Abstract
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.
Highlights
Bacillus Calmette-Guérin (BCG) immunotherapy has been effectively used in the last 30 years for the management of non-muscle-invasive bladder cancer (NMIBC)
After data normalization and quality control, principal component analysis using v3.3 Qlucore software (Qlucore Omics Explorer, Lund, Sweden) performed on 15332 methylation variable positions (MVPs) with a p-value of < 0.01 indicated grouping of the two patient cohorts, indicating that BCG responders and failures can be distinguished based on DNA methylation profiles (Figure 1A)
In order to investigate a potential biological mechanism differing between responders and failures, we evaluated protein expression of selected genes harboring Differentially methylated regions (DMRs) belonging to these pathways, such as paxillin, which is a focal adhesion protein involved in bacterial uptake [35,36,37]
Summary
Bacillus Calmette-Guérin (BCG) immunotherapy has been effectively used in the last 30 years for the management of non-muscle-invasive bladder cancer (NMIBC). It is, the standard intravesical adjuvant therapy for intermediate- and high-risk NMIBC, as it prevents/delays tumor recurrence and/or progression [1,2,3]. Despite clinical familiarity with BCG, its molecular mode of action remains only partially understood Both benign and cancerous urothelial cells, together with the local immune system, play an active role in BCG’s therapeutic anti-tumor effect. 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. We identified candidate DNA methylation biomarkers that may help to predict patient prognosis after external validation in larger, well-designed cohorts
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