European Urology Oncology

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European Urology Oncology GBX2 Methylation Is a Novel Prognostic Biomarker and Improves Prediction of Biochemical Recurrence Among Patients with Prostate Cancer Negative for Intraductal Carcinoma and Cribriform Architecture Renu Jeyapala, Andrea J. Savio, Ekaterina Olkhov-Mitsel, Shivani Kamdar, Fang Zhao, Carmelle Cuizon, Richard S.C. Liu, Alex Zlotta, Neil Fleshner, Theodorus van der Kwast, Bharati Bapat European Urology Oncology DOI: 10.1016/j.euo.2018.08.003 Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 1 Methylation profiles spanning the GBX2 gene region across different analytical platforms. (A) Gene diagram depicting the GBX2 gene and proximal regions. The promoter region (defined as the region ranging from 1500bp upstream of the transcription start site [pink triangle] to 500bp downstream) is indicated in red, and the region subjected to a MethyLight assay for validation cohort 2 is indicated in blue. The locations of probes from Agilent array data (purple circles) and the TCGA cohort (yellow circles) with significantly differential methylation are shown. The green arrow shows region exhibiting higher methylation in TET2 knockout cells. The remainder of gene region is shown in grey. (B) Representative images from the Integrative Genomics Viewer (Broad Institute, Boston, MA, USA) depicting differential methylation in TET2 knockout cell lines (CR1 and CR2) compared to parental RWPE-1 cells. Methylation tracks correspond to locations indicated on the GBX2 gene region map. The gray peak height represents the strength of the methylation signal detected via methyl-CpG binding domain 2 sequencing (MBD-seq) in representative images of a sequenced sample (top) and input control (bottom). The region showing significantly different methylation between knockout and parental samples is indicated via the lilac highlight box. (C) Methylation levels measured via MethyLight (quantitative polymerase chain reaction) in RWPE-1, CR1, and CR2 are indicated as average percentage methylated reference (PMR) values. European Urology Oncology DOI: (10.1016/j.euo.2018.08.003) Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 2 Univariate Kaplan-Meier curves for biochemical recurrence (BCR)-free survival by (A) Gleason score (GS), (B) pT stage, (C) surgical margin (SM) status, (D) IDC/C presence, (E) GBX2 methylation status, (F) combined IDC_C status and GBX2 methylation status, and (G) GBX2 methylation status for IDC_C-negative cases. Log-rank p values are shown. IDC/C=intraductal carcinoma/cribriform architecture; HM=high methylation; LM=low methylation. European Urology Oncology DOI: (10.1016/j.euo.2018.08.003) Copyright © 2018 European Association of Urology Terms and Conditions