High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies.

BACKGROUND: Epithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case. METHODS: We developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues. RESULTS: All 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive. CONCLUSION: This is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings.

Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection.

DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16(INK4a)) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP.

BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype.

INTRODUCTION: Individuals with germline mutations in the BRCA1 gene have an elevated risk of developing breast cancer, and often display characteristic clinicopathological features. We hypothesised that inactivation of BRCA1 by promoter methylation could occur as a germline or an early somatic event that predisposes to breast cancer with the phenotype normally associated with BRCA1 germline mutation. METHODS: We examined seven cases from breast-ovarian cancer families with tumours that showed BRCA1-like pathology but did not have detectable BRCA1 or BRCA2 germline mutations present. Methylation levels were tested by several quantitative techniques including MethyLight, methylation-sensitive high resolution melting (MS-HRM) and a newly developed digital MS-HRM assay. RESULTS: In one patient, methylation of 10% of the BRCA1 alleles was detected in the peripheral blood DNA, consistent with 20% of cells having one methylated allele. Buccal mucosa DNA from this individual displayed approximately 5% BRCA1 methylation. In two other patients, methylation of BRCA1 was detected in the peripheral blood at significantly lower but still readily detectable levels (approximately 1%). Tumour DNAs from these three patients were heavily methylated at BRCA1. The other patients had no detectable BRCA1 methylation in their peripheral blood. One of seven age-matched controls showed extremely low levels of methylation in their peripheral blood (approximately 0.1%). CONCLUSION: These results demonstrate that in some cases of breast cancer, low-level promoter methylation of BRCA1 occurs in normal tissues of the body and is associated with the development of BRCA1-like breast cancer.

High resolution melting for mutation scanning of TP53 exons 5-8.

BACKGROUND: p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. METHODS: We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. RESULTS: One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. CONCLUSION: HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.

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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.