Deborah Barden and Dr. from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significantKRASmutations by COLD-PCR DNA sequencing. Glycitein Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background. == Conclusions == We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay forKRASmutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing. == Background == KRAS (Kirstenratsarcoma virus homolog 2) is a membrane-anchored G-protein that acts downstream of the epidermal growth factor receptor (EGFR) to activate pro-growth and anti-apoptotic pathways, including the MAP kinase and PI3 kinase pathways [1]. Mutations in codons 12 and 13 of theKRASgene confer resistance to drugs targeted at EGFR by impairing GTPase activity, which results in constitutive EGFR-independent signaling.KRASis one of the most frequently mutated oncogenes in human cancer, Glycitein with significant mutation rates in common epithelial malignancies such as colon cancer (~40%), lung cancer (~20%), gastric cancer (~10%), and pancreatic cancer (~65%) (COSMIC database;http://www.sanger.ac.uk/genetics/CGP/cosmic/) [2,3]. The role ofKRASmutation status in clinical decision making is best defined for colon cancer. Several large randomized-controlled trials demonstrated no benefit from expensive anti-EGFR drugs such as cetuximab (Erbitux) in patients withKRAS-mutant colon cancer [4,5]. The National Comprehensive Cancer Network (NCCN) now recommendsKRAStesting prior to initiation of anti-EGFR therapy in colon cancer patients [6]. Accumulating evidence also suggests a role forKRAStesting to guide therapy in patients with non-small cell lung cancer [7,8]. Greater than 95% ofKRASmutations occur in codon 12 or codon 13. Within these codons G12 D (GGT to GAT), G12V (GGT to GTT), and G13 D (GGC to GAC) comprise ~80% of the mutations [2,4]. Less frequent mutations include G12 Glycitein S (GGT toAGT), G12C (GGT toTGT), G12R (GGT toCGT), and G12A (GGT to GCT). Silent mutations are exceedingly rarehttp://www.sanger.ac.uk/genetics/CGP/cosmic/. CO-amplification atLowerDenaturation temperature (COLD)-PCR is a recently described method to selectively amplify mutant alleles in a wild type background that does not require any additional instrumentation or reagents to Eno2 implement [9]. Two forms of COLD-PCR are described:fastCOLD-PCR andfullCOLD-PCR (reviewed in [10]). Fast COLD-PCR enriches G:C to A:T mutations that slightly, but predictably lower the melting temperature (Tm) of the PCR amplicon, by using a critical denaturation temperature (Tc) that favors PCR amplification of the mutant allele. Full COLD-PCR theoretically enhances detection of any type of mutation via conditions which promote annealing of WT:mutant pairs and selective denaturation of these heteroduplexes at an empirically determinedTc. As the name implies, fast COLD-PCR has the advantage of being more rapid than full COLD-PCR (1-2 hours of instrument time compared to 5-8 hours) and is also easier to troubleshoot and implement in our experience. Fast COLD-PCR is ideal forKRAScodon 12 and 13 mutational analysis because >90% of the mutations are G:C to A:T changes (G12 D, G12V, G12 S, G12C, G13D), and the remaining common mutations areTm-neutral G:C to G:C changes (G12R and G12A). Here, we report a novel strategy for mutation detection that combines fast COLD-PCR with two-probe melting curve analysis using the LightCyclerinstrument. By incorporating COLD-PCR we achieve >10-fold enhancement for the majority ofKRASmutations with a sensitivity of ~1% mutant allele in a wild type background. == Glycitein Methods == == Patient tumor samples and DNA extraction == All clinical specimens consisted of DNA prepared from formalin-fixed paraffin-embedded (FFPE) tissue from patients with sporadic colorectal carcinoma. De-identified residual clinical specimens were obtained in accordance with the declaration of Helsinki and ethics guidelines of the local institutional review board. We obtained a total of 61 de-identified specimens: 10 DNA specimens from the University of Texas MD Anderson Cancer Center, 4 specimens of unstained FFPE tissue sections with corresponding hematoxylin-eosin (HE) stained slides from the University of Chicago, and 47 DNA specimens prepared from FFPE tissue from colorectal cancer specimens at the University of Washington. HE-stained slides were used as a guide to manually dissect areas of tumor tissue from unstained slide sections. Genomic DNA was prepared with the.