Identification and validation of the methylated TWIST1 and NID2 genes through real-time methylation-specific polymerase chain reaction assays for the noninvasive detection of primary bladder cancer in urine samples.
BACKGROUND
Accumulating evidence suggests that DNA methylation markers could serve as sensitive and specific cancer biomarkers.


OBJECTIVE
To determine whether a panel of methylated genes would have the potential to identify primary bladder cancer (BCa) in voided urine samples.


METHODS
A pharmacologic unmasking reexpression analysis in BCa cell lines was initially undertaken to unveil candidate methylated genes, which were then evaluated in methylation-specific polymerase chain reaction (MSP) assays performed on DNA extracted from noncancerous and cancerous bladder tissues. The most frequently methylated genes in cancerous tissues, with 100% specificity, were retained for subsequent MSP analysis in DNA extracted from urine samples to build and validate a panel of potential methylated gene markers. Urine samples were prospectively collected at three urologic centres from patients with histologically proven BCa and processed for use in real-time MSP and cytologic analysis. Patients with nonmalignant urologic disorders were included as controls.


METHODS
A urine sample was classified as valid when > or = 10 copies of the gene encoding ß-actin were measured in the urine sediment genomic DNA. Sensitivity, specificity, and predictive values of the MSP and cytology tests were assessed and compared.


CONCLUSIONS
MSP assays performed on 466 of the 496 (94%) valid urine samples identified two genes, TWIST1 and NID2, that were frequently methylated in urine samples collected from BCa patients, including those with early-stage and low-grade disease. The sensitivity of this two-gene panel (90%) was significantly better than that of cytology (48%), with comparable specificity (93% and 96%, respectively). The positive predictive value and negative predictive value of the two-gene panel was 86% and 95%, respectively.


CONCLUSIONS
Detection of the methylated TWIST1 and NID2 genes in urine sediments using MSP provides a highly (> or = 90%) sensitive and specific, noninvasive approach for detecting primary BCa.


BACKGROUND
BlCa-001 study - EudraCt 2006-003303-40.
Renard
Isabelle
I
OncoMethylome Sciences SA, Liège, Belgium.
Joniau
Steven
S
van Cleynenbreugel
Ben
B
Collette
Catherine
C
Naômé
Christophe
C
Vlassenbroeck
Ilse
I
Nicolas
Hubert
H
de Leval
Jean
J
Straub
Josef
J
Van Criekinge
Wim
W
Hamida
Wissem
W
Hellel
Majed
M
Thomas
Alexandre
A
de Leval
Laurence
L
Bierau
Katja
K
Waltregny
David
D
D000199:Actins / Q000235:genetics / Q000652:urine; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000652:urine*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D051879:Twist-Related Protein 1 / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000652:urine
D000199:Actins; D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D004273:DNA, Neoplasm; C114729:NID2 protein, human; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1