Nuclear matrix proteins are non chromatin structures which play several roles from DNA replication to gene expression and contributes to the infrastructure of the cell nucleus. During replication in healthy cells, NMP22 regulates the distribution of chromatin to daughter cells and this is normally at low levels. In urothelial tumours, levels of NMPs are high due to cell turnover from tumour apoptosis. NMP22 is one of them and it is the most investigated as an assay in both diagnosis and recurrence of bladder cancer.

Two modes of detection were FDA approved for both diagnosis and surveillance. NMP22 were initially detected with quantitative ELISA in a laboratory where cut-off values were utilised. The second was a qualitative point-of-care test, the NMP22 BladderChek where monoclonal antibodies are used to detect raised NMP22 levels in BC.

In 2015, Chou et al. had done a meta-analysis identifying qualitative NMP22 which has a sensitivity of 69% and specificity of 77% and qualitative NMP22 has 83% in specificity and 70% in sensitivity (6). A meta-analysis by Wang et al. in 2017 showed a pooled sensitivity from of 56% and specificity of 88% for bladder cancer detection from 19 studies (7). However the sensitivity was low when tumour stage and grade were considered with sensitivity increasing steadily with stage of 13.68, 29.49, and 74.03% for Ta to T1 and >T2, respectively. It was also found to have a better diagnostic performance in the Asian population. NMP22 measures the cell turnover that occurs with surface shedding from bladder tumour. This process occurs in benign conditions such as inflammation, infection, bladders stones and haematuria thus resulting in false-positive results.

BTA tests detect human complement factor-H related protein in the urine which is produced by our bodies to protect cells from complement activation. It has an almost identical structure to the complement factor-H related protein produced by bladder cancer cells. There are two forms of BTA assays: (1) BTA Stat test: a “point-of-care (POC)” immunochromatographic assay which utilises five drops of urine to deliver a result within 5 min, and (2) BTA-TRAK test: standard quantitative ELISA measurement of the antigen. The FDA have approved them both for surveillance in BC in conjunction with cystoscopy only.

A meta-analysis conducted reviewing 13 studies identified specificity and sensitivity of BTA stat test to be 67 and 75%, respectively (8). Although BTA stat had shown higher sensitivity that urine cytology, the latter had better specificity. Chou et al. reviewed 22 studies identifying the sensitivity of BTA STAT was 64 % and specificity was 77%. For BTA-TRAK, four studies were evaluated and had similar results with a sensitivity of 65% and specificity was 74% (6). Similarly to other biomarkers, sensitivity had a positive correlation with increasing tumour grade of the BC.

Overall, the sensitivity and specificity of BTA Stat test ranges from 56 to 83% and 64 to 86% and with specificity up to 93% in individuals with benign conditions (9–12), and with BTA TRAK this ranges from 62 to 76% and 51 to 98% (13). Glas et al. carried out a meta-analysis and results of the bivariate analysis showed a sensitivity and specificity of cytology, BTA-Stat and BTA TRAK to be the following 55 and 94%; 70, 75, and 66%, and 65%, respectively (14). Given their lower specificities and similar issue of false positive results in benign conditions such as previous intravesical therapy, kidney stones, infection and presence of ureteric stents or nephrostomy tubes, theses tests are unable to replace cytology and can only be used concurrently as part of surveillance (9, 12, 15, 16).

UroVysion is a molecular test using multicolour fluorescence in situ hybridisation (FISH) assay to detect aneuploidy of chromosomes 3, 7, and 17 and loss of the p16 gene at the 9p21 locus which are genetic abnormalities seen in BC. A sample must have a minimum of 25 cells to be analysed and a positive test is defined by one of the following: (1) Four or more morphologically abnormal cells have polysomy of two or more chromosomes (3, 7, or 17) (2) ≥10 cells with gain of a single chromosome (3) homozygous deletion of 9p21 in 12 cells (16).

Pooled results from a meta-analysis of 13 studies showed a specificity of 83% and sensitivity of 72% in comparison to urine cytology with 96 and 42%, respectively (17). UroVysion showed the sensitivity and specificity of 75.6 and 84.8%, respectively for high grade UCC, 40.8 and 87.8%, respectively for lowgrade UCC (18). Thus it faced a similar challenge to biomarkers in detection of low grade or low stage tumours (19). However the advantage of this assay is the absence of benign conditions such as cystitis, inflammation or haematuria affecting results.

In surveillance, Yoder et al. identified over a period of 29 months that 65% of the cases with positive UroVysion but no visible lesions developed recurrence on follow-up but this was not the case for Dimashkieh et al. as where 46% (158 of 343) of patients who had positive UroVysion tests did not develop UCC during up to the 3 year follow-up (18, 20). Therefore there is variability in its clinical utility. There is also evidence to show its potential in assessing response to intravesical BCG therapy for NMIBC (21).

The Immunocyt/Ucyt+ test uses three fluorescent monoclonal antibodies (M344, LDQ10, and 19A211) which detect carcinoembryonic antigen and sulphated mucin glycoproteins on exfoliated urothelial cells in voided urine. Compoj et al. evaluated 7,244 cases and identified an overall sensitivity of 34.5% for cytology and 68.1% for uCyt+/ImmunoCyt and 97.9% for cytology, 72.3% for uCyt+/ImmunoCyt (22). There was a positive correlation with higher grade and specificity along with sensitivity as observed with other biomarkers. A meta-analysis identified a sensitivity of ImmunoCyt to be 75% and specificity was 78% and in comparison to NMP22, BTA and FISH, it had the highest pooled sensitivity (6). A split study comparing UroVysion, ImmunoCyt and cytology supported this with Immunocyst being more sensitive in detecting low grade tumours (23). Further meta-analysis also supported previous evidence to support the use of Immunocyt in combination with cytology in surveillance to reduce the frequency of follow up required in low risk cancers (24).

However, this test involves advanced technical expertise as a minimum of 500 cells need to be analysed for fluorescence to provide accurate results and thus there is interobserver variability and need for high cellularity specimens. It can be affected by benign conditions such as haematuria albeit not as easily as other biomarkers above (25).

Epigenetic alterations are part of the carcinogenesis. DNA hypermethylation of the CpG islands play a role in the promoter regions of tumour-suppressor genes. This mediates silencing of the affiliated gene which is a known phenomenon in BC. The hyper or hypomethylation of these genes can be detected in tumour cells that are shed into urine and aid diagnosis of BC. This has been reviewed in both primary and recurrent tumours. Bosschieter et al. evaluated 42 studies and identified 8 with high sensitivity and specificity with varying methodologies and heterogenous patient groups (66). Studies with promising results, had no independent validation data and Costa et al. with 94% specificity and 90% sensitivity did not report on tumour grade or stage (67). This is relevant as similarly to other reported biomarkers, results will vary with disease spectrum. As shown in Table 2 section DNA Methylation Markers studies in recurrence are listed but these are mainly small retrospective case-control studies (50). Beuker at al, as shown in Table 2, used a combination of this technique along with DNA mutation analysis with FGFR3 and TERT mutation analysis to improve detection rates showed similar results as increased sensitivity in high grade tumour cells likely due to increased shedding of BC cells (49). There is an insufficient amount of data due to variability in methodology, patient groups and gene panel selection. Studies have used it in combination with DNA mutation analysis.

Other than epigenetic changes described above, another manifestation of this is histone lysine methylation (HxKy). Histone modifications help regulate numerous cell mechanisms such as chromosome condensation, DNA repair and transcription. The site and degree of histone methylation determines the transcriptional activity. H3K9, as mentioned in Table 2 section Histone Tail Modifications are associated with repressed transcription. Other potential histone modications identified are H3K4 and H3K20 methylation which were decreased in BC compared to normal patients and global H4K20me3 levels were predictive for bladder cancer-specific survival (68).

Micro RNAs (miRNA) are short noncoding RNAs that regulate process post-transcriptionally and dysregulation leads to carcinogenesis. The aberrant expression of miRNA has led to its potential use as a biomarker. It can present in bodily fluids as they are protected by RNAse degradation because they are excreted as membrane-protected free circulating miRNAs or in extracellular vesicles (EVs) such as exosomes (69). Initially identification was done using qtPCR but now rapid profiling is done using microassays and miRNA sequencing (56).

Studies listed in Table 2 section miRNA Biomarkers are those with sensitivities of more than 80%. Multiple miRNA diagnostic assays had better sensitivity than single miRNA assays. Chen et al. carried out a meta-analysis 30 studies with 1019 BC patients and 690 controls identifying a pool sensitivity and specificity of 80 and 74%, respectively (70). The AUC for NMIBC was 0.84 and 0.76 for MIBC suggesting higher diagnostic ability in NMIBC patients. Another meta-analysis by Shi et al. evaluated 1,556 cases and 1,347 controls from 31 studies with a pooled specificity and sensitivity of 72 and 76%, respectively (71).

Most studies compared a heterogenous group of BC patients with controls. Study which explored the recurrence setting in both NMIBC and MIBC using miR-145 and miR-200a. This identified a sensitivity of 78% and specificity of 61% in NMIBC patients where lower levels of miR-145 associated with higher grade and lower levels of miR-200a independently predicted recurrence (72). Prospective trials in BC surveillance are required to validate the clinical applicability of this biomarker.

Several assays detecting mRNA biomarkers have been conducted. Table 2 section Multi-Gene Panels summarises them. CxBladder has been extensively studied and has variations which include: (1) Cxbladder® Detect to detect bladder cancer in hematuria patients with a sensitivity of 82% and specificity of 85% (62). (2) Cxbladder® Triage which is used in hematuria patients to rule out BC with a sensitivity of 95% and negative predictive value of 97% (73). (3) Cxbladder® Monitor as a complement to surveillance. It has been compared with urine cytology, NMP22 BladderChek and NMP22 ELISA with a superior SS and SP of 91/96 vs. 22/87%, 11/87 and 26/86 % (74). Koya et al. implemented CxBladder Monitor (CxBM) into local guidelines whereby low risk patients had alternate annual CxBM and cystoscopy therearfter (75). They found that 77.8% of patients were safely managed by only one cystoscopy every 2 years, reducing the total number of annual cystoscopies by 39%. This was reflected in a real world data analysis identifying this advantage of CxBM in clinical practice to have driven its increased utility (76).

Microsatellite analysis (MSA) through PCR targets highly pleomorphic short tandem repeats (STR) which occur in cancer cells with loss of heterozygosity (LOH) causing microsatellite instability. This occurs because of epigenetic silencing or inactivation of the mismatch repair gene which play an integral part in the proliferation of cancer cells. The most common LOH is in chromosome nine but this is also seen to occur in chromosome 4, 8, 11, and 17p (77–80). In comparison to urine cytology, sensitivity was 97 vs. 79% with 95–100% in low grade tumours in a small study of 34 cancer patients with 21 cancer-free subjects (81). A prospective study of 228 patients undergoing BC surveillance had a specificity and sensitivity of 58 and 74%, respectively (82). A further prospective study of 91 patients evaluating MSA in combination with cytology had a sensitivity of 72% in G1–2 and 96% in G3. They found using LOH analysis to improve specificity and all recurrence cases were identified (83). Overall, this test has good sensitivity but difficult to incorporate into present laboratories due to its complexity. Larger prospective studies which include a validation cohort to assess feasibility is required.