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Comparative Study
, 18 (1), 1236

Liquid Biopsy Using the Supernatant of a Pleural Effusion for EGFR Genotyping in Pulmonary Adenocarcinoma Patients: A Comparison Between Cell-Free DNA and Extracellular Vesicle-Derived DNA

Affiliations
Comparative Study

Liquid Biopsy Using the Supernatant of a Pleural Effusion for EGFR Genotyping in Pulmonary Adenocarcinoma Patients: A Comparison Between Cell-Free DNA and Extracellular Vesicle-Derived DNA

Jong Sik Lee et al. BMC Cancer.

Abstract

Background: EGFR genotyping in pulmonary adenocarcinoma patients who develop pleural effusions is mostly performed using cytology or cell block slides with low sensitivity. Liquid biopsy using the supernatant of pleural effusions may be more effective because they contain many components released by cancer cells. Extracellular vesicles (EVs) are known to carry oncogenic double-stranded DNA that is considered a notable biomarker. Here, we investigate the efficiency of liquid biopsy using cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (EV-derived DNA) from the supernatant of pleural effusions for EGFR genotyping in patients with pulmonary adenocarcinoma.

Methods: Fifty pleural effusion samples from patients with pulmonary adenocarcinoma were evaluated. The supernatant, after removing the cell pellet by centrifugation, was used for liquid biopsy, and EVs were isolated from the pleural effusion by ultracentrifugation. EV-derived DNA and cfDNA were extracted separately, and EGFR genotyping was performed by the PNA clamping method.

Results: Among 32 patients who were EGFR-tyrosine kinase inhibitor (TKI) naïve with a known tissue EGFR genotype, liquid biopsy using EV-derived DNA from the pleural effusion supernatant showed 100% matching results with tissue EGFR genotyping in 19 EGFR mutant cases and detected three additional EGFR mutations in patients with wild-type (WT) tissue. Liquid biopsy using cfDNA from pleural effusion supernatants missed two cases of tissue-based EGFR mutations and found two additional EGFR mutation cases. In 18 patients who acquired resistance to EGFR-TKI, EGFR genotyping using EV-derived DNA from the pleural effusion supernatant detected the T790 M mutation in 13 of 18 (72.2%) patients, and this mutation was detected in 11 (61.1%) patients using cfDNA. By contrast, only three patients were found to present the T790 M mutation when using cell block or cytology slides.

Conclusions: Liquid biopsy using the supernatant of pleural effusions showed significantly improved results for EGFR genotyping compared to those using conventional cell block or cytology samples. Liquid biopsy using EV-derived DNA is promising for EGFR genotyping, including T790 M detection in pulmonary adenocarcinoma patients who develop pleural effusions.

Keywords: EGFR mutation; Extracellular vesicles; Liquid biopsy; Pleural effusion; Pulmonary adenocarcinoma.

Conflict of interest statement

Ethics approval and consent to participate

The study protocol was approved by the Institutional Review Board of Konkuk University Medical Center and Asan Medical Center and was conducted in accordance with the amended Declaration of Helsinki. All participants provided written informed consent before enrollment.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Agreement of EGFR genotyping in cell-free and EV-derived DNA from the supernatant of pleural effusions compared with tumor tissue from EGFR-TKI naïve patients. cfDNA = cell-free DNA; EV-derived DNA = extracellular vesicle-derived DNA; a, c = follow-up loss after diagnosis; b = TKI response could not be evaluated as the patient had expired during TKI treatment
Fig. 2
Fig. 2
Comparison of the EGFR genotyping between cell block or cytology, cell-free DNA and EV-derived DNA from the supernatant of pleural effusions in EGFR-TKI acquired-resistance patients. a Detection of the T790 M mutation using cell block or cytology, cfDNA and EV-derived DNA of pleural effusions. The detection rate of the T790 M mutation is 17, 61, 72% in cell block or cytology, cfDNA, and EV-derived DNA of pleural effusions, respectively. b EGFR genotyping in accordance with tissue using a cell block or cytology, cfDNA and EV-derived DNA of pleural effusion are 39, 94, and 100%, respectively. cfDNA = cell-free DNA; EV-derived DNA = extracellular vesicle-derived DNA

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