DNA methylation biomarkers for blood-based colorectal cancer screening.
BACKGROUND
Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma.


METHODS
We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range.


RESULTS
Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%).


CONCLUSIONS
The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.
Lofton-Day
Catherine
C
Epigenomics, Inc., Seattle, WA, USA. clofton@us.epigenomics.com
Model
Fabian
F
Devos
Theo
T
Tetzner
Reimo
R
Distler
Juergen
J
Schuster
Matthias
M
Song
Xiaoling
X
Lesche
Ralf
R
Liebenberg
Volker
V
Ebert
Matthias
M
Molnar
Bela
B
Grützmann
Robert
R
Pilarsky
Christian
C
Sledziewski
Andrew
A
D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D003106:Colon / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D004247:DNA / Q000097:blood*; D019175:DNA Methylation; D005260:Female; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016015:Logistic Models; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D015999:Multivariate Analysis; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D058112:Septins
D014408:Biomarkers, Tumor; D008565:Membrane Proteins; C484065:NGFR protein, human; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D017475:Receptors, Nerve Growth Factor; C423567:TMEFF2 protein, human; D004247:DNA; D020558:GTP Phosphohydrolases; C474315:SEPTIN9 protein, human; D058112:Septins