Analysis of cancer-related mutations in extracellular vesicles RNA by Droplet Digital™ PCR

Extracellular vesicles (EVs) are taking their place as potential biomarkers in the field of liquid biopsy. In this study, EVs were isolated from plasma samples of 31 patients with colorectal cancer and melanoma via differential centrifugation and Droplet Digital (TM) PCR (Bio-Rad, CA, USA) was used to profile BRAF V600E/K, KRAS G12A/C/D/V and KRAS G13D mutations from EV-derived cDNA. The concordance rates with corresponding tissue were 54% and 44% in the colorectal cancer and melanoma cohort, respectively. Two patients displayed mutations in EVs not previously detected in tissue as evidence for emerging molecular resistance to anti-EGFR and BRAF/MEK inhibitor therapy prior to radiological evidence of tumor progression. We concluded that EV-derived nucleic acids may provide clinically relevant diagnostic information and mirror evolution of the disease. METHOD SUMMARY Extracellular vesicles (EVs) derived from patient plasma were isolated via serial centrifugation, followed by EV-RNA isolation and EV-cDNA amplification. Amplified EV-cDNAs were analyzed with the Bio-Rad QX200(TM) ddPCR system (CA, USA). EVs were also morphologically visualized with transmission electron microscopy, while their proteomic content was verified by western blotting.