Combination analysis of hypermethylated Wnt-antagonist family genes as a novel epigenetic biomarker panel for bladder cancer detection.
OBJECTIVE
Aberrant promoter hypermethylation of Wnt-antagonist genes contributes to the pathogenesis of several cancers. We hypothesized that combined methylation analysis of Wnt-antagonist genes could improve their use as a panel of biomarkers for diagnosing and staging of bladder cancers.


METHODS
Samples (54 total) of bladder tumor and corresponding normal bladder mucosa were analyzed for the methylation and expression levels of six Wnt-antagonist genes (sFRP-1, sFRP-2, sFRP-4, and sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of bladder tumor detection, the methylation score (M score), a new method for multigene methylation analysis, was developed. The M score of each sample was calculated as the sum of the corresponding log hazard ratio coefficients derived from multivariate logistic regression analysis of the methylation status for each Wnt-antagonist gene. Receiver operator characteristic (ROC) curve analysis was used to determine the optimal sensitivity/specificity of the M score. Urine DNA from 24 matched patients with bladder tumor and 20 cancer-free volunteers was also used to investigate the methylation status of Wnt-antagonist genes.


RESULTS
The methylation levels of Wnt-antagonists were significantly higher and mRNA levels were significantly lower in bladder tumor than in bladder mucosa. Each methylation level was inversely correlated with the corresponding mRNA level. In multivariate regression analysis, the methylation levels of sFRP-2 and Dkk-3 were significant independent predictors of bladder tumor (P < 0.05 and P < 0.01, respectively), whereas with sFRP-1, sFRP-5, and Wif-1 there was a trend towards significance as independent predictors. The M score of Wnt-antagonist genes was significantly higher in bladder tumor than in bladder mucosa (P < 0.05). Overall, the M score had a sensitivity of 77.2% and a specificity of 66.7% as a diagnostic biomarker (areas under the curve, 0.763). The M score could distinguish superficial from invasive bladder tumors with a sensitivity of 72.2% and a specificity of 61.1% as a staging biomarker (areas under the curve, 0.671). In patients with bladder tumor, 80.6% of the methylation-specific PCR results had identical methylation in samples of tumor- and urine-derived DNA. Most urine DNA in normal controls showed no aberrant methylation of the Wnt-antagonist genes.


CONCLUSIONS
Hypermethylation of Wnt-antagonist genes plays an important role in the pathogenesis of bladder tumor and can be detected using cellular DNA extracted from urine samples. This is the first report demonstrating that M score analysis of Wnt-antagonist genes could serve as an excellent epigenetic biomarker panel for bladder tumors.
Urakami
Shinji
S
Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, California, USA.
Shiina
Hiroaki
H
Enokida
Hideki
H
Kawakami
Toshifumi
T
Kawamoto
Ken
K
Hirata
Hiroshi
H
Tanaka
Yuichiro
Y
Kikuno
Nobuyuki
N
Nakagawa
Masayuki
M
Igawa
Mikio
M
Dahiya
Rajvir
R
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics / Q000652:urine; D002295:Carcinoma, Transitional Cell / Q000175:diagnosis* / Q000235:genetics / Q000652:urine; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000652:urine; D018925:Chemokines; D019175:DNA Methylation; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics / Q000652:urine; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics / Q000652:urine; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000652:urine; D015999:Multivariate Analysis; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics / Q000652:urine; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics / Q000652:urine; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D001743:Urinary Bladder / Q000502:physiology; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000652:urine; D000094742:Secreted Frizzled-Related Proteins
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D018925:Chemokines; D005136:Eye Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; C498624:DKK3 protein, human; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins; C109618:SFRP2 protein, human; C118723:SFRP4 protein, human; C118511:SFRP5 protein, human; C485569:WIF1 protein, human