Extracellular vesicle-based EGFR genotyping in bronchoalveolar lavage fluid from treatment-naive non-small cell lung cancer patients

Abstract

Background: Extracellular vesicles (EV) have been proven to contain double-stranded DNA reflecting the mutational status of the parental tumor cells in non-small cell lung cancer (NSCLC), which can be translated into clinically useful EV-based liquid biopsy for Epidermal growth factor receptor (EGFR) genotyping using bronchoalveolar lavage fluid (BALF) obtained from tumor site.

Methods: Patients subjected for an initial lung cancer work-up underwent bronchoscopy and BALF was obtained from tumor site. After isolating EVs from BALF by ultracentrifugation, EV-derived DNA (EV DNA) was extracted for subsequent EGFR genotyping performed through peptide nucleic acid (PNA)-mediated Real-Time PCR. The sensitivity, specificity, and concordance rate of BALF EV-based EGFR genotyping were calculated in comparison to tissue genotyping.

Results: The average sensitivity and specificity of BALF EV-based EGFR genotyping were 76% and 87%, respectively, while the sensitivity significantly increased as the stage progressed. Especially, in stage IV, BALF EV-based EGFR typing identified all tissue-proven EGFR mutant cases (n=31) and detected 6 additional mutant cases. The concordance rate was 79% in stage I, 100% in stage II, 74% in stage III, and 92% in stage IV. As TNM stage advanced, especially in the presence of metastasis, concordance rate significantly increased (P<0.05).

Conclusions: The use of BALF for the collection of EV DNA in lung cancer patients resulted in a highly accurate diagnosis. The establishment of a fast and reliable method to identify target genes using EV DNA illustrated that it can overcome the problems of low sensitivity and instability in using cell-free DNA (cfDNA).

Keywords: EGFR genotyping; Extracellular vesicles (EV); bronchoalveolar lavage fluid (BALF); liquid biopsy; non-small cell lung cancer (NSCLC).

Figure 1

Characterization of isolated EVs and EV DNA. (A) The correlation between EV DNA concentration and EGFR copy number; (B) the correlation between EV concentration and EGFR copy number; (C) the correlation between EV concentration and T stage.

Figure 2

Sensitivity trends of BALF EV-based EGFR genotyping according to TNM stage. (A) T stage; (B) N stage; (C) M stage. *, indicates significant differences after the Bonferroni correction.

Figure 3

Comparison of EGFR genotyping using BALF EV and tumor tissue/cytology. ^a, exon 20 insertion or T790M was regarded as wild type.

Figure 4

EGFR mutation detecting rate comparison according to each stage. *P<0.05.

Figure S1

Electron microscopy (EM) image of BALF EVs. Samples for EM analysis were negatively stained. BALF, bronchoalveolar lavage fluid; EV, extracellular vesicles.

Figure S2

Size distribution of BALF EV. Sizes of purified EVs were determined using Nanosight NS300. Average size distribution from three separate measurements is plotted in concentration (particles/mL) according to their size. BALF, bronchoalveolar lavage fluid; EV, extracellular vesicles.

Figure S3

Gel-like images of EV DNA from BALF. Gel-like images show the size and amount of EV DNA determined using the TapeStation. First lane shows the standard size ladder distribution, and numbers on the left indicate corresponding sizes (bp).BALF, bronchoalveolar lavage fluid; EV, extracellular vesicles.

Figure S4

Turn-around time (TAT): a case demonstration of 57-year-old female metastatic EGFR mutant adenocarcinoma. BALF, bronchoalveolar lavage fluid; EV, extracellular vesicles.