BACKGROUND: Increasing evidence points to a negative correlation between K-ras mutations and patient's response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed. METHODS: We designed a high resolution melting (HRM) technology-based approach followed by direct sequencing to determine K-ras exon 1 (codons 12/13) tumour genotype. RESULTS: Reconstruction experiments demonstrated an analytical sensitivity of the K-ras exon 1 HRM assay following sequencing of 1.5-2.5% of mutated DNA in a background of wild-type DNA. Assay reproducibility and accuracy were 100%. Application of the HRM assay following sequencing onto genomic DNA isolated from formalin-fixed paraffin-embedded tumour specimens of non-small cell lung cancer (n=91) and colorectal cancer (n=7) patients revealed nucleotide substitutions at codons 12 or 13, including a homozygous mutation, in 33 (34%) and 5 (5%) cases, respectively. Comparison to conventional nested-PCR following cycle-sequencing showed an overall high agreement in genotype findings (kappa value of 0.96), with more mutations detected by the HRM assay following sequencing. CONCLUSIONS: HRM allows rapid, reliable and sensitive pre-screening of routine diagnostic specimens for subsequent genotyping of K-ras mutations, even if present at low abundance or homozygosity, and may considerably facilitate personalized therapy planning.
BACKGROUND: Increasing evidence points to a negative correlation between K-ras mutations and patient's response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed. METHODS: We designed a high resolution melting (HRM) technology-based approach followed by direct sequencing to determine K-ras exon 1 (codons 12/13) tumour genotype. RESULTS: Reconstruction experiments demonstrated an analytical sensitivity of the K-ras exon 1 HRM assay following sequencing of 1.5-2.5% of mutated DNA in a background of wild-type DNA. Assay reproducibility and accuracy were 100%. Application of the HRM assay following sequencing onto genomic DNA isolated from formalin-fixed paraffin-embedded tumour specimens of non-small cell lung cancer (n=91) and colorectal cancer (n=7) patients revealed nucleotide substitutions at codons 12 or 13, including a homozygous mutation, in 33 (34%) and 5 (5%) cases, respectively. Comparison to conventional nested-PCR following cycle-sequencing showed an overall high agreement in genotype findings (kappa value of 0.96), with more mutations detected by the HRM assay following sequencing. CONCLUSIONS: HRM allows rapid, reliable and sensitive pre-screening of routine diagnostic specimens for subsequent genotyping of K-ras mutations, even if present at low abundance or homozygosity, and may considerably facilitate personalized therapy planning.
Authors: Daoud Sie; Peter J F Snijders; Gerrit A Meijer; Marije W Doeleman; Marinda I H van Moorsel; Hendrik F van Essen; Paul P Eijk; Katrien Grünberg; Nicole C T van Grieken; Erik Thunnissen; Henk M Verheul; Egbert F Smit; Bauke Ylstra; Daniëlle A M Heideman Journal: Cell Oncol (Dordr) Date: 2014-09-11 Impact factor: 6.730
Authors: Leonard Da Silva; Peter T Simpson; Chanel E Smart; Sibylle Cocciardi; Nic Waddell; Annette Lane; Brian J Morrison; Ana Cristina Vargas; Sue Healey; Jonathan Beesley; Pria Pakkiri; Suzanne Parry; Nyoman Kurniawan; Lynne Reid; Patricia Keith; Paulo Faria; Emilio Pereira; Alena Skalova; Michael Bilous; Rosemary L Balleine; Hongdo Do; Alexander Dobrovic; Stephen Fox; Marcello Franco; Brent Reynolds; Kum Kum Khanna; Margaret Cummings; Georgia Chenevix-Trench; Sunil R Lakhani Journal: Breast Cancer Res Date: 2010-07-06 Impact factor: 6.466
Authors: J R Dijkstra; D A M Heideman; G A Meijer; J E Boers; N A 't Hart; J Diebold; A Hirschmann; G Hoefler; G Winter; G Miltenberger-Miltenyi; S V Pereira; S D Richman; P Quirke; E L Rouleau; J M Guinebretiere; S Tejpar; B Biesmans; J H J M van Krieken Journal: Virchows Arch Date: 2012-12-15 Impact factor: 4.064
Authors: Erik Thunnissen; Evan Boers; Daniëlle A M Heideman; Katrien Grünberg; Dirk J Kuik; Arnold Noorduin; Matthijs van Oosterhout; Divera Pronk; Cees Seldenrijk; Hannie Sietsma; Egbert F Smit; Robertjan van Suylen; Jan von der Thusen; Bart Vrugt; Anne Wiersma; Birgit I Witte; Michael den Bakker Journal: Virchows Arch Date: 2012-10-12 Impact factor: 4.064
Authors: V Deschoolmeester; C Boeckx; M Baay; J Weyler; W Wuyts; E Van Marck; M Peeters; F Lardon; J B Vermorken Journal: Br J Cancer Date: 2010-10-19 Impact factor: 7.640
Authors: N C T van Grieken; T Aoyama; T Aoyma; P A Chambers; D Bottomley; L C Ward; I Inam; T E Buffart; K Das; T Lim; B Pang; S L Zhang; I B Tan; B Carvalho; D A M Heideman; Y Miyagi; Y Kameda; T Arai; G A Meijer; A Tsuburaya; P Tan; T Yoshikawa; H I Grabsch Journal: Br J Cancer Date: 2013-03-19 Impact factor: 7.640