Table 7.

Table presenting data for others biomarkers.

Biomarker	Purpose	Number of Patients	Method	Diagnostic Value	Prognostic Value	Predictive Capacity	Type of Study	Reference
VPAC	diagnostic	n = 103 (group 1 = 65, LG = 30, HG = 35; group 2 = 38, NMIBC)	Urine samples collected from 103 patients were treated with 5-aimnolevulinic acid and then tested for protoporhyrin IX using Nikon ECLIPSE NI fluorescent microscope (Nikon Corporation, Tokyo, Japan). TP4303 solution was used to identify VPAC receptors under fluorescent microscope. All patients underwent cystoscopy/biopsy/TURBT and surgical samples were examined by a pathologist and then compared to the results of conventional cytology, 5-ALA-induced fluorescent cytology, and fluorescent microscopic VPAC receptors examination. Data analysis was performed using Microsoft Excel 2019.	Detection of BCa	n/a	Se. = 89.23%, Sp. = 100%	prospective	[54]
utDNA	Prognostic and diagnostic	n = 57 (BCa = 42, MIBC = 32, NMIBC = 10, control group = 15)	Urine and blood sample of 57 patients acquired on the day of RT. cfDNA was isolated from urine and purified by AMPure XP (Beckman Coulter Life Sciences, Indianapolis, IN, USA) and then analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). utDNA detection using urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq). Data analysis performed with RStudio v1.1.463 environment (RStudio, Boston, MA, USA) and Prism 8 (GraphPad Software, San Diego, CA, USA).	MRD detection	Predicting FPS and OS	AUC = 0.78, PPV = 88%, NPV = 72%, Se. = 81%, Sp. = 81%	Retrospective	[55]
CP	diagnostic	n = 273 (cohort 1 = 97; cohort 2 = 176)	Urine collected from 273 patients was measured for CP levels using commercial ELISA kit (R&D systems, Minneapolis, MN, USA). Data analysis was performed using StatView (v 5.0, Abacus Concepts, Berkeley, CA, USA)		BCa progression	No data	retrospective	[56]
MTC-PCR	prognostic	n = 123 (LG = 37, HG = 86)	Urine samples of 123 patients collected after TURBT and before initiation of BCG instillation, and every year after the last BCG instillation, including induction and maintenance for up to 10 yr. DNA was purified from the pellet using a QIAamp DNA kit (Qiagen, Hilden, Germany). MTC-PCR was used to detect the presence of mycobacterial DNA in urine samples. Statistical analysis performed with R software v.3.5.1 (R Foundation for Statistical Computing, Vienna, Austria).		BCa progression and recurrence	Progression AUC = 0.875 Se. = 83.3%, Sp. = 91.5% Recurrence AUC = 0.868, Se. = 75%, Sp. = 100%		[57]
Multiple Chromatographic Analysis (Fluorescent peak F)	diagnostic	n = 47 (BCa = 23, LG = 19, HG = 4; NMHU = 24)	Urine collected from 47 patients was filtered using a 0.22um nylon membrane filter-LLG Syringe Filter PTFE (AZ chrome, Bratislava, Slovak Republic). Urines samples were analyzed using RP-HPLC system Prominence 20A (Shimadzu Co., Kyoto, Japan). Data analysis was performed using Software LC solution (Shimadzu Co., Kyoto, Japan).	BCa vs. NMHU differentiation		AUC = 0.824, PPV = 78%, NPV = 88%, Se. = 90%, Sp. = 74%	retrospective	[59]
miR-34a-5p, miR-205-3p, miR-210-3p	diagnostic	n = 147 (prospective: BCa = 15, NMIBC = 12, MIBC = 3, LG = 8, HG = 7; control group = 16; retrospective: BCa = 66, NMIBC = 56, MIBC = 10, LG = 25, HG = 41; control group = 50)	BenchMark XT immunostainer (Ventana Medycal Sysems, Tucson, AZ, USA) was used for immunohistochemical staining of tissue samples. The stains were inspected using an Olympus BX50 and Olympus BX46 mucroscopes (Olympus Europe) by two pathologists. Total RNA was extracted from urine supernatant samples of 116 patients using the Urine microRNA Purification kit (Norgen Biotek, Thorold, Canada). RNA concentration was quantified by Invitrogen Qubit® 4 Fluorometer with Qubit® microRNA Assay Kit (Invitrogen, Milan, Italy). Small RNA transcripts were converted into barcoded cDNA libraries with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA) and run on Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA). Differential expression analysis was performed with DESeq2 Bioconductor’s package (version 1.22.2). miRNA biomarkers were replicated using miRCURY LNA miRNA PCR Assays (Qiagen, Milan, Italy). Reverse transcription was performed using the miRCURY LNA RT kit (Qiagen, Milan, Italy). Functional enrichment analysis of miRNA target genes was performed using RBiomirGS v0.2.12. Data analysis was performed using Graphpad Prism 8 sofware (Graphpad Software, San Diego, CA, USA) and Quasar-Orange software (Bioinformatics Laboratory of the University of Ljubljana).	BCa detection		AUC = 0.92	Prospective and retrospective	[60]
Urinary VOC analysis	Diagnostic and surveillance	n = 305 (BCa = 96, Control group = 209)	Urine samples of 305 patients were collected before cystoscopy. Sulphuric acid solution (Fisher Scientific, Waltham, MA, USA) was added to urine. A PerkinElmer Clarus 500 GC-MS single quadrupole system (PerkinElmer, Waltham, MA, USA) and PAL COMBI-xt autosampler (CTC Analytics, Zwingen, Switzerland) were used to analyse samples. Data analysis performed with Metaboanalyst.	BCa detection		Eight-Voc diagnostic biomarker AUC = 0.77, Se. = 71%, Sp. = 72% Six-VOC surveillace biomarker AUC = 0.80, Se. = 71%, Sp. = 80%	retrospective	[61]

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, NPV—negative predictive value, Se.—sensitivity, Sp.—specificity, cfDNA—cell-free DNA, utDNA—urine tumour DNA RP-HPLC—reverse-phase high-performance liquid chromatography, ELISA—enzyme-linked immunosorbent assay, uCAPP-Seq—urine Cancer Personalized Profiling by Deep Sequencing, VPAC—Combined vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide family of cell surface receptors, utDNA—urine tumour DNA, CP—Ceruloplasmin, MTC-PCR—Mycobacterium tuberculosis complex polymerase chain reaction, miR—microRNA, VOC—Volatile Organic Compounds.