Abstract 4023: Genome-wide analysis of CpG island methylation identified OTX1, OSR1 and ONECUT2 as biomarkers for recurrent bladder cancer detection in voided urine
Authors: Kandimalla Raju , Masius Roy , Van Tilborg Angela A.G , Beukers Willemien , Orntoft Torben , Dyrskjot Lars , Bangma Chris H. , Zwarthoff Ellen C. 
Published: August 2012

Abstract
Cancers of the urinary bladder (BC) present as muscle-invasive (MIBC) or non-muscle invasive (NMIBC). Major problems with NMIBC are that 70% of the tumors will recur and 10-20% will eventually progress to MIBC. Therefore the patients are monitored by cystoscopy every 3-6 months after transurethral resection of the tumor in order to spot potential recurrences. DNA methylation has been shown to contribute to the pathogenesis cancer and may serve as useful biomarker. Recent studies showed promising urine methylation biomarkers for BC, but none of these were specifically tested for detection of recurrent BCs which are often smaller and hence more difficult to detect than primary tumors. Therefore we aimed to develop an assay specific for the diagnosis of recurrent bladder tumors in voided urine. From our earlier genome-wide study, we selected 8 candidate CGIs (CpG islands) methylated in BC to screen for the detection of recurrent bladder tumors in voided urine. We first screened these 8 CGIs on an independent set of 50 FFPE bladder tumors and 70 urines from age matched individuals without any history of BC as controls using BS-SNaPshot (Bisulfite specific single nucleotide primer extension assay). Subsequently, the 8 CGIs were investigated in a test set of 100 preTUR (before Trans Urethral Resection) urines associated with a concomitant recurrent tumor. We analyzed the sensitivity, specificity, AUC (area under the curve), PPV and NPV of all the individual markers and five best combinations. We then validated this on a separate cohort of 100 preTUR urines samples. Single marker OTX1 identified recurrent bladder tumors in voided urine with a sensitivity of 71% at a specificity of 90% with an AUC of 0.85 (CI: 0.80-0.91, P&lt;0.0001). A 3 gene methylation panel OTX1, ONECUT2 and OSR1 showed a sensitivity of 73% at a specificity of 90% with an AUC of 0.86 (CI: 0.80-0.91, P&lt; 0.0001). Combining this 3-gene panel methylation assay with FGFR3 (fibroblast growth factor receptor 3) mutation assay achieved a sensitivity of 78% at a specificity of 90% with an AUC of 0.87 (CI: 0.82-0.93, P&lt; 0.0001). This panel of markers showed a sensitivity of 100% in detecting bladder tumor tissue DNA. These markers were also investigated on 40 urines collected from patients who are recurrence free for a period of longer than 6 months. A total of 9/40 (22%) urines were positive in these recurrence free patients. The lower specificity of these samples could be due to the anticipatory effect that has been attributed to urine tests, i.e. urines test sees the tumor earlier than cystocopy. We also observed that multiple tumors from a patient are highly concordant in their methylation percentage, which underlines the usefulness of the markers. In summary the panel biomarkers that are identified will be able to detect the recurrent bladder tumors in voided urine, thereby helping in surveillance of patients with bladder cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4023. doi:1538-7445.AM2012-4023