A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.

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.

Epidermal growth factor receptor (EGFR) gene copy number detection in non-small-cell lung cancer; a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization.

AIMS: The epidermal growth factor receptor (EGFR) is an important target for anticancer therapy. In non-small-cell lung cancer (NSCLC), mutations in the tyrosine kinase domain of EGFR and EGFR gene copy number have been demonstrated to identify patients most likely to benefit from EGFR tyrosine kinase inhibitors. EGFR gene copy number has been assessed mainly by fluorescence in situ hybridization (FISH), a method requiring the use of a fluorescence microscope and often hampered by the rapid fading of the fluorescent signal. These limitations of FISH can be overcome by using chromogenic in situ hybridization (CISH). To test the applicability of CISH for EGFR gene copy number testing in NSCLC, a comparison of CISH and FISH was performed. METHODS AND RESULTS: A total of 58 formalin-fixed, frozen NSCLC tissue samples were collected on which both CISH and FISH were performed. High concordance was found in the assessment of EGFR copy number between observers and between techniques (kappa coefficient = 0.64-0.76). CISH seemed the ideal technique for paraffin sections, whereas FISH was favourable for frozen material. CONCLUSIONS: CISH is a suitable alternative strategy to FISH in determining EGFR gene copy number in NSCLC patients.