title: Optimization of routine KRAS mutation PCR-based testing procedure for rational individualized first-line-targeted therapy selection in metastatic colorectal cancer.
abstract: KRAS mutation detection represents a crucial issue in metastatic colorectal cancer (mCRC). The optimization of KRAS mutation detection delay enabling rational prescription of first-line treatment in mCRC including anti-EGFR-targeted therapy requires robust and rapid molecular biology techniques. Routine analysis of mutations in codons 12 and 13 on 674 paraffin-embedded tissue specimens of mCRC has been performed for KRAS mutations detection using three molecular biology techniques, that is, high-resolution melting (HRM), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and allelic discrimination PCR (TaqMan PCR). Discordant cases were assessed with COBAS 4800 KRAS CE-IVD assay. Among the 674 tumor specimens, 1.5% (10/674) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 38.0% (256/674) of the analysable specimens (82.4% in codon 12 and 17.6% in codon 13). Among 613 specimens in whom all three techniques were used, 12 (2.0%) cases of discordance between the three techniques were observed. 83.3% (10/12) of the discordances were due to PCR-RFLP as confirmed by COBAS 4800 retrospective analysis. The three techniques were statistically comparable (κ > 0.9; P < 0.001). From these results, optimization of the routine procedure consisted of proceeding to systematic KRAS detection using HRM and TaqMan and PCR-RFLP in case of discordance and allowed significant decrease in delays. The results showed an excellent correlation between the three techniques. Using HRM and TaqMan warrants high-quality and rapid-routine KRAS mutation detection in paraffin-embedded tumor specimens. The new procedure allowed a significant decrease in delays for reporting results, enabling rational prescription of first-line-targeted therapy in mCRC.
authors.affiliation: Service de Biopathologie, Centre Alexis Vautrin 6 Avenue de Bourgogne, 54519, Vandœuvre-lès-Nancy, France ; CNRS, UMR 7039 CRAN Nancy, France ; Université de Lorraine Nancy, France.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Chretien
authors.forename: Alexandre
authors.initials: A
authors.lastname: Harlé
authors.forename: Magali
authors.initials: M
authors.lastname: Meyer-Lefebvre
authors.forename: Marie
authors.initials: M
authors.lastname: Rouyer
authors.forename: Marie
authors.initials: M
authors.lastname: Husson
authors.forename: Carole
authors.initials: C
authors.lastname: Ramacci
authors.forename: Valentin
authors.initials: V
authors.lastname: Harter
authors.forename: Pascal
authors.initials: P
authors.lastname: Genin
authors.forename: Agnès
authors.initials: A
authors.lastname: Leroux
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Merlin
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D058990:Molecular Targeted Therapy; D009154:Mutation; D009362:Neoplasm Metastasis; D009691:Nucleic Acid Denaturation; D018579:Patient Selection; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D055815:Young Adult; 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
keywords: Colorectal cancer; HRM; KRAS; PCR-RFLP; TaqMan PCR