title: DNA methylation biomarkers for blood-based colorectal cancer screening.
abstract: 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.
authors: Epigenomics, Inc., Seattle, WA, USA. clofton@us.epigenomics.com
authors: Catherine
authors: C
authors: Lofton-Day
authors: Fabian
authors: F
authors: Model
authors: Theo
authors: T
authors: Devos
authors: Reimo
authors: R
authors: Tetzner
authors: Juergen
authors: J
authors: Distler
authors: Matthias
authors: M
authors: Schuster
authors: Xiaoling
authors: X
authors: Song
authors: Ralf
authors: R
authors: Lesche
authors: Volker
authors: V
authors: Liebenberg
authors: Matthias
authors: M
authors: Ebert
authors: Bela
authors: B
authors: Molnar
authors: Robert
authors: R
authors: Grützmann
authors: Christian
authors: C
authors: Pilarsky
authors: Andrew
authors: A
authors: Sledziewski
mesh_terms: 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
chemical_list: 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