Wnt antagonist family genes as biomarkers for diagnosis, staging, and prognosis of renal cell carcinoma using tumor and serum DNA.
OBJECTIVE
We hypothesized that combined methylation analysis of Wnt antagonist genes could serve as a panel of biomarkers for diagnosis, staging, and prognosis in renal cell carcinoma (RCC).


METHODS
Samples (n = 62) of RCC and corresponding normal renal tissue (NRT) were analyzed using methylation-specific PCR for methylation of six Wnt antagonist genes (sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of RCC detection, the methylation score (M score) for multigene methylation analysis was developed. Receiver operator characteristic curve analysis was used to determine the optimal sensitivity/specificity of the M score. In addition, the M score was compared with the clinicopathologic outcome. Thirty-three serum DNA samples were also used to investigate the methylation status of Wnt antagonist genes.


RESULTS
The methylation levels of all Wnt antagonists were significantly higher in RCC than in NRT. In multivariate regression analysis, the methylation level of sFRP-1 was a significant independent predictor of RCC, whereas for sFRP-2 and sFRP-4 there was a trend toward significance as independent predictors. The M score of Wnt antagonist genes was significantly higher in RCC than in NRT. Overall, the M score had a sensitivity of 79.0% and a specificity of 75.8% (area under the curve, 0.808) as a diagnostic biomarker. In addition, the M score could significantly distinguish grade, pT category, M category, and overall survival of RCC patients. The M score was independent of age and gender in predicting overall survival by the Cox proportional hazards model. In RCC patients, 72.7% of the methylation-specific PCR results had identical methylation in samples of tumor and serum DNA. No serum DNA in normal controls showed aberrant methylation of the Wnt antagonist genes. In addition, the methylation status of Wnt antagonist genes in serum DNA was significantly correlated with tumor grade and stage.


CONCLUSIONS
This is the first report showing that M score analysis of Wnt antagonist genes can serve as an excellent epigenetic biomarker panel for detection, staging, and prognosis of RCC using serum DNA.
Urakami
Shinji
S
Department of Urology, Veterans Affairs Medical Center, University of California, San Francisco, California 94121, USA.
Shiina
Hiroaki
H
Enokida
Hideki
H
Hirata
Hiroshi
H
Kawamoto
Ken
K
Kawakami
Toshifumi
T
Kikuno
Nobuyuki
N
Tanaka
Yuichiro
Y
Majid
Shahana
S
Nakagawa
Masayuki
M
Igawa
Mikio
M
Dahiya
Rajvir
R
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D002292:Carcinoma, Renal Cell / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D018925:Chemokines; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood*; D005136:Eye Proteins / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D007680:Kidney Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D015999:Multivariate Analysis; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction / Q000379:methods; D011237:Predictive Value of Tests; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D000094742:Secreted Frizzled-Related Proteins
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D018925:Chemokines; D004273:DNA, Neoplasm; 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