OBJECTIVES: To identify a better set of DNA methylation markers to detect 
superficial, low grade cancer cell in urine sediment for improving cancer 
treatment, morbidity, and mortality.
MATERIALS AND METHODS: Methylation-specific PCR (MSP) assay was used to detect 
promoter hypermethylation in 4 genes (E-cadherin, p16, p14, and RASSF1A) to 
identify reliable biomarkers for bladder cancer diagnosis in primary tumor DNA 
and urine sediment DNA from 57 bladder cancer patients. Urine DNA was compared 
with 20 healthy controls.
RESULTS: Fifty-one (90%) tumor DNA and 47 urine DNA (83%) samples from bladder 
cancer patients revealed hypermethylation in at least 1 of the 4 analyzed genes, 
whereas all urine samples from normal controls were negative. The sensitivity of 
MSP assay for detecting E-cadherin, p16, p14 and RASSF1A in tumor cells in 
voided urine was 35%, 35%, 33%, and 65%, respectively. Diagnostic sensitivity 
was 75% for combining RASSF1A and p14, and 83% for RASSF1A, p14 and E-cadherin. 
Urine cytology, however, detect only 13 (28%) cases of cancer or suspicious 
cancer. For detecting superficial and invasive bladder tumor, urine cytology 
revealed a sensitivity of 23% (6/26) and 35% (7/20), respectively. In contrast, 
MSP detected hypermethylation in the urine of 80% (37/46) bladder cancer 
patients. Moreover, hypermethylation analysis of E-cadherin, p14 or RASSF1A 
genes in urine sediment DNA detected in 85% (22/26) of superficial, 85% (11/13) 
of low grade, 75% (15/20) of invasive and 79% (26/33) of high grade bladder 
cancers. Importantly, hypermethylation was detected in the urine DNA of 90% 
(18/20) superficial tumors with negative or atypia cytology.
CONCLUSIONS: Hypermethylation of E-cadherin, p14 or RASSF1A in urine sediment 
DNA is a potential biomarker for detecting superficial, low grade cancer. 
Besides, hypermethylation of these 3 genes is a valuable adjunct diagnostic 
marker to urine cytology, which can enhance the diagnostic accuracy and 
follow-up treatment of bladder cancer patients.