Abstract 690: Potential biomarkers for the prediction of urothelial cell carcinoma recurrence.
Authors: Michailidi Christina , Maldonado Leonel , Brait Mariana , Schultz Luciana , Park Jong Chul , Sidransky David , Netto George , Hoque Mohammad O. 
Published: August 2014

Background: Over 90% of bladder cancer cases in the western world present as urothelial cell carcinoma (UCC). Superficial UCC is the most common subtype at the time of detection (around 75%) and usually treated by trans-urethral resection (TUR). Twenty-percent of the previous 75% will be cured after TUR and up to 70% will recur at least once every 5 years even after complete TUR; the remaining progress to muscle invasion and higher stages with a poor prognosis. Currently there are no well validated markers that can discern the tumors that will recur from those that will not. The purpose of this study is to identify methylation based biomarkers to stratify patients with a higher risk of recurrence.

Materials and Methods: Methylation biomarker analysis was performed retrospectively in tumors from 38 UCC patients;18 non-recurrent and 20 recurrent. Using Quantitative Methylation Specific PCR (QMSP), we analyzed the promoter region of 9 genes (ARF, TIMP3, RAR-β, NID2, CCNA1, VGF, AIM1, CALCA and CCND2) in primary UCC tissues. The association of promoter methylation of these genes to recurrence was performed using Fisher Exact and/or Chi-square test. In vitro functional studies of two highly relevant genes (CCNA1 and CCND2) in several urothelial cancer cell lines were performed to have a better understanding of the role of these genes in UCC genesis.

Results: By establishing an empiric cutoff value, the frequencies of methylation in recurrent and non-recurrent UCC respectively are: CCND2 11/19 (58%) and 1/18 (5.5%) (p=0.001,); CCNA1 10/20 (50%) and 3/18 (16%) (p=0.043); NID2 13/20 (65%) and 4/18 (22%) (p=0.01); and, CALCA 8/20 (40%) and 2/18 (11%) (p=0.067). Functional studies of CCNA1 and CCND2 revealed that these genes are inactivated by promoter methylation and have anti-proliferative properties. To assess the potential use of these genes as biomarkers in bodily fluids, we analyzed their promoter methylation in urines from UCC patients and controls. By establishing an empiric cutoff, we found CCND2, CCNA1 and CALCA being significantly more methylated in urine of UCC patients than controls. The methylation frequency of CCND2, CCNA1 and CALCA is 51/148 (34%), 57/82 (69%) and 99/148 (67%), respectively for UCC patients urines, and 3/65(5%), 10/60 (17%) and 20/65(31%) for control urines, respectively.

Conclusion: Our findings depict the relevance of promoter methylation of CCND2, CCNA1, NID2 and CALCA genes for predicting recurrence in patients with UCC and the potential utility for detecting UCC in urine. Future studies using a larger cohort will provide us a better understanding of the molecular alterations associated with UCC in order to develop a reliable tool for outcome prediction and recurrence.

Citation Format: Christina Michailidi, Leonel Maldonado, Mariana Brait, Luciana Schultz, Jong Chul Park, David Sidransky, George Netto, Mohammad O. Hoque. Potential biomarkers for the prediction of urothelial cell carcinoma recurrence. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 690. doi:10.1158/1538-7445.AM2013-690