title: KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology.
abstract: KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (kappa values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.
authors.affiliation: Institut für Pathologie, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. wilko.weichert@charite.de
authors.forename: Wilko
authors.initials: W
authors.lastname: Weichert
authors.forename: Christiane
authors.initials: C
authors.lastname: Schewe
authors.forename: Annika
authors.initials: A
authors.lastname: Lehmann
authors.forename: Christine
authors.initials: C
authors.lastname: Sers
authors.forename: Carsten
authors.initials: C
authors.lastname: Denkert
authors.forename: Jan
authors.initials: J
authors.lastname: Budczies
authors.forename: Albrecht
authors.initials: A
authors.lastname: Stenzinger
authors.forename: Hans
authors.initials: H
authors.lastname: Joos
authors.forename: Olfert
authors.initials: O
authors.lastname: Landt
authors.forename: Volker
authors.initials: V
authors.lastname: Heiser
authors.forename: Christoph
authors.initials: C
authors.lastname: Röcken
authors.forename: Manfred
authors.initials: M
authors.lastname: Dietel
mesh_terms: D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods; D005557:Formaldehyde; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D005838:Genotype; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins