Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients

Davide Zocco,Maria Teresa Fierro,Simona Bernardi,Mauro Novelli,Ottavia Malavenda,Chiara Astrua,Chiara Foroni,Natasa Zarovni,Antonio Chiesi,Pietro Quaglino,Domenico Russo,Paolo Fava,Francesco M Carpi,Laura Bianciardi

doi:10.1038/s41598-020-72834-6

Abstract

Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

Highlights

Melanoma is the most serious and aggressive form of skin cancer, accounting for 79% of all skin cancer deaths[1]
These methods were compared to UC for their ability to capture BRAFV600E–positive Extracellular vesicles (EVs) derived from a melanoma cell line and spiked into healthy donor (HD) plasma (Fig. 1A)
We found that peptide-based affinity isolation (PA) recovered more EV-derived BRAFV600E mutation than Chemical precipitation (CP), UC and IA using antitetraspanin-CD63 and anti-CD9-antibody-coated beads (56%, 47%, 25% or 4% respectively; Fig. 1B)

Summary

Introduction

Melanoma is the most serious and aggressive form of skin cancer, accounting for 79% of all skin cancer deaths[1]. Selective inhibition of B-Raf with vemurafenib (Zelboraf; Roche) or dabrafenib (Tafinlar; Novartis) currently used in association with comibenitib (Cotellic; Roche) and trametinib (Mekinist; Novartis), has revolutionized the treatment of unresectable or metastatic melanoma (MM) patients bearing BRAF mutations[2,3]. These patients are routinely identified with molecular diagnostic testing Sequencing, PCR) using DNA specimens obtained from primary and/or metastatic tumor tissues This procedure has two major shortcomings: (a) it is invasive and cannot be used to re-assess or monitor the BRAF gene status overtime; (b) a significant number of mutation-positive patients are missed due to tumor genomic h eterogeneity[4]. High prevalence of KRAS mutation was found in EV-associated DNA (EV-DNA) from early stage pancreatic cancer patients, further supporting the role of EVs as a carrier of clinically valuable genomic variants[22]

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