Table 1.

Table presenting data for methylation biomarkers.

Biomarker	Purpose	Number of Patients	Method	Diagnostic Value	Prognostic Value	Predictive Capacity	Type of Study	Reference
VIM, OSTM1, SLC4A10, AC092805.1, ONECUT2	Prognostic	Cohort 1 = 192 (116 BCa, MIBC = 47, NMIBC = 68; 76 non-BCa)	Urine samples collected from 464 patients before cystoscopy or surgery. Genomic DNA was extracted using the QIAamp DNA blood Mini Kit (Qiagen, Germany, Catalog No. 51106) and quantified by the Qubit Assay (Thermo Fisher Scientific, USA, Catalog No. Q32851). Bisulfite treatment was performed on genomic DNA with the EZ-96-DNA Methylation-Direct MagPrep Kit (Zymo Research, USA, Catalog No. D5044). The methylation of bisulfite-treated DNA was analyzed by a 22-marker BCA DNA (AnchorDX, China, Catalog No. UME043) on the QuantStudio 3 Real-Time PCR System (Thermo Fisher, Waltham, Ma, USA). EpiTect PCR Control DNA Set (Qiagen, Germany, Catalog No. 59695) was used as positive and negative controls. DNA quantified by the Meth-Quant Master Mix (AnchorDx, China, Catalog No. UME043-01 ) and the 22-marker BCA Detect Panel (AnchorDx, China, Catalog No. UME043-02).		Preoperative risk stratification of BCa	Non-BCa AUC = 0.78, PPV = 88.6%, NPV = 81.8%, Se. = 87.8%, Sp. = 82.9%	Retrospective	[19]
						LMR-NMIBC AUC = 0.78, PPV = 54.5%, NPV = 91.7%, Se. = 46.2%, Sp. = 93.9%
						NMIBC+MIBC AUC = 0.78, PPV = 80.4%, NPV = 84.8%, Se. = 83.1%, Sp. = 82.4%
		Cohort 2 = 98 (59 BCa, MIBC = 22, NMIBC = 35; 39 non-BCa)				Non-BCa AUC = 0.821, PPV = 81.1%, NPV = 87.2%, Se. = 91.1% Sp. = 87.2%
						LMR-NMIBC AUC = 0.821, PPV = 54.5%, NPV = 90.5%, Se. = 42.9%, Sp. = 93.8%
						NMIBC+MIBC AUC = 0.821, PPV = 84.4%, NPV = 92.0%, Se. = 90.5%, Sp. = 86.8%
ONECUT2, VIM	Diagnostic and prognostic	Cohort 1 = 192 (116 BCa, MIBC = 47, NMIBC = 68; 76 non-BCa)		BCa detection		AUC = 0.898, PPV = 88.6%, NPV = 80.8%, Se. = 87.1%, Sp. = 82.9%	Retrospective
		Cohort 2 = 98 (59 BCA, MIBC = 22, NMIBC = 35; 39 non-BCa)				AUC = 0.921, PPV = 92.9%, NPV = 83.3%, Se. = 88.1%, Sp. = 89.7%
		Cohort 3 = 174 (35 BCa, MIBC = 5, NMIBC = 29; 147 non-BCa)				AUC = 0.935, PPV = 60.8%, NPV = 97.6%, Se. = 91.2%, Sp. = 85.7%	Prospective
FAM19A4	diagnostic	n = 208 (BCa = 108; LG = 45, HG = 63; Control group = 100, haematuria = 34, benign urological conditions = 43, healthy = 23)	Urine samples of 208 patients were collected before cystoscopy or TURBT. DNA was isolated from urine using QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). DNA concentrations were measured with NanoDrop 1000 (ThermoFisher Scientific, Waltham, MA, USA). EZ DNA Methylation™ Kit (Zymo Research, Orange, CA, USA) was used for bisulphite conversion. qMSP was performed to identify methylation values of targeted biomarkers. Data analysis were performed with R Statistical Software (v.3.6.1, R Foundation for Statistical Computing, Vienna, Austria).	BCa detection		AUC = 0.72	Retrospective	[20]
GHSR						GHSR AUC = 0.89, MAL AUC = 0.85, GHSR/MAL AUC = 0.89, Se. = 80%, Sp. = 93%
MAL
miR-129						AUC = 0.83
miR-935						AUC = 0.79
PHACTR3						AUC = 0.69
PRDM14						AUC = 0.88
SST						AUC = 0.84
ZIC1						AUC = 0.88
Altogether						Se. = 81%, Sp. = 95%
Bladder epicheck	diagnostic	n = 205 (HG NMIBC = 135, T1G3 = 86, T1G2 = 49, CIS = 70)	Urine samples collected from 205 patients were centrifuged twice. DNA was extracted using Bladder EpiCheck DNA extraction kit and prepared for the PCR assay using the Bladder EpiCheck test kit. Data analysis performed with GraphPad-Prism 5 software (Graphpad Software-Prism 5 software, San Diego, CA, USA) and MedCalc version 10.2.0.0 (MedCalc Software, Mariakerke, Belgium).	BCa detection		AUC = 0.94, PPV = 60%, NPV = 97.7%, Se. = 94.3%, Sp. = 79.6%	Prospective	[21]
DNA hypermethylation (RASSF1, RARB, DAPK, TERT, APC)	diagnostic	n = 85 (BCa = 50, NMIBC = 37, MIBC = 5; control group = 35)	Urine samples were collected form 85 patients before cystoscopy. DNA was extracted from urine using the Cells and Tissue DNA Isolation Kit (Norgen Biotek Corp., Thorold, Canada). Sodium bisulfite modification of DNA was performed with the EZ DNA Methylation-GoldKit (Zymo Research, Orange, CA). Quantification of the percentage of methylation of DNA was performed with Luna Universal Probe qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) and on Applied Biosystems StepOnePlus Real Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA).	BCa detection		AUC = 0.76, PPV = 90%, NPV = 53%, Se. = 61.4%, Sp. = 86.4%	Prospective	[22]
PCDH17, POU4F2, PENK	diagnostic	n = 252 (panel design: BCa = 18, control group = 15; validation: BCa = 107, control group = 100)	DNA from the urinary cell pellets was extracted using QIAamp DNA Mini Kit (Qiagen). DNA quality and quantity were assessed using a NanoDrop2000 (Thermo Scientific, Wilmington, DE, USA). Bisulfite conversion was performed with a ZYMO EZ DNA Methylation-Gold Kit (ZYMO, Irvine, CA, USA). Sequencing was performed on an Illumina Hiseq platform. The bisulfite modification reaction was executed by 96-Well GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). DNA amplification was performed on 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).	BCa detection		AUC = 0.96, Se. = 87%, Sp. = 97%	Retrospective	[23]

Abbreviations: AUC—Area under the ROC Curve, n—number of patients participating in study, HG—high grade, LG—low grade, BCa—bladder cancer, PCR—polymerase chain reaction, PPV—positive predictive value, qPCR—quantitative polymerase chain reaction, NPV—negative predictive value, Se.—sensitivity, Sp.—specificity, VIM—Vimentin, OSTM1—Osteoclastogenesis Associated Transmembrane Protein 1, SLC4A10—Solute Carrier Family 4 Member 10, AC092805.1—, ONECUT2—One Cut Homeobox 2, FAM19A4—TAFA chemokine like family member 4, PHACTR3—Phosphatase And Actin Regulator 3, PRDM14—PR/SET Domain 14, SST—Somatostatin, ZIC1—Zic Family Member 1, RARB—Retinoic Acid Receptor Beta, DAPK—Death Associated Protein Kinase 1, TERT—Telomerase Reverse Transcriptase, APC—Adenomatous Polyposis Coli, miR—microRNA.