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

Catherine Taylor,Stephen M Lewis,Jacynthe Lacroix,Anirban Ghosh,Rodney J Ouellette,Alexander Macpherson,Gabriel Wajnberg,Nicolas Crapoulet,Nicholas Finn,Simi Chacko,Michelle Davey

doi:10.3390/ijms21239083

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.

Highlights

Lung cancer is the leading cause of cancer deaths worldwide [1] and non-small cell lung cancer (NSCLC) accounts for approximately 90% of all lung cancer cases [2]
Testing for oncogenic activation of tyrosine kinases, such as those mediated by epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements, has become standard practice during diagnostic evaluation of NSCLC adenocarcinoma patients and has led to wide adoption of targeted therapies and greatly improved outcomes for patients with these types of genetic modifications [35]
L-extracellular vesicles (EVs) in particular are shed by cancer cells and are much more abundant in plasma from cancer patients compared to healthy donors and have been reported to contain histone-associated dsDNA [31]

Summary

Introduction

Lung cancer is the leading cause of cancer deaths worldwide [1] and non-small cell lung cancer (NSCLC) accounts for approximately 90% of all lung cancer cases [2]. Circulating tumour DNA (ctDNA) isolated from plasma has been demonstrated to have high concordance with molecular alterations observed in the primary tumour [8,9] It can be useful for selecting an appropriate targeted therapy, to assess the emergence of drug resistance mutations, and to monitor tumour burden during treatment; there is a great degree of variability in the amount of ctDNA present in plasma and the levels can be very low, representing 0.01% to 10% of the total cell-free DNA (cf-DNA) in the sample [10,11]. The cobas® EGFR Mutation Test v2 is the first FDA-approved liquid biopsy test for lung cancer patients Despite their many potential benefits, liquid biopsy approaches for tumour profiling in NSCLC still suffer from a lack of standardized techniques [13], limited coverage of relevant genes and pathogenic mutation hotspots, lack of sensitivity, and insufficient clinical validation [14]

Methods

Results

Conclusion