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.

Michael Krypuy,Alexander Dobrovic,Genni M Newnham,David M Thomas,Matthew Conron

doi:10.1186/1471-2407-6-295

Abstract

BackgroundThe 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.MethodsWe 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.ResultsKnown 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.ConclusionHRM 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.

Highlights

The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers
High resolution melting (HRM) was able to discriminate between wild-type DNA and the different mutations present in the cell line DNAs using the 189 bp amplicon flanking exon 2
We have shown that SYTO 9 is a suitable dye for mutation scanning with HRM, as it was able to sensitively detect KRAS mutations in cell line controls and in tumour samples

Summary

Introduction

The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. In nonsmall 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 clinically important. High resolution melting (HRM) is an emerging technique for detection of nucleic acid sequence variation [1] that has enormous potential to meet these clinical demands. We have used detection of codon 12 and 13 mutations in the KRAS gene to establish that HRM is a viable methodology that is readily performed both in a research setting and in a routine molecular pathology laboratory. Mutated ras proteins have impaired GTPase activity removing the "off switch" thereby resulting in a continual stimulus for cellular proliferation

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