Table 4.

Table presenting data for mRNA biomarkers.

Biomarker	Purpose	Number of Patients	Method	Diagnostic Value	Prognostic Value	Predictive capacity	Type of Study	Reference
ROBO1, CRH, IGF2	diagnostic	n = 177 (screening = 95; surveillance = 76; both = 6)	Urine collected from 177 patients and then evaluated d for ROBO1, WNT5A, CDC42BPB, ABL1, CRH, IGF2, ANXA10, and UPK1B expression using GeneXpert Dx (Cepheid, Sunnyvale, CA, USA) automated multiplex RT-PCR platform. Statistical analysis was performed using statistical software R 3.5 (R Foundation for Statistical Computing).	BCa screening and surveillance (detection)	Risk stratification	AUC = 0.923, PPV = 47.1%, NPV = 97.4%, Se. = 92.5%, Sp. = 73.5%	retrospective	[39]
S100A6, TRAM1	diagnostic	n = 113 (HR patients = 66; control group = 47)	RNeasy Midi Kit (QIAGEN, Hilden, Germany) was used to isolate RNA from urine samples of 113 patients which then were quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Measuring of concentration and integrity of pooled urinary RNA was done with Agilent 2100 Bioanalyzer and Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA). cDNA synthesis was done with SMARTer Stranded Total RNA-Seq Kit—Pico Input Mammalian (TaKaRa Bio INC., Kusatsu, prefecture Shiga, Japan) and then purified with Agencourt AMPure XP PCR purification system (Beckman Coulter, Brea, CA, USA). The double-stranded cDNA libraries were quantified with Qubit dsDNA HS Assay Kit and the Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA was sequenced by GATC Biotech AG (GATC Biotech AG, Konstanz, Germany). qPCR was performed using SYBR green and TaqMan Systems. Data analysis was performed with SDS 2.1 software (Applied Biosystems, Foster City, CA, USA).	BCa detection		No data	retrospective	[40]
Xpert BCa Monitor (ABL1, ANXA10, CRH, IGF2, UPK1B)	diagnostic	n = 500 (LG = 287, HG = 194)	Cepheid GeneXpert Instrument System was used for sample processing, nucleic acid amplification, and detection of the target sequences. XLSTAT version 2020.2.2 (Addinsoft) was used for data analysis.	BCa recurrence detection		AUC = 0.73, PPV = 21.3%, NPV = 96.5%, Se. = 72.7%, Sp. = 73.7%	retrospective	[41]
Xpert analysis	Diagnostic and prognostic	n = 254 (LG = 60, HG = 194)	GeneXpert system (Cepheid, Synnyvale, CA, USA) was used to detect target mRNA sequences (ABL1, ANXA, UPK1B, CRH, IGF2) in urine samples of 254 patients using RT-PCR. Data analysis performed with IBM statistica software v.20.	BCa detection		PPV = 66.4%, NPV = 88.9%, Se. = 85.9%, Sp. = 72.3%	prospective	[42]
CYR61	Prognostic and diagnostic	n = 303 (screening set = 30, LG = 12, HG = 18; validation set = 54, LG = 20, HG = 34; FFPE set = 115, LG43, HG = 32; Urine set = 104, LG = 57, HG = 47)	Total RNA was extracted from frozen tissue using TRIzol (Invitrogen, Carlsbad, CA, USA). RNA concentration was determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA’s integrity was determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). qRT-PCR was performed with SYBR PREMIX ex Taq (TaKaRa, Dalian, China) and Mx3005p thermal cycler (Stratagene, La Jolla, CA, USA). Mouse monoclonal antibodies against CYR61 (ab80112; dilution 1:400, Abcam, Cambridge, UK) were used to stain CYR61. Urine samples of 303 patients were tested for CYR61 levels using commercial ELISA test (DY4055, R&D Systems, MN, USA). Data analysis performed with SPSS statistical package, v 17.0 (SPSS Inc., Chicago, IL, USA).	MIBC vs. NMIBC differentiation	BCa progression	AUC = 0.883, Se. = 72.7%, Sp. = 86.0%	retrospective	[43]
Xpert BCa monitor (ABL1, CRH, IGF2, UPK1B, ANXA10)	diagnostic	n = 139 (LG = 62, HG = 63)	Urine samples collected from 139 patients were tested with the XBCM. Data analysis peformed with Excel (Microsoft Corp., Redmond, WA, USA).	BCa detection		AUC = 0.79, PPV = 51%, NPV = 92%, Se. = 58%, Sp. = 89%	retrospective	[44]
	Diagnostic and surveillance	n = 139 (LG = 139)	Urine samples of 139 patients were tested with Xpert BCa Monitor (Cepheid, Sunnyvale, CA, USA). Data analysis performed with STATA® (IC 16.1; StataCorp LLC, College Station, TX, USA).	BCa detection		No data	prospective	[45]
CxBladder (MDK, HOXA13, CDC2, IGFBP5, CXCR2)	diagnostic	n = 28 (LG = 10, HG = 18)	Urine samples collected from 28 patients were sent for urine cytology and CxBladder test. Statistical analysis performed with Microsoft Excel.	BCa detection		PPV = 62%, NPV = 100%, Se. = 100%, Sp. = 75%	prospective	[46]
	diagnostic	n = 1411 (Development data set = 863, LG = 43, HG = 46, haematuria = 774; Independent data = 548, LG = 5, HG = 9, haematuria 534)	Quantitative reverse transcription polymerase chain reaction was used on urine of to measure the expression of 5 genotypic biomarkers (MDK, CDK1, IGFBP5, HOXA13, CXCR2). Data analysis was performed with R 3.5.1 software (R Foundation for Statistical Computing, Vienna, Austria).	Stratification of patients at low and high probability of BCa		NPV = 99.4%, Se. = 92.4%, Sp. = 93.8%	prospective	[47]

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, qRT-PCR—Real-Time Quantitative Reverse Transcription PCR, NPV—negative predictive value, Se.—sensitivity, Sp.—specificity, ROBO1—Roundabout Guidance Receptor 1, CRH—Corticotropin Releasing Hormone, IGF2—Insulin Like Growth Factor 2, S100A6—S100 Calcium Binding Protein A6, TRAM1—Translocation Associated Membrane Protein 1, ABL1—ABL Proto-Oncogene 1, ANXA10—Annexin A10, CRH—Corticotropin Releasing Hormone, UPK1B—Uroplakin 1B, CYR61—Cysteine-rich Angiogenic Inducer 61, MDK—Midkine, HOXA13—Homeobox A13, CDC2—Cell Division Cycle 2 (Cyclin Dependent Kinase 1), IGFBP5—Insulin Like Growth Factor Binding Protein 5, CXCR2—C-X-C Motif Chemokine Receptor 2.