Genomic profiling of extracellular vesicle-derived DNA from bronchoalveolar lavage fluid of patients with lung adenocarcinoma

Abstract

Background: Extracellular vesicles (EVs) are membrane-bound and nanometer-sized particles released from most types of cells, containing double-stranded DNA reflecting mutational status of the parental tumor cells. Furthermore, epidermal growth factor receptor (EGFR) genotyping using EV-derived DNA (EV DNA) in bronchoalveolar lavage fluid (BALF) showed almost 100% sensitivity in patients with advanced non-small cell lung cancer (NSCLC).

Methods: We assessed the technical performance of DNA derived from BALF-EV (BALF EV DNA) in targeted next-generation sequencing (NGS) for detection and quantification of mutations compared with the matching tissue DNA in 20 lung adenocarcinomas.

Results: DNA yields, tumor purity, and depth of coverage were higher using the tissue DNA than using the BALF EV DNA. However, estimated library size was not significantly different between the two samples, and BALF EV DNA yielded longer fragments than tissue DNA. Overall mutation concordance between the two samples were 56% for nonsynonymous somatic mutations and increased to 81% for clinically significant mutations. By-variant sensitivity for clinically significant somatic mutations increased from 62% to 83% in the NGS of BALF EV DNA. Allele frequencies of EGFR and TP53 were higher in tissue DNA (10-25%) than in BALF EV DNA (<5%). Tumor mutation burden of BALF EV DNA correlated with that of tissue DNA.

Conclusions: Our findings demonstrate, for the first time, that BALF EV DNA in patients with NSCLC can be a reliable DNA source for targeted NGS for the identification of actionable genetic alterations and that this approach has high clinical feasibility and utility.

Keywords: Extracellular vesicles (EV); bronchoalveolar lavage fluid (BALF); liquid biopsy; next-generation sequencing (NGS); non-small cell lung cancer (NSCLC).

Figure 1

Quality parameters of tissue and BALF EV DNAs. BALF EV DNA (EV) was inferior to tissue DNA (tissue) in terms of total DNA amount (A), mean read depth (B), uniformity (C), and estimated tumor purity (D), but was of comparable quality in terms of library size (E) and fragment length (F). BALF, bronchoalveolar lavage fluid; EV, extracellular vesicle.

Figure 2

Mutation concordance and sensitivity. (A) Overall mutation concordance between tissue and BALF EV DNAs was 56% for nonsynonymous mutations and increased to 81% for clinically significant mutations. (B) With mutations detected in tissue DNA as reference, by-variant sensitivity of nonsynonymous mutations in BALF EV DNA was 62% and increased to 83% for clinically significant mutations. BALF, bronchoalveolar lavage fluid; EV, extracellular vesicle.

Figure 3

VAF of clinically significant putative somatic mutations. (A) VAF of clinically significant putative somatic variants detected in tissue and BALF EV DNAs. (B) Correlation between VAF of mutations identified in tissue and BALF EV DNAs. VAF, variant allele frequencies; BALF, bronchoalveolar lavage fluid; EV, extracellular vesicle.

Figure 4

Schematic overview of overall mutational profile of 20 pairs of tissue and BALF EV DNAs. Each column represents a case. The top two panels show the stage of tumors and collection time of BALF EV DNA. The bottom panels show the distribution of clinically significant putative somatic mutations. The three variant types (tissue only, EV only, and both) are distinguished by blue, green, and bright blue, respectively. The right panel represents the overall frequencies of variants in tissue and BALF EV DNAs. BALF, bronchoalveolar lavage fluid; EV, extracellular vesicle.

Figure 5

VAFs of EGFR and TP53. (A) Distribution of VAFs of EGFR and TP53. (B) The frequencies for EGFR and TP53 collectively in tissue and BALF EV DNAs samples were 10–25% and <5%, respectively. VAF, variant allele frequencies; BALF, bronchoalveolar lavage fluid; EV, extracellular vesicle.

Figure 6

Tumor mutation burden. The tumor mutation burden determined by liquid biopsy performed using EV DNA correlated with that obtained by tumor tissue biopsy (Pearson correlation coefficient: 0.64, P<0.001). TMB, tumor mutation burden; BALF, bronchoalveolar lavage fluid; EV, extracellular vesicle.