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

Abstract

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, RASS-FIA, 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 agematched 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.

Methylational urinalysis: a prospective study of bladder cancer patients and age stratified benign controls

“…This is the only study, to our knowledge, assessing methylation in an age stratified control population and comparing it to methylation in a group of patients with malignancy. In support of these recent findings (Friedrich et al, 2004), we have also found TSG methylation is often present in the exfoliated urinary cells from both cancer and control patients. Furthermore, the frequency of methylation at several loci increases with age and malignancy, supporting the description in other reports (Issa et al, 1994;Ahuja et al, 1998).…”

“…While our study was limited by the small panel of genes used for methylation analysis and the size of the various investigated groups, two conclusions may be drawn. Firstly, from our results and those of Friedrich et al (2004) it appears that TSG methylation can be found in exfoliated urinary cells from patients without cancer and increases in frequency with ageing. Thus, while methylational urinalysis has a number of attractive features, each targeted locus should be thoroughly investigated in a large control population (of mixed aged volunteers) for tumour specificity, before clinical introduction.…”

“…Silencing of tumour suppressor genes (TSG) associated with aberrant hypermethylation has been shown to be a molecular pathway for human cancer development (Herman and Baylin, 2003). As promoter hypermethylation occurs frequently in UC (Catto et al, 2005), several authors have investigated its detection in exfoliated urinary cells as a novel diagnostic test (Maruyama et al, 2001;Chan et al, 2002;Dulaimi et al, 2004;Friedrich et al, 2004). Recently, Dulaimi et al (2004) found that analysis of hypermethylation using a panel of TSG's yielded superior results to cytology in the detection of bladder cancer.…”

“…This method may fail to detect low concentrations of methylated alleles, unlike QMSP which can detect up to 1/1000 methylated alleles (own data not shown and Harnden et al, 2003). Support for this explanation comes from work by Friedrich et al (2004), who analysed the methylation status of 12 apoptosis-associated genes including RASSF1a and DAPK in 127 UC samples and 37 samples of adjacent normal mucosa, using the quantitative fluorometric MethyLight technique. Urine DNA was analysed for 20 cancer free volunteers and 37 UC patients.…”

Hypermethylation of tumor-suppressor gene CpG islands in small-cell carcinoma of the urinary bladder

“…23,24 The rate of methylation of RASSF1 that we detect in pure TCC and TCC with SCBC are similar to those in previous reports. 37,[46][47][48][49] However, the frequency of MGMT methylation in TCC with SCBC that we observed was significantly higher than the 2-5% rates reported in two prior studies comprising 196 combined specimens of pure TCC. 36,37 We believe this reflects an innate propensity for TCCs harboring MGMT methylation to develop clones of SCBC.…”