title: Metastatic colorectal cancer KRAS genotyping in routine practice: results and pitfalls.
abstract: KRAS genotyping is mandatory before anti-epidermal growth factor receptor monoclonal antibody therapy in metastatic colorectal cancer, which is the second leading cause of cancer-related death in the United States and in Europe. Thus, large-scale KRAS mutation screening is needed for efficient patient management and in the future metastatic colorectal cancer genotyping might also include the detection of the BRAF V600E mutation, which is a very strong negative prognostic factor in colorectal cancer. We report our experience of routine KRAS/BRAF mutation screening practice performed on 1130 formalin-fixed paraffin-embedded tumor samples from 992 colorectal cancer patients. DNA was extracted from macrodissected tumor areas highlighted by a pathologist, KRAS codons 12/13 and BRAF V600E mutations were assessed in a single SNaPshot® multiplex assay and each mutation was confirmed by an independent analysis. KRAS and BRAF mutations were, respectively, present in 41.8 and 6.5% of the tumor samples. If KRAS and BRAF mutations were mutually exclusive, four samples presented two concomitant KRAS mutations. Genotyping of paired primary tumors and metastases from 44 patients indicated that 5 patients (11.4%) presented discordant KRAS mutational status. KRAS genotype heterogeneity was also observed within primary tumor sites in seven cases. Non-reproducible KRAS artefactual mutations were detected in 53 samples (4.7%). We found that the prominent mechanism leading to these artefactual mutations was the fragmentation of DNA occurring during tissue processing. Routine KRAS genotyping performed on formalin-fixed paraffin-embedded tissues requires, therefore, the development of quality control scheme for molecular pathology, especially because of DNA damages induced by formalin fixation. The tumor heterogeneity observed in some patients indicates that it should be more appropriate to perform KRAS genotyping on metastases if sample is available.
authors.affiliation: Laboratory of Tumor Genetics, University Hospital, Rouen, France. aude.lamy@chu-rouen.fr
authors.forename: Aude
authors.initials: A
authors.lastname: Lamy
authors.forename: France
authors.initials: F
authors.lastname: Blanchard
authors.forename: Florence
authors.initials: F
authors.lastname: Le Pessot
authors.forename: Richard
authors.initials: R
authors.lastname: Sesboüé
authors.forename: Frédéric
authors.initials: F
authors.lastname: Di Fiore
authors.forename: Jessie
authors.initials: J
authors.lastname: Bossut
authors.forename: Elodie
authors.initials: E
authors.lastname: Fiant
authors.forename: Thierry
authors.initials: T
authors.lastname: Frébourg
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Sabourin
mesh_terms: D016477:Artifacts; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000302:isolation & purification; D005557:Formaldehyde; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D009362:Neoplasm Metastasis; D016612:Paraffin Embedding; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins