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

Abstract

Tumour suppressor gene (TSG) methylation has been proposed as a diagnostic marker for urothelial cancer (UC). Here, we compare the frequency of urinary TSG methylation in young and elderly patients, with and without UC. Urine samples were obtained prospectively from 35 UC patients, 35 benign controls over the age of 70 years and 34 healthy volunteers under the age of 40 years. Methylation analysis was performed for eight gene promoters using quantitative methylation-specific PCR. Methylation was detected in urine DNA from all three patient groups. The highest frequencies were seen in UC patients. Significantly less methylation was present in control samples than UC cases for RASSF1a and APC (Po0.034). The 'methylation index' and level of methylation was highest in the UC group and lowest in the young control group. A marker panel of RASSF1a, E-cad and APC generated a sensitivity of 69%, a specificity of 60% and a diagnostic accuracy of 86%. TSG methylation is detectable in urine DNA from patients with and without bladder cancer. The frequency and extent of methylation appears to increase with age and malignancy. The lack of tumour specificity suggests that further investigation is required before this test is introduced into clinical practice.

Multiplexed Methylation Profiles of Tumor Suppressor Genes in Bladder Cancer

“…The most frequent methylated genes previously reported in urinary specimens of patients with bladder cancer were BRCA1, 38 RARB, 39 and CDH13 38 ; these genes also occurred in bladder cancer cell lines and tumors. The detection of distinct methylation profiles between noninvasive and invasive bladder tumors is in concordance with genes previously reported as methylated in bladder cancer by standard MS-PCR, [32][33][34][35][36][37][38][39] despite the fact that different CpG dinucleotides could be targeted by the MS-MLPA and MS-PCR methods. The MS-MLPA method allowed identification of several new and possibly interesting epigenetic alterations (eg, MSH6, WT1, and THBS1 genes), serving to gain more insight into the development of bladder carcinomas.…”

“…Having information of epigenetic alterations reported for several genes included in the MS-MLPA under analyses in bladder tumors, and even in urinary specimens by an independent method, [32][33][34][35][36][37][38][39] served to support our findings in urinary specimens. A correlation of epigenetic aberrations with bladder cancer histopathological variables was reported in numerous studies 8 -12,32-39 using techniques other than MS-MLPA.…”

“…In addition to the interindividual variation, the differences in sensitivity found between the sets and previous reports describing urinary meth- CDH13, 35,38 or BRCA1 38 ) could be attributed to several issues. Aberrant methylation was defined as needing to meet the cutoff ratio of 30% or greater set by the mathematical algorithm designed to distinguish legitimate methylation peaks.…”

“…15 The advantages of the MS-MLPA technique as an alter- native for MS-PCR include allowing screening of more than 25 predefined promoter methylation candidates in one single step using a low amount of DNA (100 to 200 ng), 16 which is feasible using DNA extracted from tissue (even in formalin-fixed material) 18 ; providing semiquantitative data; and requiring only standard laboratory equipment. Furthermore, the (potentially) troublesome bisulfite conversion of unmethylated cytosines required for MS-PCR [31][32][33][34][35][36][37][38][39] can be omitted in MS-MLPA using methylationsensitive digestion. The present study showed that these generally acknowledged advantages can also be translated to investigate methylation in body fluids in a multiplexed manner.…”

Epigenetic Biomarkers in Bladder Cancer

“…The evaluation of this panel yielded superior results compared to those of cytology in the detection of bladder cancer. Yates et al (2006) also investigated the APC, RASSF1A and CDH1 genes in urine. This panel generated a lower sensitivity (69%) and specificity (60%) than the former; however, the diagnostic accuracy was 86%.…”