[en] BACKGROUND: Highly sensitive and specific urinary biomarkers for the early detection of bladder cancer (BC) to improve the performance of urinary cytology are needed. OBJECTIVE: To investigate the usefulness of methylation markers in voided urine to identify BC presence and grade. DESIGN, SETTINGS, AND PARTICIPANTS: Using genome-wide methylation strategies in Toronto, Canada and Liège, Belgium, we have identified differentially methylated genes (TWIST1, RUNX3, GATA4, NID2, and FOXE1) in low-grade vs. high-grade BC tissue and urine. We accrued urine samples from 313 patients using a 2:1 ratio in a case-control setting from Toronto, Canada, Halifax, Canada, and Zurich, Switzerland. We studied the usefulness of these 5 methylated genes to identify BC and discriminate cancer grade in voided urine specimens. Urinary cell sediment DNA was evaluated using qPCR-based MethyLight assay. Multivariable logistic regression prediction models were created. RESULTS AND LIMITATIONS: We included 211 BC patients (180 nonmuscle invasive) and 102 controls. In univariate analyses, all methylated genes significantly predicted BC vs. no BC, and high grade vs. low grade (all P < 0.05). In multivariable analysis, NID2, TWIST1, and age were independent predictors of BC (all P < 0.05). Sensitivity of NID2 and TWIST1 to predict BC and BC grade was 76.2% and 77.6%, respectively, whereas specificity was 83.3% and 61.1%, respectively. Multivariable models predicting BC overall and discriminating between high-grade and low-grade BC reached area under the receiver operating characteristics curves of 0.89 and 0.78, respectively. CONCLUSIONS: This multi-centric study in a real life scenario (different countries, techniques, and pathologists) supports the promise of epigenetic urinary markers in noninvasively detecting BC. With sensitivities and specificities in the range of 80%, the overall performance characteristics of this panel of methylated genes probably does not allow such signature to significantly alter clinical care at this stage but is worth further studying for instance in BC surveillance or screening in high-risk populations.
Research center :
Service d'Urologie
Disciplines :
Urology & nephrology
Author, co-author :
Hermanns, Thomas
Savio, Andrea J.
Olkhov-Mitsel, Ekaterina
Mari, Andrea
Wettstein, Marian S.
Saba, Karim
Bhindi, Bimal
Kuk, Cynthia
Poyet, Cédric
Wild, Peter J.
Noon, Aidan
Bashir, Shaheena
Juvet, Tristan
Rendon, Ricardo A.
Waltregny, David ; Université de Liège - ULiège > Département des sciences cliniques > Urologie
Burger, M, Catto, JWF, Dalbagni, G, et al. Epidemiology and risk factors of urothelial bladder cancer. Eur Urol 63:2 (2013), 234–241.
Zlotta, AR, Roumeguere, T, Kuk, C, et al. Select screening in a specific high-risk population of patients suggests a stage migration toward detection of non-muscle-invasive bladder cancer. Eur Urol 59:6 (2011), 1026–1031.
Larré, S1, Catto, JW, Cookson, MS, et al. Screening for bladder cancer: rationale, limitations, whom to target, and perspectives. Eur Urol 63:6 (2013), 1049–1058.
Robertson, AG, Kim, J, Al-Ahmadie, H, et al. Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell 171:3 (2017), 540–556.
Kandimalla, R, van Tilborg, AA, Zwarthoff, EC, DNA methylation-based biomarkers in bladder cancer. Nat Rev Urol 10:6 (2013), 327–335.
Tilki, D, Burger, M, Dalbagni, G, et al. Urine markers for detection and surveillance of non-muscle invasive bladder cancer: a systematic review. Eur Urol 60:3 (2011), 484–492.
Catto, JW, Azzouzi, AR, Rehman, I, et al. Promoter hypermethylation is associated with tumor location, stage, and subsequent progression in transitional cell carcinoma. J Clin Oncol 23:13 (2005), 2903–2910.
García-Baquero, R, Puerta, P, Beltran, M, et al. Methylation of a novel panel of tumor suppressor genes in urine moves forward noninvasive diagnosis and prognosis of bladder cancer: a 2-center prospective study. J Urol 190:2 (2013), 723–730.
Witjes, JA, Morote, J, Cornel, EB, et al. Performance of the Bladder EpiCheck™ methylation test for patients under surveillance for non-muscle-invasive bladder cancer: results of a multicenter, prospective, blinded clinical trial. Eur Urol Oncol 1:4 (2018), 307–313.
Olkhov-Mitsel, E, Savio, AJ, Kron, KJ, et al. Epigenome-wide DNA methylation profiling identifies differential methylation biomarkers in high-grade bladder cancer. Transl Oncol 10:2 (2017), 168–177.
Renard, I, Joniau, S, van Cleynenbreugel, B, et al. Identification and validation of the methylated TWIST1 and NID2 genes through real-time methylation-specific polymerase chain reaction assays for the noninvasive detection of primary bladder cancer in urine samples. Eur Urol 58:1 (2010), 96–104.
Olkhov-Mitsel, E, Zdravic, D, Kron, K, van der Kwast, T, Fleshner, N, Bapat, B, Novel multiplex MethyLight protocol for detection of DNA methylation in patient tissues and body fluids. Sci Rep, 4, 2014, 4432.
Bosschieter, J, Lutz, C, Segerink, L, et al. The diagnostic accuracy of methylation markers in urine for the detection of bladder cancer: a systematic review. Epigenomics 10:5 (2018), 673–687.
Zhao, F, Olkhov-Mitsel, E, van der Kwast, T, et al. Urinary DNA methylation biomarkers for noninvasive prediction of aggressive disease in patients with prostate cancer on active surveillance. J Urol 197:2 (2017), 335–341.
Bosschieter, J, Hentschel, A, Savci-Heijink, CD, et al. Reproducibility and prognostic performance of the 1973 and 2004 World Health Organization classifications for grade in non-muscle-invasive bladder cancer: a multicenter study in 328 bladder tumors. Clin Genitourin Cancer 16:5 (2018), e985–e992.
Van Kessel, KE, Beukers, W, Lurkin, I, et al. Validation of a DNA methylation-mutation urine assay to select patients with hematuria for cystoscopy. J Urol 193:3 Pt 1 (2017), 590–595.
van der Heyden, AG, Mengual, L, Ingelmo-Torres, M, et al. Urine cell-based DNA methylation classifier for monitoring bladder cancer. Clin Epigenetics, 10, 2018, 71.
Karnes, RJ, Fernandez, CA, Shuber, AP, A noninvasive multianalyte urine-based diagnostic assay for urothelial cancer of the bladder in the evaluation of hematuria. Mayo Clin Proc 87:9 (2012), 835–842.
Roperch, JP, Grandchamp, B, Desgrandchamps, F, et al. Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer. BMC Cancer, 16, 2016, 704.
Abern, MR, Owusu, R, Inman, BA, Clinical performance and utility of a DNA methylation urine test for bladder cancer. Urol Oncol, 32(1), 2014 51,e21-6.
Fantony, JJ, Abern, MR, Gopalakrishna, A, et al. Multi-institutional external validation of urinary TWIST1 and NID2 methylation as a diagnostic test for bladder cancer. Urol Oncol, 33(9), 2015, 387.
Fantony, JJ, Longo, TA, Gopalakrishna, A, et al. Urinary NID2 and TWIST1 methylation to augment conventional urine cytology for the detection of bladder cancer. Cancer Biomark 18:4 (2017), 381–387.
Mandic, S, Go, C, Aggarwal, I, Myers, J, Froelicher, VF, Relationship of predictive modeling to receiver operating characteristics. J Cardiopulm Rehabil Prev 28:6 (2008), 415–419.
Oeyen, E, Hoekx, L, De Wachter, S, Baldewijns, M, Ameye, F, Mertens, I, Bladder cancer diagnosis and follow-up: the current status and possible role of extracellular vesicles. Int J Mol Sci, 20(4), 2019 pii: E821.
McCroskey, Z, Pambuccian, SE, Kleitherms, S, et al. Accuracy and interobserver variability of the cytologic diagnosis of low-grade urothelial carcinoma in instrumented urinary tract cytology specimens. Am J Clin Pathol 144:6 (2015), 902–908.
Chandrasekar, T, Zlotta, AR, Shen, J, et al. RNA sequencing identifies 3 different molecular grades and immune checkpoint cascades with distinct clinical behaviour in non muscle invasive bladder cancer. J Urol Suppl, 199(4), 2018, e863.
