title: Detection of KRAS, NRAS and BRAF by mass spectrometry - a sensitive, reliable, fast and cost-effective technique.
abstract: BACKGROUND
  According to current clinical guidelines mutational analysis for KRAS and NRAS is recommended prior to EGFR-directed therapy of colorectal cancer (CRC) in the metastatic setting. Therefore, reliable, fast, sensitive and cost-effective methods for routine tissue based molecular diagnostics are required that allow the assessment of the CRC mutational status in a high throughput fashion.
  
  
  METHODS
  We have developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY, Agena Bioscience) analysis to test the presence/absence of 18 KRAS, 14 NRAS and 4 BRAF mutations. We have applied this assay to 93 samples from patients with CRC and have compared the results with Sanger sequencing and a chip hybridization assay (KRAS LCD-array Kit, Chipron). In cases with discordant results, next-generation sequencing (NGS) was performed.
  
  
  RESULTS
  MS detected a KRAS mutation in 46/93 (49%), a NRAS mutation in 2/93 (2%) and a BRAF mutation in 1/93 (1%) of the cases. MS results were in agreement with results obtained by combination of the two other methods in 92 (99%) of 93 cases. In 1/93 (1%) of the cases a G12V mutation has been detected by Sanger sequencing and MS, but not by the chip assay. In this case, NGS has confirmed the G12V mutation in KRAS.
  
  
  CONCLUSIONS
  Mutational analysis by MS is a reliable method for routine diagnostic use, which can be easily extended for testing of additional mutations.
authors.affiliation: Institute of Pathology, University of Heidelberg, INF 224, Heidelberg, Germany. mark.kriegsmann@med.uni-heidelberg.de.
authors.forename: Mark
authors.initials: M
authors.lastname: Kriegsmann
authors.affiliation: Institute of Molecular Pathology, Trier, Germany. norbert.arens@molekularpatho-trier.de.
authors.forename: Norbert
authors.initials: N
authors.lastname: Arens
authors.affiliation: Institute of Pathology, University of Heidelberg, INF 224, Heidelberg, Germany. Volker.endris@med.uni-heidelberg.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Endris
authors.affiliation: Institute of Pathology, University of Heidelberg, INF 224, Heidelberg, Germany. Wilko.weichert@med.uni-heidelberg.de.|National Center of Tumor Diseases, Heidelberg, Germany. Wilko.weichert@med.uni-heidelberg.de.|German Cancer Consortium (DKTK), Heidelberg, Germany. Wilko.weichert@med.uni-heidelberg.de.
authors.forename: Wilko
authors.initials: W
authors.lastname: Weichert
authors.affiliation: Institute of Molecular Pathology, Trier, Germany. Kriegsmann@patho-trier.de.|MVZ for Histology, Cytology and Molecular Diagnostics, Trier, Germany. Kriegsmann@patho-trier.de.
authors.forename: Jörg
authors.initials: J
authors.lastname: Kriegsmann
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D020558:GTP Phosphohydrolases / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000191:economics / Q000379:methods; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
chemical_list: C117307:KRAS protein, human; D008565:Membrane Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)