1. Multiplex Enrichment and Detection of Rare KRAS Mutations in Liquid Biopsy Samples using Digital Droplet Pre-Amplification
- Author
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Diane M. Simeone, Kara Schradle, Robert W. Cowan, Heather Cameron, Sara L. Manning, David Bing Zhen, Erica D. Pratt, Andrew D. Rhim, and Edmund Qiao
- Subjects
Male ,Mutant ,Early detection ,010402 general chemistry ,medicine.disease_cause ,Polymerase Chain Reaction ,01 natural sciences ,Circulating Tumor DNA ,Analytical Chemistry ,law.invention ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Gene Frequency ,Limit of Detection ,law ,medicine ,Humans ,Multiplex ,Liquid biopsy ,Allele frequency ,Polymerase chain reaction ,030304 developmental biology ,Aged ,Fluorescent Dyes ,Aged, 80 and over ,Detection limit ,0303 health sciences ,Mutation ,010401 analytical chemistry ,Dna concentration ,Liquid Biopsy ,Cancer ,Lipid Droplets ,Middle Aged ,medicine.disease ,Molecular biology ,0104 chemical sciences ,Pancreatic Neoplasms ,chemistry ,030220 oncology & carcinogenesis ,Case-Control Studies ,Female ,KRAS ,DNA - Abstract
Oncology research is increasingly incorporating molecular detection of circulating tumor DNA (ctDNA) as a tool for cancer surveillance and early detection. However, non-invasive 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 com-bines single-molecule emulsification and short-round PCR preamplification with digital droplet PCR (ddPCR) 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 3 mutant copies in wild-type DNA concentrations ranging from 5 to 50ng. 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
- Published
- 2019
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