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1	Despite these efforts, the greatest advance in bladder cancer diagnosis over the last decade remains blue light cystoscopy. A new body of work in the field has been identified where photodynamic diagnostic is used ex vivo in combination with microdevices.	K.M. Chan	+ 1470 word(s)	1470	2020-06-17 08:55:40	\|
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Chan, K.M.; Gleadle, J.; Li, J.; Vasilev, K.; Macgregor, M. Urinary Biomarkers for Bladder Cancer. Encyclopedia. Available online: https://encyclopedia.pub/entry/2174 (accessed on 19 December 2024).

Chan KM, Gleadle J, Li J, Vasilev K, Macgregor M. Urinary Biomarkers for Bladder Cancer. Encyclopedia. Available at: https://encyclopedia.pub/entry/2174. Accessed December 19, 2024.

Chan, Kit Man, Jonathan Gleadle, Jordan Li, Krasimir Vasilev, Melanie Macgregor. "Urinary Biomarkers for Bladder Cancer" Encyclopedia, https://encyclopedia.pub/entry/2174 (accessed December 19, 2024).

Chan, K.M., Gleadle, J., Li, J., Vasilev, K., & Macgregor, M. (2020, September 25). Urinary Biomarkers for Bladder Cancer. In Encyclopedia. https://encyclopedia.pub/entry/2174

Chan, Kit Man, et al. "Urinary Biomarkers for Bladder Cancer." Encyclopedia. Web. 25 September, 2020.

Urinary Biomarkers for Bladder Cancer

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This entry is adapted from the peer-reviewed paper 10.3390/diagnostics10060383

Blue light cystoscopy (BLC) is the most recent clinical approach in the detection and diagnosis of bladder cancer, a common type of cancer with a high rate of recurrence. Representing a significant advance over previous approaches, this photodynamic diagnostic technique uses a photosensitiser prodrug as an adjunct to white light cystoscopy to enhance the in vivo detection of malignant tissues in the bladder based on their distinctive fluorescence. Whilst it does improve detection rates, BLC remains an invasive and costly procedure. Meanwhile, a variety of noninvasive urine detection methods and related microdevices have been developed. In the following section, we provide the current context for urinary biomarker testing, including commercially available tests and recent development involving microdevices.

noninvasive urinary markers urine microdevices bladder cancer biomarkers

1. Current noninvasive test

New noninvasive tests based on the detection of cancer-specific biomarkers in urine have been in development over the last decades. The different types of target biomarkers found in urine are summarised in Figure 1 below.

Figure 1. Infographic illustrating the currently available (yellow boxes) and potential microdevices (colour bordered boxes) for urinary bladder cancer diagnosis, as described in Tables 1 and 2 below.

Table 1 provides a nonexhaustive list of studies involving these biomarkers that have resulted in commercially available tests for bladder cancer diagnosis. Most efforts have focused on detecting molecular biomarkers, i.e., tumour-specific proteins such as complement factor H-related protein, nuclear matrix protein (NMP) or UBC specific glycoproteins, primarily via immunochemical methods ^[1]. Several urine-based tests that detect these protein biomarkers have been commercialised and six of those approved by the FDA (BTA stat, BTA TRAK, NMP22, NMP22 BladderChek, uCyt+/ImmunoCyt and UroVysion) ^[2]^[3]. Other urinary tests under development that are not, to date, recommended for diagnostic use, include UBC-Rapid/ELISA test, CYFRA 21-1 and BLCA-1/BLCA-4, which assay proteins predominantly present in metastatic cells. These urine-based assays have the advantage of being noninvasive and rapid. They also have higher sensitivity than urine cytology but tend to be less specific and many suffer from variable performance ^[2]^[3]^[4] (Table 1). In addition, they have a lower sensitivity than white light cystoscopy for lower grade tumours (30–60%), with specificity ranging from 60% to 90%, and false-positive results in patients with inflammatory conditions ^[5]. The sensitivity and specificity values reported (Table 1) are highly dependent upon the clinical setting of the studies and discrepancy, therefore, arises from differences in patient cohort (selection criteria and size, tumour grades examined) and study design (primary or recurrent tumours; initial diagnosis or surveillance). Although some of these urine-based tests have been commercialised, their sensitivities and specificities have not been sufficient to justify changes in diagnostic or surveillance protocols. So far, the application of these new urine tests tends not to improve the identification of the disease but merely increase the associated costs ^[6].

In the quest for an accurate urinary biomarker for bladder cancer, many new—omics biomarkers have been reported ^[7]^[8], as recently summarised in comprehensive reviews ^[1]^[9]^[10]. Tests targeting genomic biomarkers that are commercially available are provided in Table 1. These tests typically detect DNA methylation, mutation or mRNA expression using PCR, SAGE and/or mass spectrometry methods. The detection of next-generation “omics” biomarkers may be more accurate but has the disadvantage of relying on expensive reagents and complex analytical platforms.

Table 1. Summary of available tests based on the detection of urinary biomarkers ^[2]^[3]. A non-systematic literature research was performed using the PubMed/Medline database. Searched by using the following keywords: “bladder cancer”, “urinary markers”, “biomarkers”, “diagnosis”, “detection”, “urine biomarkers”, “NMIBC”, “surveillance”. The search was conducted in 2020.

Test (Manufacturer)	Detected Biomarker	Assay type	Sensitivity %	Specificity %	Development Stage*	FDA Approved	Ref.
Urine Cytology	Atypical urothelial cells	Microscopy	33.3	100	Clinical practice	NA	^[11]
NMP22/BladderChek^® (Abbott Laboratories, IL, USA)	Nuclear mitotic apparatus proteins (Nuclear matrix protein-22)	Sandwich ELISA/point-of-care test	33–77	75–97	FDA approved diagnosis and follow-up	1996/2002	^[4]
uCyt+™/Immunocyt™ (Scimedx Corporation, NJ, USA)	Bladder tumour cell associated mucins/carcinoembryonic antigen (antibodies19A211, LDQ10 and M344)	Immunocytochemistry	78–90	77–87	FDA approved follow-up	2000	^[4]
UroVysion ™ (Abbott Laboratories, IL, USA)	Aneuploidy and loss of loci (chromosomes 3, 7, 17 and 9p21 loci)	Multicoloured and multiprobed FISH	50–88	87–98	FDA approved diagnosis and follow-up	2002	^[4]
BTA stat^®/TRAK^® (Polymedco Inc., NY, USA)	Complement factor H-related protein	Dipstick immunoassay/sandwich ELISA	61–87	38–87	FDA approved diagnosis and follow-up	1998/1997	^[4]
UBC-Rapid/ELISA test (IDL Biotech AB, Bromma, Sweden)	Cytoskeletal protein (cytokeratin 8 and 18)	Sandwich ELISA/point-of-care test	48.7–70.5	64.5–79.3	Clinical laboratory research		^[11]
CYFRA 21-1 (Roche Diagnostics, IN, USA)	Cytoskeletal protein (cytokeratin 19)	Electrochemiluminescent immunoassay/ELISA/immunoradiometric assay	82	80	Clinical laboratory research		^[12]
BLCA-4 (Eichrom Technologies, IL, USA)	Nuclear matrix protein (BLCA-4)	Sandwich ELISA	96.4	100	Clinical laboratory research		^[13]
Survivin (Fujirebio Diagnostics Inc., PA, USA)	Inhibitor of apoptosis gene	Bio-dot test	64	93	Clinical trial		^[14]
Cx Bladder (Pacific Edge Diagnostics, PA, USA)	mRNA expression of genes (IGF, HOXA, MDK, CDC and IL8R)	RT-qPCR	91	96	Clinical trial		^[15]
AssureMDx (MDxHealth, CA, USA)	Methylation analysis (OTX1, ONECUT2 and TWIST)/mutation analysis (FGFR3, TERT and HRAS)	Methylation/mutation analysis	57–83	59	Clinical laboratory research		^[16]
Xpert^® bladder cancer monitor (Cepheid Inc., CA, USA)	mRNA expression of genes (CRH, IGF2, UPK1B, ANXA10 and ABL1)	RT-qPCR	73	77–90	Clinical trial		^[17]
UroMark (Kelly:Feber Lab, UCL, UK)	Targeted loci DNA methylation (150 CpG loci)	Microdroplet-based PCR and NGS	98	97	Clinical trial		^[18]
CellDetect ^® (Micromedic Technologies Ltd., Tel Aviv, Israel)	Atypical urothelial cells	Microscopy	94	89	Clinical trial		^[19]
ELISA: enzyme-linked immunosorbent assay; UBC: urinary bladder cancer; RT-qPCR: reverse transcription quantitative polymerase chain reaction; NGS: next-generation sequencing

* According to the ClinicalTrials.gov, a source provided by the U.S. National Library of Medicine ^[20]. Development stages are considered as “Clinical laboratory research” and “Clinical trial”.

2. Novel microdevices for bladder cancer detection

In attempts to reduce the operating complexity of urinary tests without compromising their efficiency, existing (e.g., ELISA) and novel (e.g., cell membrane capacitance) detection approaches have been integrated into microdevices (Table 2). Most of these devices are still at a development stage and have not been rigorously assessed for clinical sensitivity and specificity. They target all types of bladder cancer biomarkers, including protein ^[21]^[22], DNA ^[23], extracellular vesicles ^[24]^[25] and whole cells ^[26]^[27]^[28] (Figure 1) but use advanced materials and nanotechnology to reduce analysis time and sample volumes.

Table 2. Types of microdevices for bladder cancer detection in urine.

Microdevices	Detected Marker	Assay Type	Ref.
Negative pressure-driven microfluidic chip	APOA1 protein via antibody capture on magnetic microbead	ELISA	^[21]
Magnetic nanoprobes with lectins platform	Glycoproteins via Glycoproteomics and CD44 expression	Slot-blot analysis, immunohistochemistry	^[22]
Microfluidic multiplex electrochemical sensor	cfDNA via DNA hairpins bound to electrode, DNA methylation	SPR/EIS	^[23]
Microfluidic antibody capture platform	Cancer cell capture via EpCAM on POx coating	Point-of-care test	^[26]
Antibody conjugated nanoprobes immunosensor	Intracellular Gal-1 protein via immunosensor	Point-of-care test	^[27]
Microfluidic label-free silicon-based electrical approach	Whole cells via membrane capacitance difference	Flow cytometry	^[28]
Microfluidic double filtration	Extracellular vesicles via size filtration	ELISA	^[24]^[25]

APOA1: apolipoprotein 1; SPR: surface plasmon resonance; EIS: electrochemical impedance spectroscopy; cfDNA: cell-free deoxyribonucleic acid; EVs: extracellular vesicles; EpCAM: epithelial cell adhesion molecule; Gal-1: galectin-1; POx: polyoxazoline.

For instance, a negative pressure-driven microchip integrating magnetic microbead-assisted immunocapture of bladder cancer biomarker apolipoprotein A1 (APOA1), report a measurement time of 40 min which is six times faster than a conventional ELISA test ^[21]. The detection of cancer-specific nucleic acid has been achieved using electrochemical impedance spectroscopy (EIS) and Surface plasmon resonance (SPR) within a microdevice containing porphyrin-tagged DNA probes ^[23]. Methods capable of detecting whole bladder cancer cells shed in urine are typically based on cell size, cellular features or the expression of specific proteins (e.g., intracellular galectin-1 or EpCAM). These microdevice-assisted approaches provide real-time detection in microliter volumes of urine ^[27] and reported specificity and sensitivity above 95% for the detection of cancer cells in spiked urine samples ^[26]. Microdevices provide an opportunity for the detection of novel biomarkers such as extracellular vesicles (EV) ^[29]^[30]. Tumour-derived EVs exist in various biological fluids, including urine, and carry cancer-specific proteins and nucleic acids. Technological approaches which capture and isolate bladder cancer EV through double-nanofiltration have been developed ^[24]. One of these approaches reported a sensitivity of 81% and a specificity of 90% in a modest cohort of 16 bladder cancer and eight healthy patients’ urine samples ^[25]. The advantages of these microfluidic devices over traditional EV isolation are that they require less processing steps and are, therefore, simpler and quicker (30 min). Furthermore, the final product contains nucleic acids and proteins that can be further used for genetic research which may provide personalised insight into the tumour heterogeneity. However, microdevice-based testing generally suffers from variations in the chemical and cellular composition of urine, as well as interpatient variability, more than conventional tests ^[31] because of the particularly low volume of the sample tested.

Overall, no urinary test based on urinary biomarker detection has yet replaced cystoscopy in screening and primary detection for NMIBC bladder cancer, according to current oncological guidelines (the American Urological Association (AUA)/Society of Urologic Oncology (SUO) ^[32], National Institute for Health and Care Excellence (NICE) ^[33], European Association of Urology (EAU) ^[34], and National Comprehensive Cancer Network (NCCN) ^[35]. Their use is not recommended for routine testing of low-risk NMIBC follow-up patients, and while they may be considered for the surveillance of high-risk NMIBC follow-up cases, the health care management plan for bladder cancer survivors still recommends including frequent cystoscopy and cytology.

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