Highly sensitive somatic mutation detection in tumor samples by hi-res melting    on the LightScanner Free

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The goal of this project was to identify somatic mutations in primary tumors using a high sensitivity DNA screening assay. The discovery of somatic variants is vital for understanding the basis of tumorigenesis and potentially for patient selection. The primary technical challenge encountered in detecting somatic variants is the cellular heterogeneity that exists in tumor samples, e.g. a mutant allele may only be present at ~ 5-10% the level of the wild-type allele. We have used Hi-Res Melting on the LightScanner^® instrument, a 96-well plate-based platform, as a high sensitivity prescreen of tumor DNA samples for sequence variants.

The platform was used to scan for variants in a diverse set of solid tumor types. Seventy-six assays for 77 exons from 18 genes, including AXL, BRAF, EGFR, ERBB2, FGF3R, JAK1, JAK2, KIT, MET, PDGFRA, PDPK1, PIK3CA and RET, were developed and used to screen approximately 600 tumors. Overall, ~ 45,000 reactions were scanned and Hi-Res Melting detected over 600 variants including base (single and multiple) substitutions and indels. The majority (~ 65%) of the variants detected were determined to be germline based on Sanger resequencing of both tumor & normal samples. The dominant type of somatic mutations were missense mutations (85%). Also found were splice site, indels, nonsense and silent changes. Based on resequencing data, the total Hi-Res Melting false positive rate was estimated to be less than 0.2%.

The sensitivity and specificity of Hi-Res Melting was determined by a blinded Sanger re-sequencing study of 6 exons, 4 of which had a high prevalence of variants, using independently designed amplicons. A total of 695 amplicons were sequenced in both directions. Two independent reviewers identified 90 variants. All 90 of these variants were detected by Hi-Res Melting, demonstrating 100% sensitivity. In addition to these 90 variants identified by re-sequencing, 25 more Hi-Res Melting variant calls were made. Eighteen of these variants were observed upon more comprehensive manual review of sequence data. This indicates Hi-Res Melting was more sensitive than sequencing with a specificity of at least 99%.

This study demonstrates that Hi-Res Melting using the LightScanner^® instrument is a highly sensitive and specific, high throughput method that can be used to screen for somatic mutations in heterogeneous tumor samples.