{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T17:09:00Z","timestamp":1787677740968,"version":"build-2784847793"},"reference-count":48,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2024,1,6]],"date-time":"2024-01-06T00:00:00Z","timestamp":1704499200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"German Network for Bioinformatics Infrastructure (de.NBI)","award":["FKZ 031 A 534A"],"award-info":[{"award-number":["FKZ 031 A 534A"]}]},{"name":"PURE and VALIBIO","award":["FKZ 031 A 534A"],"award-info":[{"award-number":["FKZ 031 A 534A"]}]},{"name":"CUBiMed.RUB","award":["FKZ 031 A 534A"],"award-info":[{"award-number":["FKZ 031 A 534A"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Diagnosing urothelial cancer (UCa) via invasive cystoscopy is painful, specifically in men, and can cause infection and bleeding. Because the UCa risk is higher for male patients, urinary non-invasive UCa biomarkers are highly desired to stratify men for invasive cystoscopy. We previously identified multiple DNA methylation sites in urine samples that detect UCa with a high sensitivity and specificity in men. Here, we identified the most relevant markers by employing multiple statistical approaches and machine learning (random forest, boosted trees, LASSO) using a dataset of 251 male UCa patients and 111 controls. Three CpG sites located in ALOX5, TRPS1 and an intergenic region on chromosome 16 have been concordantly selected by all approaches, and their combination in a single decision matrix for clinical use was tested based on their respective thresholds of the individual CpGs. The combination of ALOX5 and TRPS1 yielded the best overall sensitivity (61%) at a pre-set specificity of 95%. This combination exceeded both the diagnostic performance of the most sensitive bioinformatic approach and that of the best single CpG. In summary, we showed that overlap analysis of multiple statistical approaches identifies the most reliable biomarkers for UCa in a male collective. The results may assist in stratifying men for cystoscopy.<\/jats:p>","DOI":"10.3390\/ijms25020738","type":"journal-article","created":{"date-parts":[[2024,1,8]],"date-time":"2024-01-08T07:59:20Z","timestamp":1704700760000},"page":"738","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Use of Multiple Machine Learning Approaches for Selecting Urothelial Cancer-Specific DNA Methylation Biomarkers in Urine"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4345-2213","authenticated-orcid":false,"given":"Christina U.","family":"K\u00f6hler","sequence":"first","affiliation":[{"name":"Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Ruhr University Bochum (IPA), B\u00fcrkle-de-la-Camp Platz 1, 44789 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3756-4347","authenticated-orcid":false,"given":"Karin","family":"Schork","sequence":"additional","affiliation":[{"name":"Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum and Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Gesundheitscampus 4, 44081 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0737-1114","authenticated-orcid":false,"given":"Michael","family":"Turewicz","sequence":"additional","affiliation":[{"name":"Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum and Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Gesundheitscampus 4, 44081 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2687-7444","authenticated-orcid":false,"given":"Martin","family":"Eisenacher","sequence":"additional","affiliation":[{"name":"Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum and Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Gesundheitscampus 4, 44081 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0299-489X","authenticated-orcid":false,"given":"Florian","family":"Roghmann","sequence":"additional","affiliation":[{"name":"Department of Urology, Marien Hospital Herne, University Hospital of the Ruhr University Bochum, H\u00f6lkeskampring 40, 44625 Herne, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joachim","family":"Noldus","sequence":"additional","affiliation":[{"name":"Department of Urology, Marien Hospital Herne, University Hospital of the Ruhr University Bochum, H\u00f6lkeskampring 40, 44625 Herne, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3313-8845","authenticated-orcid":false,"given":"Katrin","family":"Marcus","sequence":"additional","affiliation":[{"name":"Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum and Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Gesundheitscampus 4, 44081 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Br\u00fcning","sequence":"additional","affiliation":[{"name":"Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Ruhr University Bochum (IPA), B\u00fcrkle-de-la-Camp Platz 1, 44789 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heiko U.","family":"K\u00e4fferlein","sequence":"additional","affiliation":[{"name":"Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Ruhr University Bochum (IPA), B\u00fcrkle-de-la-Camp Platz 1, 44789 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,1,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"11","DOI":"10.3322\/caac.21166","article-title":"Cancer statistics, 2013","volume":"63","author":"Siegel","year":"2013","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"9.e19","DOI":"10.1016\/j.urolonc.2021.05.009","article-title":"Discomfort and relieving factors among patients with bladder cancer undergoing office-based cystoscopy","volume":"40","author":"Kukreja","year":"2022","journal-title":"Urol. Oncol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/S0090-4295(02)02136-2","article-title":"Sensitivity and specificity of commonly available bladder tumor markers versus cytology: Results of a comprehensive literature review and meta-analyses","volume":"61","author":"Lotan","year":"2003","journal-title":"Urology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1136\/jcp.48.4.314","article-title":"The clinical value of urinary cytology: 12 years of experience with 615 patients","volume":"48","author":"Maier","year":"1995","journal-title":"J. Clin. Pathol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2787","DOI":"10.21037\/tau-20-1327","article-title":"Trends in urine biomarker discovery for urothelial bladder cancer: DNA, RNA, or protein?","volume":"10","author":"Ward","year":"2021","journal-title":"Transl. Androl. Urol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.ctrv.2018.05.012","article-title":"Novel urinary biomarkers for the detection of bladder cancer: A systematic review","volume":"69","author":"Tan","year":"2018","journal-title":"Cancer Treat. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Larsen, L.K., Lind, G.E., Guldberg, P., and Dahl, C. (2019). DNA-Methylation-Based Detection of Urological Cancer in Urine: Overview of Biomarkers and Considerations on Biomarker Design, Source of DNA, and Detection Technologies. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20112657"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Lozano, F., Raventos, C.X., Carrion, A., Trilla, E., and Morote, J. (2020). Current status of genetic urinary biomarkers for surveillance of non-muscle invasive bladder cancer: A systematic review. BMC Urol., 20.","DOI":"10.1186\/s12894-020-00670-x"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"927","DOI":"10.1016\/j.euo.2021.10.003","article-title":"Diagnostic Accuracy of Novel Urinary Biomarker Tests in Non-muscle-invasive Bladder Cancer: A Systematic Review and Network Meta-analysis","volume":"4","author":"Laukhtina","year":"2021","journal-title":"Eur. Urol. Oncol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Malinaric, R., Mantica, G., Lo Monaco, L., Mariano, F., Leonardi, R., Simonato, A., van der Merwe, A., and Terrone, C. (2022). The Role of Novel Bladder Cancer Diagnostic and Surveillance Biomarkers-What Should a Urologist Really Know?. Int. J. Environ. Res. Public Health, 19.","DOI":"10.3390\/ijerph19159648"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Matuszczak, M., Kilja\u0144czyk, A., and Salagierski, M. (2022). A Liquid Biopsy in Bladder Cancer-The Current Landscape in Urinary Biomarkers. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23158597"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2861","DOI":"10.1002\/ijc.32356","article-title":"Noninvasive diagnosis of urothelial cancer in urine using DNA hypermethylation signatures-Gender matters","volume":"145","author":"Bonberg","year":"2019","journal-title":"Int. J. Cancer"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Tan, W.S., Feber, A., Dong, L., Sarpong, R., Rezaee, S., Rodney, S., Khetrapal, P., de Winter, P., Ocampo, F., and Jalil, R. (2017). DETECT I & DETECT II: A study protocol for a prospective multicentre observational study to validate the UroMark assay for the detection of bladder cancer from urinary cells. BMC Cancer, 17.","DOI":"10.1186\/s12885-017-3758-7"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1002\/pros.23968","article-title":"Development of a multivariable risk model integrating urinary cell DNA methylation and cell-free RNA data for the detection of significant prostate cancer","volume":"80","author":"Connell","year":"2020","journal-title":"Prostate"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.3389\/fonc.2019.01281","article-title":"A Diagnostic Panel of DNA Methylation Biomarkers for Lung Adenocarcinoma","volume":"9","author":"Shen","year":"2019","journal-title":"Front. Oncol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Ren, J., Zhang, B., Wei, D., and Zhang, Z. (2020). Identification of Methylated Gene Biomarkers in Patients with Alzheimer\u2019s Disease Based on Machine Learning. BioMed Res. Int., 2020.","DOI":"10.1155\/2020\/8348147"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e9654","DOI":"10.7717\/peerj.9654","article-title":"Identification of DNA methylation patterns and biomarkers for clear-cell renal cell carcinoma by multi-omics data analysis","volume":"8","author":"Liu","year":"2020","journal-title":"PeerJ"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"758926","DOI":"10.3389\/fgene.2021.758926","article-title":"Recognition of DNA Methylation Molecular Features for Diagnosis and Prognosis in Gastric Cancer","volume":"12","author":"Liu","year":"2021","journal-title":"Front. Genet."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"17592","DOI":"10.18632\/aging.203249","article-title":"DNA methylation biomarkers for diagnosis of primary liver cancer and distinguishing hepatocellular","volume":"13","author":"Bai","year":"2021","journal-title":"Aging"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Sprang, M., Paret, C., and Faber, J. (2020). CpG-Islands as Markers for Liquid Biopsies of Cancer Patients. Cells, 9.","DOI":"10.3390\/cells9081820"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Wu, S.P., Cooper, B.T., Bu, F., Bowman, C.J., Killian, J.K., Serrano, J., Wang, S., Jackson, T.M., Gorovets, D., and Shukla, N. (2017). DNA Methylation-Based Classifier for Accurate Molecular Diagnosis of Bone Sarcomas. JCO Precis. Oncol., 2017.","DOI":"10.1200\/PO.17.00031"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"398","DOI":"10.1093\/bioinformatics\/btx622","article-title":"Tumor origin detection with tissue-specific miRNA and DNA methylation markers","volume":"34","author":"Tang","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1038\/s41596-019-0251-6","article-title":"Machine learning workflows to estimate class probabilities for precision cancer diagnostics on DNA methylation microarray data","volume":"15","author":"Maros","year":"2020","journal-title":"Nat. Protoc."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1341","DOI":"10.1158\/1078-0432.CCR-20-3281","article-title":"Genome Methylation Accurately Predicts Neuroendocrine Tumor Origin: An Online Tool","volume":"27","author":"Hackeng","year":"2021","journal-title":"Clin. Cancer Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1111\/1556-4029.14872","article-title":"A novel multiplex assay system based on 10 methylation markers for forensic identification of body fluids","volume":"67","author":"Huang","year":"2022","journal-title":"J. Forensic Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1080\/15592294.2021.1890885","article-title":"DNA-methylation for the detection and distinction of 19 human malignancies","volume":"17","author":"Danilova","year":"2022","journal-title":"Epigenetics"},{"key":"ref_27","first-page":"47","article-title":"Detection of bladder cancer in urine sediments by a hypermethylation panel of selected tumor suppressor genes","volume":"18","author":"Pietrusinski","year":"2017","journal-title":"Cancer Biomark. Sect. A Dis. Markers"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1089\/dna.2013.2030","article-title":"Hypermethylation of TWIST1 and NID2 in tumor tissues and voided urine in urinary bladder cancer patients","volume":"32","author":"Yegin","year":"2013","journal-title":"DNA Cell Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1483","DOI":"10.1158\/1055-9965.EPI-11-0067","article-title":"Detection of bladder cancer using novel DNA methylation biomarkers in urine sediments","volume":"20","author":"Chung","year":"2011","journal-title":"Cancer Epidemiol. Biomark. Prev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"104946","DOI":"10.18632\/oncotarget.20737","article-title":"Soluble chemokine (C-X-C motif) ligand 16 (CXCL16) in urine as a novel biomarker candidate to identify high grade and muscle invasive urothelial carcinomas","volume":"8","author":"Lang","year":"2017","journal-title":"Oncotarget"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"7457","DOI":"10.1158\/1078-0432.CCR-04-0930","article-title":"Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients","volume":"10","author":"Friedrich","year":"2004","journal-title":"Clin. Cancer Res."},{"key":"ref_32","first-page":"99","article-title":"Study of arachidonic Acid pathway in human bladder tumor","volume":"3","author":"Matsuyama","year":"2009","journal-title":"Subst. Abus. Res. Treat."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Liu, J., Ma, H., Meng, L., Liu, X., Lv, Z., Zhang, Y., and Wang, J. (2021). Construction and External Validation of a Ferroptosis-Related Gene Signature of Predictive Value for the Overall Survival in Bladder Cancer. Front. Mol. Biosci., 8.","DOI":"10.3389\/fmolb.2021.675651"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1994","DOI":"10.1097\/01.ju.0000080296.54262.c8","article-title":"Expression of lipoxygenase in human bladder carcinoma and growth inhibition by its inhibitors","volume":"170","author":"Yoshimura","year":"2003","journal-title":"J. Urol."},{"key":"ref_35","first-page":"1086","article-title":"Inhibition of 5-lipoxygenase pathway suppresses the growth of bladder cancer cells","volume":"13","author":"Hayashi","year":"2006","journal-title":"Int. J. Urol. Off. J. Jpn. Urol. Assoc."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"710","DOI":"10.1038\/s41379-020-00692-8","article-title":"TRPS1: A highly sensitive and specific marker for breast carcinoma, especially for triple-negative breast cancer","volume":"34","author":"Ai","year":"2021","journal-title":"Mod. Pathol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1978","DOI":"10.1158\/1078-0432.CCR-13-2637","article-title":"A panel of three markers hyper- and hypomethylated in urine sediments accurately predicts bladder cancer recurrence","volume":"20","author":"Su","year":"2014","journal-title":"Clin. Cancer Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/s13148-016-0303-5","article-title":"UroMark-a urinary biomarker assay for the detection of bladder cancer","volume":"9","author":"Feber","year":"2017","journal-title":"Clin. Epigenetics"},{"key":"ref_39","unstructured":"Eble, J.N. (2004). Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs, IARC Press."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e119","DOI":"10.1093\/nar\/gkm662","article-title":"Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: Critical evaluation and improvements","volume":"35","author":"Coolen","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"15785","DOI":"10.1073\/pnas.0507816102","article-title":"Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry","volume":"102","author":"Ehrich","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1023\/A:1010933404324","article-title":"Random Forests","volume":"45","author":"Breiman","year":"2001","journal-title":"Mach. Learn."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Chen, T., and Guestrin, C. (2016, January 13\u201317). Xgboost: A scalable tree boosting system. Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, San Francisco, CA, USA.","DOI":"10.1145\/2939672.2939785"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2249","DOI":"10.1016\/j.csda.2007.08.015","article-title":"Empirical characterization of random forest variable importance measures","volume":"52","author":"Archer","year":"2008","journal-title":"Comput. Stat. Data Anal."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1111\/j.2517-6161.1996.tb02080.x","article-title":"Regression shrinkage and selection via the lasso","volume":"58","author":"Tibshirani","year":"1996","journal-title":"J. R. Stat. Soc. Ser. B Stat. Methodol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Jordahl, K.M., Phipps, A.I., Randolph, T.W., Tinker, L.F., Nassir, R., Hou, L., Anderson, G.L., Kelsey, K.T., White, E., and Bhatti, P. (2020). Mediation by differential DNA methylation of known associations between single nucleotide polymorphisms and bladder cancer risk. BMC Med. Genet., 21.","DOI":"10.1186\/s12881-020-01172-1"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1332","DOI":"10.1093\/carcin\/bgz128","article-title":"DNA methylation patterns in bladder tumors of African American patients point to distinct alterations in xenobiotic metabolism","volume":"40","author":"Vantaku","year":"2019","journal-title":"Carcinogenesis"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.gene.2013.10.027","article-title":"E-cadherin gene promoter hypermethylation may contribute to the risk of bladder cancer among Asian populations","volume":"534","author":"Li","year":"2014","journal-title":"Gene"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/2\/738\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:41:10Z","timestamp":1760103670000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/2\/738"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,6]]},"references-count":48,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2024,1]]}},"alternative-id":["ijms25020738"],"URL":"https:\/\/doi.org\/10.3390\/ijms25020738","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,1,6]]}}}