title: Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer.
abstract: BACKGROUND
  Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC) a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR). Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples.
  
  
  METHODS
  We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA). The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit.
  
  
  RESULTS
  The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing.
  
  
  CONCLUSIONS
  The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.
authors.affiliation: Department of Biomedicine, University of Aarhus, Bartholin Building, Wilhelm Meyers Allé 4, DK-8000 Aarhus C, Denmark. lasse@hum-gen.au.dk
authors.forename: Lasse Sommer
authors.initials: LS
authors.lastname: Kristensen
authors.forename: Tina Ellegaard
authors.initials: TE
authors.lastname: Kjeldsen
authors.forename: Henrik
authors.initials: H
authors.lastname: Hager
authors.forename: Lise Lotte
authors.initials: LL
authors.lastname: Hansen
mesh_terms: D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D018631:ras Proteins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins