With the advent of personalised medicine, there is a compelling need for rapid and accurate methods for detection of nucleic acid sequence changes in clinical specimens. An ideal technology should be sensitive enough to accommodate significant amounts of stromal and normal cell contamination, robust and simple enough to be readily implemented in a diagnostic laboratory, rapid enough to provide important therapeutic information in a clinical timeframe, and cost-efficient. 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.

The ras family genes were originally identified as oncogenes in acutely transforming retroviruses 2. Three highly homologous ras proteins are encoded by the KRAS, HRAS and NRAS genes. A high frequency of ras mutations has been found in many tumour types. Ras mutations are generally restricted to codons 12 and 13 in exon 2 (Figure 1) and codons 59 and 61 in exon 3, all of which result in constitutive activation of the ras protein 34. Mutated ras proteins have impaired GTPase activity removing the "off switch" thereby resulting in a continual stimulus for cellular proliferation.

While the frequency of mutations and the ras family gene that is mutated varies by cancer type, approximately 30% of all human cancers harbour a mutation in a ras gene with mutations most frequently occurring in KRAS. KRAS mutations occur in more than 90% of pancreatic adenocarcinomas, in approximately 40% of colorectal cancers and 33% of non- small cell lung carcinomas (NSCLC) 4.

KRAS mutations have a mutually exclusive relationship with EGFR mutations in NSCLC 56. Mutations in the tyrosine kinase domain of the EGFR gene have been observed in NSCLC patients with a response to tyrosine kinase inhibitors such as gefitinib and erlotinib 789. In contrast to EGFR mutations, NSCLC patients that have mutations in KRAS do not respond to tyrosine kinase inhibitors 10. NSCLC patients with EGFR mutations seem to have a favourable prognosis 1112 whereas those with KRAS mutations have poor prognosis 13141516171819. Mutant KRAS is thus both a predictive marker and a prognostic marker in NSCLC.

KRAS mutation is also a predictive and prognostic marker in other tumour types. In a recent study of metastatic colorectal cancer treated with the monoclonal antibody cetuximab, patients with KRAS mutations were resistant to the drug and had a poorer prognosis compared with those without mutations 20. A review of the literature concluded that KRAS, but not p53, mutations, were associated with poorer overall survival in colorectal cancer patients 21. KRAS mutations in pancreatic cancer are also associated with poor prognosis 22232425.

Detection of KRAS mutations has often been the primary validation for many of the numerous polymerase chain reaction (PCR) based mutation detection methods. Methodologies described in the literature include single-strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (DHPLC), denaturant gradient gel electrophoresis, denaturant capillary electrophoresis, automated constant denaturant capillary electrophoresis and temperature denaturant capillary electrophoresis, restriction digestion, allele specific PCR, allele specific nucleotide hybridisation, PCR-reverse dot blot, activated RAS-GTP specific biosensor, oligonucleotide hybridisation assay, mass spectrometry, peptide nucleic acid PCR, and array based techniques 2627282930313233343536373839404142. All these methods have their advantages and disadvantages in terms of simplicity, performance, sensitivity, turn-around time and cost.

DNA sequencing 43 has been considered the "gold standard" technique because it can identify the specific mutation that may be present. However it has the disadvantages of high relative cost and limited sensitivity. Pyrosequencing has the advantage of greater sensitivity and lower cost 44, but the relatively high cost of the required apparatus has limited its uptake.

In mutation scanning methodologies such as SSCP and DHPLC, samples demonstrating variant profiles are selected for confirmatory analysis with mutation identifying methods such as dideoxy sequencing. An advantage of scanning methodologies is that the amount of sequencing performed is significantly reduced, thereby reducing laboratory costs and analysis time. The range of denaturant capillary electrophoresis methods have many advantages including sensitivity, identification of mutant samples through characteristic peak patterns and high throughput when using a 96-array capillary electrophoresis instrument 303132. A disadvantage of the denaturant capillary electrophoresis methods is that access to a dedicated capillary electrophoresis instrument is required.

High resolution melting (HRM) involves the precise monitoring of the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured by increasing temperature. Developments in instrumentation and the use of fully saturating intercalating dyes have made HRM analysis possible 1. An important advantage of HRM over many of the above methods is that it is an in-tube method in which the analysis is performed immediately after the amplification and is thus particularly suitable for medium to high-throughput applications. Methods that require amplified PCR product to be removed from the tube for analysis are inevitably more laborious and require stringent precautions to prevent crossover of PCR products. The HRM methodology has major potential for the rapid and inexpensive detection of DNA sequence variations such as polymorphisms and germline mutations in DNA 4546.

A particularly desirable HRM application is the detection of cancer specific mutations in tumour biopsies. HRM has been used to identify somatic mutations in the c-KIT, BRAF, HER2 and EGFR genes 47484950515253. In this study, we set out to develop a sensitive, clinically useful HRM assay to detect somatic mutations in the KRAS gene. We validated the assay with cell lines with known KRAS mutations, and with a panel of NSCLC biopsies that were also tested for KRAS mutations by sequencing.

A wide range of mutation detection methodologies exist, of which sequencing has been considered the gold standard because of its ability to identify the specific DNA sequence change that has occurred. However, dideoxy sequencing is rarely sensitive below a 10% mutant allele frequency, which corresponds to a threshold of 20% tumour cells heterozygous for a mutation 44. The sensitivity of sequencing can be increased by microdissection (ranging from relatively crude scraping to laser capture) to enrich for tumour cells, but this adds to the complexity of genetic testing. The variety of methodologies used for mutation detection probably account for much of the wide divergence of mutation frequencies often reported for particular genes in given cancer types.

High resolution melting (HRM) methodology represents a significant advance for mutation detection in tumour specimens. The developments in intercalating dye technology have played a major role in the emergence of HRM methodology. Different base substitutions produce slight differences in melting behaviour and the resolution of these melting differences requires an appropriate intercalating dye. SYBR green, the most widely used intercalating dye for monitoring PCR, is not suitable for HRM analysis because it can only be used at non-saturating concentrations. Thus, SYBR green molecules that dissociate from DNA during melting can re-intercalate into regions of un-melted double stranded DNA 55. This phenomenon of dye redistribution can mask the small differences in melting behaviour. New generation intercalating dyes such as LC Green and LC Green PLUS allow accurate genotyping of single-nucleotide polymorphisms and sensitive mutation scanning 156. Monis et al. (2005) showed that SYTO 9 was suitable for fluorescence monitoring of PCR and standard melting analysis 57. In this study, 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.

An advantage of performing HRM analysis on a real time PCR machine with HRM capability (for which this is the first report), is that the PCR amplification and HRM analysis are performed in the one run and the results are available for analysis at the end of the run. This allows assessment of amplification for all samples before HRM analysis as a quality control measure. In our experience, samples with poor or late amplification must be treated with some caution in HRM analysis. Use of a single instrument also minimises the amount of manual handling which improves turnaround times.

We observed a degree of variation of the melting plots within wild-type samples, which imposed a lower limit on the sensitivity of mutation detection with the tumour sample set. The presence of small amounts of non-specific amplicons, differing salt or inhibitor concentrations, or differences in PCR amplification between samples may contribute to the variation. It is clear that as the spread of wild-type curves increases, the sensitivity of mutation detection decreases. Thus instrumentation with greater temperature uniformity across the samples assayed may have an advantage in mutation scanning.

Difference plots are visually the best way to compare melting profiles. However, difference plots emphasise small variations in the melting profiles between different samples giving rise to a wild-type spread. Nevertheless, wild-type sample plots were quite distinct from samples with mutations. We chose a sample from the centre of the distribution of the wild-type samples as the normalisation sample for the construction of the difference plots to standardise the interpretation. The presence of mutated sequence, even in low proportion, produced difference plots that were readily discriminated from wild-type samples as seen in the sensitivity testing of the assay (Figure 3).

In a study that used HRM to screen for mutations in the RET proto-oncogene, it was found that some mutations could be directly genotyped from the melting curve shapes 58. However, the applicability of shape analysis in tumour samples will be compromised by the influence on the shape of the melting profile of varying degrees of heteroduplex formation due to the proportion of normal DNA present in the tumour biopsy. Determination of the precise mutation will depend on more complex methods such as allele-specific competitive blocker PCR, dideoxy sequencing or pyrosequencing.

Sensitivity testing is an essential step in the development of HRM assays for mutation detection and different sensitivities may be achieved with certain mutations or as in this report, with particular PCR amplicons. Liew et al. previously examined the role of the size of the PCR product in SNP genotyping and found that smaller amplicons gave better differentiation between genotypes 59. Consistent with those results, we found that use of a smaller amplicon increased the sensitivity of mutation detection for KRAS mutations in the cell lines tested. The 189 bp and 92 bp PCR amplicons have a G+C content of 37% and 43% respectively and this may also have played a role in the altered sensitivity of mutation resolution.

Identifying mutations will depend on being able to confidently differentiate the aberrant curves from the wild-type samples. The cell line dilution results were assessed by several independent observers in a blinded fashion and the results indicate that 5–6% tumour cell line DNA in a background of normal DNA could be detected using the 92 bp PCR amplicon. This will allow confident screening for mutations in samples that have at least 10% tumour cells.

The results of our cell line dilution studies indicate that in some cases, mutations detected by HRM will not be detectable by dideoxy sequencing. These situations must be considered when setting up mutation detection algorithms. Pyrosequencing may be the confirmatory sequencing method of choice as it has been shown to be sensitive to 5% mutant sequence 44 and minimises the amount of reactions needed compared to allele-specific PCR methods. We did not observe a discrepancy between HRM and dideoxy sequencing in our study. This is likely to be due to the pathological review and dissection of areas consisting of at least 50% tumour cells. This argues for a testing algorithm consisting of a pathological review to mark out sections of interest and a crude microdissection to purify the tumour component.

In the case of KRAS codon 12 and 13 mutations, allele-specific competitive blocker PCR may be a cost-effective sensitive method to identify mutations detected by HRM which has the ability to identify low level mutations identified by HRM not detectable by dideoxy sequencing 60. Allele-specific competitive blocker PCR has been shown to be sensitive down to 0.1% mutant sequence in a background of normal DNA 61.

Somatic mutations of KRAS occurring at bases 34, 35, 37 and 38 from the start codon give rise to amino acid substitutions at codon 12 and 13. We observed six of the possible 12 mutations, five of which were seen in the patient specimens. The G12S (35G > A) and G12R (35G > C) mutations, which were not observed in our relatively small patient set, are the rarest codon 12 mutations in lung cancer 62. G13D was the only codon 13 mutation observed but all of the codon 13 mutations are rare with only G13C (37G > T) and G13D (38G > A) occurring at any appreciable frequency. All of the samples were sequenced and no other mutations were detected that had not been detected by HRM. It might be considered that a limitation of this study is that all possible KRAS mutations were not tested. However, similar sensitivities would be achieved for the other mutations because the lower melting temperature of heteroduplexes is the basis of the sensitivity of mutation detection using HRM.

The frequency of KRAS mutations in this small sample set was higher than many reports in the literature. There were 7 mutations in 22 (32%) in the adenocarcinomas and 2 mutations in 8 (25%) large cell carcinomas but these frequencies may change substantially when larger numbers of specimens are analysed. As previously observed by others 56, there was a mutually exclusive relationship between KRAS and EGFR mutation status for our sample set. The adenocarcinoma samples that had EGFR mutations did not present with KRAS mutations. NSCLC patients with mutated KRAS do not respond to tyrosine kinase inhibitors and have a poorer prognosis than patients without mutations 1013141516171819. Early identification of these patients will allow appropriate and cost-effective treatment decisions to be made by clinicians.

In conclusion, we have presented a robust assay for screening for KRAS codon 12 and 13 mutations that is applicable to clinical samples. The main limitation of HRM is that the precise mutation cannot be readily identified and it thus needs to be used in conjunction with a sequencing method. Nevertheless, because HRM is both inexpensive and high throughput, we believe that HRM is now the method of choice for screening tumour samples for somatic mutations prior to sequencing, especially when the mutations are likely to occur at low frequency. As targeted therapies focus increasingly on key mutations in oncogenic pathways, it will be necessary to screen not only for multiple exons within candidate genes, but also multiple genes within key pathways to confirm or exclude involvement of that pathway. The rate limiting step in genetic analysis of tumours is now the collection of samples and the preparation of the DNA.

MK carried out the molecular genetic studies, data analysis and wrote the manuscript. AD conceived the study, designed the primers, participated in the study design and coordination and co-wrote the manuscript. GN, MC, DT were responsible for human research ethic committee submissions to acquire the samples used in the study, their storage and processing, participated in the study design and coordination and contributed their ideas to the manuscript. All authors read and approved the final manuscript.