title: Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer.
abstract: BACKGROUND
  DNA methylation is a well-known epigenetic mechanism involved in epigenetic gene regulation. Several genes were reported hypermethylated in CRC, althought no gene marker was proven to be individually of sufficient sensitivity or specificity in routine clinical practice. Here, we identified novel epigenetic markers and assessed their combined use for diagnostic accuracy.
  
  
  METHODS
  We used methylation arrays on samples from several effluents to characterize methylation profiles in CRC samples and controls, as established by colonoscopy and pathology findings, and selected two differentially methylated candidate epigenetic genes (NPY, PENK). To this gene panel we added WIF, on the basis of being reported in literature as silenced by promoter hypermethylation in several cancers, including CRC. We measured their methylation degrees by quantitative multiplex-methylation specific PCR (QM-MSP) on 15 paired carcinomas and adjacent non-cancerous colorectal tissues and we subsequently performed a clinical validation on two different series of 266 serums, subdivided in 32 CRC, 26 polyps, 47 other cancers and 161 with normal colonoscopy. We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.
  
  
  RESULTS
  We obtained CRC detection on tissues with both sensitivity and specificity of 100%. On serum CRC samples, we obtained sensitivity/specificity values of, e.g., 87%/80%, 78%/90% and 59%/95%, and negative predictive value/positive predictive value figures of 97%/47%, 95%/61% and 92%/70%. On serum samples from other cancers we obtained sensitivity/specificity of, e.g, 89%/25%, 43%/80% and 28%/91%.
  
  
  CONCLUSIONS
  We showed the potential of NPY, PENK, and WIF1 as combined epigenetic markers for CRC diagnosis, both in tissue and serum and tested their use as serum biomarkers in other cancers. We optimized a QM-MSP for simultaneously quantifying their methylation levels. Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination. This panel of markers, if validated, can also be a cost effective screening tool for the detection of asymptomatic cancer patients for colonoscopy.
authors.affiliation: Profilome SAS, Paris Biotech 24 rue du Faubourg St Jacques, Paris 75014, France. jp.roperch@profilome-sas.com.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Roperch
authors.forename: Roberto
authors.initials: R
authors.lastname: Incitti
authors.forename: Solène
authors.initials: S
authors.lastname: Forbin
authors.forename: Floriane
authors.initials: F
authors.lastname: Bard
authors.forename: Hicham
authors.initials: H
authors.lastname: Mansour
authors.forename: Farida
authors.initials: F
authors.lastname: Mesli
authors.forename: Isabelle
authors.initials: I
authors.lastname: Baumgaertner
authors.forename: Francesco
authors.initials: F
authors.lastname: Brunetti
authors.forename: Iradj
authors.initials: I
authors.lastname: Sobhani
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000230:Adenocarcinoma / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D004247:DNA / Q000097:blood / Q000235:genetics; D019175:DNA Methylation; D004745:Enkephalins / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D009367:Neoplasm Staging; D009478:Neuropeptide Y / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D012372:ROC Curve; D012097:Repressor Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D004745:Enkephalins; D009478:Neuropeptide Y; D011498:Protein Precursors; D012097:Repressor Proteins; C485569:WIF1 protein, human; C029992:proenkephalin; D004247:DNA