Multiplex Enrichment and Detection of Rare KRAS Mutations in Liquid Biopsy Samples using Digital Droplet Pre-Amplification
Year: 2019
Type: article
Abstract: Oncology research is increasingly incorporating molecular detection of circulating tumor DNA (ctDNA) as a tool for cancer surveillance and early detection. However, noninvasive monitoring of conditions with low tumor burden remains challenging, as the diagnostic sensitivity of most ctDNA assays is inversely correlated with total DNA concentration and ctDNA abundance. Here we present the Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp) method, which combines single-molecule emulsification and short-round polymerase chain reaction (PCR) preamplification with digital droplet PCR detection of mutant DNA template. The MED-Amp assay increased mutant signal by over 50-fold with minimal distortion in allelic frequency. We demonstrate detection of as few as three mutant copies in wild-type DNA concentrations ranging from 5 to 50 ng. The MED-Amp assay successfully detected KRAS mutant ctDNA in 86% plasma samples obtained from patients with metastatic pancreatic ductal adenocarcinoma. This assay for high-sensitivity rare variant detection is appropriate for liquid biopsy samples or other limited clinical biospecimens.
Source: Analytical Chemistry
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: green
Funders: Doris Duke Charitable Foundation, Cancer Prevention and Research Institute of Texas, University of Michigan, University of Texas at Austin, Andrew Sabin Family Foundation, National Institutes of Health, University of Texas MD Anderson Cancer Center, National Cancer Institute, National Institute of Diabetes and Digestive and Kidney Diseases
OpenAlex ID: W2949983989