Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity

Abstract

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.

Keywords

• extracellular vesicles
• non-small cell lung cancer
• <i>EGFR</i> T790M
• genotyping
• size exclusion chromatography
• intravesicular DNA