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Detection of Methylated Apoptosis-Associated Genes in Urine Sediments of Bladder Cancer Patients

Departments of ¹ Urology, ² Clinical Pathology, and ³ Preventive Medicine, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California; ⁴ Department of Urology, University Hospital Hamburg, Hamburg, Germany; ⁵ James Buchanan Brady Urological Institute, The Johns Hopkins Medical Institutions, Baltimore, Maryland; and ⁶ Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, New York

Purpose: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments.

Experimental Design: We analyzed the methylation status of the 5' regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients.

Results: The 5' regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals.

Conclusions: Our results indicate that methylation of the 5' region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

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