladder EpiCheck were compared with a reference standard defined as the results of cystoscopy, cytology, and/or histology. The definition of the reference standard is critical for evaluating the results of Bladder EpiCheck in terms of positivity or negativity for this test. Histological examination was performed to better define cases with positive or suspected cystoscopy results for carcinoma recurrence, both on endoscopic biopsy and transurethral resection of the bladder (TURB). Mapping biopsies were planned for patients with positive or suspicious cytology and negative cystoscopy. Negative white-light cystoscopy, negative urinary cytology, and negative histology are the criteria for defining a patient as negative for recurrence of urothelial carcinoma. All studies used “The Paris System for Reporting Urinary Cytology” (TPS) for cytological specimen classification [36], and a negative urinary cytology was defined when the result was “Negative for High Grade Carcinoma” (NHGUC), while a diagnosis of “Atypical Urothelial Cells” (AUC) and “Suspicious for High Grade Carcinoma” (SHGUC) identified suspicious cases of recurrence of urothelial carcinoma. In cases with positive urinary cytology and negative cystoscopy and histology, a follow-up was performed with cystoscopy and multiple random biopsies or target biopsies when cystoscopy showed a suspicious area [32].

In the study by Pierconti et al. [32], a cytological diagnosis of AUC was considered positive, and patients underwent cystoscopy within three months with multiple random biopsies. In the study by Witjes et al. [21], a histological diagnosis of bladder carcinoma represented the only criterion for defining a recurrence of the neoplasm; in fact, in cases with histology negative for carcinoma and a cytological diagnosis positive for high-grade carcinoma, or in cases with positive or suspicious cystoscopy without a confirmatory biopsy, the sample was considered inconclusive and excluded from the analysis.

The follow-up of patients after bladder resection varies from 3 months [34] to a median follow-up of 3 years [33]. In the Witjes study, follow-up data from Bladder EpiCheck were not available [21].

All studies analyzed the sensitivity of Bladder EpiCheck to evaluate the performance of the test in detecting low- and high-grade urothelial carcinoma. A Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC) were used to measure the EpiScore continuous variables. The positive predictive value (PPV), negative predictive value (NPV), and ROC curve were used to evaluate the accuracy of Bladder EpiCheck. To predict the presence of bladder carcinoma, D’Andrea et al. [27] generated two nomograms using the association between Bladder EpiCheck results and disease recurrence. The authors then used the Bladder EpiCheck results to make decisions in routine clinical practice.