High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer

The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis (HRM) to screen for KRAS mutations in clinical cancer samples. In non-small cell lung cancer...

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Vydané v:BMC cancer Ročník 6; číslo 1; s. 295
Hlavní autori: Krypuy, Michael, Newnham, Genni M, Thomas, David M, Conron, Matthew, Dobrovic, Alexander
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: England BioMed Central 21.12.2006
BMC
Predmet:
Adenocarcinoma - genetics
Carcinoma, Large Cell - genetics
Carcinoma, Non-Small-Cell Lung - genetics
Codon - genetics
DNA, Neoplasm - genetics
DNA, Neoplasm - isolation & purification
Humans
Lung Neoplasms - genetics
Mutation
Nucleic Acid Denaturation
Polymerase Chain Reaction
Proto-Oncogene Proteins - genetics
Proto-Oncogene Proteins p21(ras)
ras Proteins
ISSN:1471-2407, 1471-2407
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Shrnutí:The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis (HRM) to screen for KRAS mutations in clinical cancer samples. In non-small cell lung cancer, KRAS mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important. We developed a high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for KRAS mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in EGFR exons 18-21. Known KRAS mutations in cell lines (A549, HCT116 and RPMI8226) were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5-6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had KRAS mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in KRAS and EGFR were mutually exclusive. HRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.
Bibliografia:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:1471-2407
1471-2407
DOI:10.1186/1471-2407-6-295