Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

Abstract

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

Enriching extracellular vesicles for mass spectrometry

“…In addition to antibody reagents, vesicles have been selected using peptides (Ghosh et al, 2014; Knol et al, 2016; Taylor et al, 2020) or heparin (Balaj et al, 2015; Reiter et al, 2019; Wang & Turko, 2018). The most widely reported use of a peptide affinity reagent is Vn96, a peptide designed to have high affinity for heat shock proteins (Griffiths et al, 2017, 2019).…”

“…The limitation of the Vn96 affinity enrichment strategy is that the presence of heat shock proteins on exosome vesicles has not been established to be a universal feature. Vn96 affinity enrichment has been used to preferentially enrich samples in vesicles from cancer cell lines as vesicles from those cells are reported to have HSP proteins on the exterior (Griffiths et al, 2017; Taylor et al, 2020). Enrichment of vesicles on heparin coated beads is also likely due to affinity of heparin for proteins on the vesicles (Balaj et al, 2015).…”

Feasibility of Leukemia-Derived Exosome Enrichment and Co-isolated dsDNA Sequencing in Acute Myeloid Leukemia Patients: A Proof of Concept for New Leukemia Biomarkers Detection

“…We tested the possibility of taking advantage of the application of a user-friendly commercial kit certifying the selection of EVs with endosomal origin. The peptide-based enrichment of EVs derived from malicious cells is expected to really improve the sensitivity of the approach, as experienced by Taylor and colleagues [ 33 ], and result in an increased quality and quantity of exosomal dsDNA. Similar approaches were explored in solid tumors with impressive results [ 34 ].…”

Recent developments in isolating methods for exosomes

“…Bathini S et al proposed a magnetic particle-based chip to isolate EVs by using the synthetic peptide Vn96 (Bathini et al, 2021). Vn96 can bind to the heat shock protein (HSP) expressed on the surface of EVs (Taylor et al, 2020;Roy et al, 2021). A 3D mixer and a sedimentation unit are included in the chip.…”