BACKGROUND: Bacillus Calmette-Guérin (BCG) immunotherapy, the standard adjuvant intravesical therapy for some intermediate and most high-risk non-muscle invasive bladder cancers (NMIBCs), suffers from a heterogenous response rate. Molecular markers to help guide responses are scarce and currently not used in the clinical setting. METHODS: To identify novel biomarkers and pathways involved in response to BCG immunotherapy, we performed a genome-wide DNA methylation analysis of NMIBCs before BCG therapy. Genome-wide DNA methylation profiles of DNA isolated from tumors of 26 BCG responders and 27 failures were obtained using the Infinium MethylationEPIC BeadChip. RESULTS: Distinct DNA methylation patterns were found by genome-wide analysis in the two groups. Differentially methylated CpG sites were predominantly located in gene promoters and gene bodies associated with bacterial invasion of epithelial cells, chemokine signaling, endocytosis, and focal adhesion. In total, 40 genomic regions with a significant difference in methylation between responders and failures were detected. The differential methylation state of six of these regions, localized in the promoters of the genes GPR158, KLF8, C12orf42, WDR44, FLT1, and CHST11, were internally validated by bisulfite-sequencing. GPR158 promoter hypermethylation was the best predictor of BCG failure with an AUC of 0.809 (p-value < 0.001). CONCLUSIONS: Tumors from BCG responders and BCG failures harbor distinct DNA methylation profiles. Differentially methylated DNA regions were detected in genes related to pathways involved in bacterial invasion of cells or focal adhesion. We identified candidate DNA methylation biomarkers that may help to predict patient prognosis after external validation in larger, well-designed cohorts.

AIT Austrian Institute of Technology GmbH Health and Environment Department Molecular Diagnostics 1210 Vienna Austria

Clinical Institute of Pathology Medical University of Vienna Vienna 1090 Austria

College of Health Care Professions Claudiana Research Claudiana 39100 Bolzano Italy

Department of Urology 2nd Faculty of Medicine Charles University 150 06 Prague Czech Republic

Department of Urology Central Hospital of Bolzano Bozen 39100 Bozen Italy

Department of Urology CHRU Tours Francois Rabelais University 37000 Tours France

Department of Urology Jikei University School of Medicine Tokyo 105 8461 Japan

Department of Urology Medical University of Vienna 1090 Vienna Austria

Department of Urology University of Texas Southwestern Medical Center Dallas TX 75390 USA

Department of Urology Weill Cornell Medical College New York NY 10065 USA

Division of Pathology Department of Medical Sciences University of Studies of Torino 10124 Turin Italy

Division of Urology Department of Special Surgery The University of Jordan Amman 11942 Jordan

Division of Urology Department of Surgical Sciences San Giovanni Battista Hospital University of Studies of Torino 10124 Turin Italy

Institute for Urology and Reproductive Health 1 M Sechenov 1st Moscow State Medical University 119992 Moscow Russia

Karl Landsteiner Institute of Urology and Andrology 3100 St Poelten Austria

Ludwig Boltzmann Institute Applied Diagnostics Währinger Gürtel 18 20 1090 Vienna Austria

Sigmund Freud Private University Medical University 1020 Vienna Austria

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