A 3-Plex Methylation Assay Combined with the FGFR3 Mutation Assay Sensitively Detects Recurrent Bladder Cancer in Voided Urine

Abstract

Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non-muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study is to determine the sensitivity and specificity of a urine assay for the diagnosis of recurrences in patients with a previous primary NMIBC G1/G2 by using cystoscopy as the reference standard.Experimental Design: We selected eight CpG islands (CGI) methylated in bladder cancer from our earlier genome-wide study. Sensitivity of the CGIs for recurrences detection was investigated on a test set of 101 preTUR urines. Specificity was determined on 70 urines from healthy males aged more than 50 years. A 3-plex assay for the best combination was developed and validated on an independent set of 95 preTUR, recurrence free, and nonmalignant urines (n ¼ 130).Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57%, respectively.Conclusion: The combination of methylation and FGFR3 assays efficiently detects recurrent bladder cancer without the need for stratification of patients regarding methylation/mutation status of the primary tumor. We conclude that the sensitivity of this combination is in the same range as cystoscopy and paves the way for a subsequent study that investigates a modified surveillance protocol consisting of the urine test followed by cystoscopy only when the urine test is positive.

Development and Validation of Urine-based Peptide Biomarker Panels for Detecting Bladder Cancer in a Multi-center Study

“…A 10-biomarker ELISA-based assay (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SDC1, and SERPINE1) provided an overall AUC of 0.85 (95% CI, 0.80-0.91) in discriminating urothelial bladder cancer patients from healthy and benign controls (45), slightly lower than the rates received from the CE-MS classifier for primary urothelial bladder cancer (AUC ¼ 0.87; 95% CI, 0.83-0.91). A three-gene methylation panel (OTX1, ONECUT2, and OSR1) detected low/intermediate risk urothelial bladder cancer with a sensitivity of 74% and specificity of 90% (43). In the current study, when investigating the subpopulation of low/intermediate-risk patients (NMIBC G 1 -G 2 ; n ¼ 26), the CE-MS-based classifier provided a sensitivity of 89% in urothelial bladder cancer detection at the preselected cut-off (AUC ¼ 0.72).…”

The Diagnostic and Prognostic Performance of Urinary FGFR3 Mutation Analysis in Bladder Cancer Surveillance: A Prospective Multicenter Study

“…A number of noninvasive tests for detection of urinary non-muscle invasive B-TCC have been developed in order to overcome the low sensitivity of cytology and to reduce the number of cystoscopies, and have been reviewed by Tomasini et al (7). Following the pioneering work of Sidransky and colleagues (8), several reports have suggested the diagnostic utility of genetic and epigenetic markers (9)(10)(11). Mutations in fibroblast growth factor 3 (FGFR3) are found in a large proportion of non-muscle invasive bladder cancers (12) and may M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 2 therefore represent useful biomarkers for the follow-up of patients with B-TCC.…”

“…It has been shown that urine-based FGFR3 mutation analysis can detect recurrence, although its sensitivity may be limited if samples have few tumor cells (13)(14)(15). Recently, several studies have evaluated the sensitivity and specificity of FGFR3 mutation analysis in voided urine for detection of recurrence of superficial bladder cancer (9,(16)(17)(18). In these studies, screening for mutated FGFR3 was performed using various assays (9,(16)(17)(18).…”

Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer

“…Furthermore, the SnapShot method proved to be highly reproducible. 27 A limitation of this study is the retrospective design. Because of the low incidence of progression in low-grade pTa disease a prospective study design is difficult to establish.…”

“…Determining DNA methylation is a highly reproducible method as we showed previously. 27 Hence these markers present a valuable tool for assessing risk of progression in patients presenting with TaG1/G2 tumors.…”