title: Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.
abstract: Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy.
authors.affiliation: Digestive Oncology Unit, Rouen University Hospital, France; Inserm U1079, Iron group, IRIB, University of Rouen, Normandy University, Rouen University Hospital, Normandy Center for Genomic and Personalized Medicine, Rouen, France.
authors.forename: David
authors.initials: D
authors.lastname: Sefrioui
authors.affiliation: Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Mauger
authors.affiliation: Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Leclere
authors.affiliation: Inserm U1079, Iron group, IRIB, University of Rouen, Normandy University, Rouen University Hospital, Normandy Center for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Ludivine
authors.initials: L
authors.lastname: Beaussire
authors.affiliation: Digestive Oncology Unit, Rouen University Hospital, France; Inserm U1079, Iron group, IRIB, University of Rouen, Normandy University, Rouen University Hospital, Normandy Center for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Di Fiore
authors.affiliation: Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Deleuze
authors.affiliation: Inserm U1079, Iron group, IRIB, University of Rouen, Normandy University, Rouen University Hospital, Normandy Center for Genomic and Personalized Medicine, Rouen, France.
authors.forename: Nasrin
authors.initials: N
authors.lastname: Sarafan-Vasseur
authors.affiliation: Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France. Electronic address: tost@cng.fr.
authors.forename: Jörg
authors.initials: J
authors.lastname: Tost
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004247:DNA / Q000032:analysis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Circulating cell-free DNA; Colorectal cancer; Digital PCR; E-ice-COLD-PCR; KRAS; Mutation enrichment; Quantification