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).

Liquid biopsy using extracellular vesicle–derived DNA in lung adenocarcinoma

“…This study also highlighted the ongoing diagnostic and prognostic ability of EV DNA by showing that the mutations identifiable by EV DNA liquid biopsy included the acquired resistance specific mutation T790M. Furthermore, the specificity and sensitivity of liquid biopsy increased even more with the use of BALF EV DNA [16,76]. Similar to NSCLC, analysis of serum EV DNA from pancreatic cancer patients also demonstrated the presence of clinically relevant KRAS-specific mutations [14,77,78].…”

“…In a study performed with pathologically confirmed NSCLC patients, the sensitivity and specificity of BALF EV-based EGFR genotyping were high, and this assay showed an even better mutation detection rate than tissue/cytology-based typing. Also, the sensitivity increased as the stage progressed, reaching 100% with stage IV patient samples [76]. These results seem to suggest BALF EV-based EGFR genotyping is a highly promising liquid biopsy method, and particularly efficient for patients that require repeat biopsies throughout progression of the disease.…”

“…Recent studies have demonstrated that EVs successfully isolated from the BALF of lung cancer patients contain an abundant amount of dsDNAs, and that liquid biopsy for EGFR genotyping using BALF is tissue-specific and extremely sensitive compared to using cfDNAs [16,76]. In a study performed with pathologically confirmed NSCLC patients, the sensitivity and specificity of BALF EV-based EGFR genotyping were high, and this assay showed an even better mutation detection rate than tissue/cytology-based typing.…”

MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods

“…Obtaining these kinds of liquid biopsies might be beneficial since body fluids derived from the proximity of the primary tumor bear the potential to better represent the molecular landscape of the tumor which would increase sensitivity and specificity of the diagnosis [87,96,103]. For example, EGFR genotyping of EVs of bronchoalveolar lavage fluid of NSCLC patients showed superior sensitivity and accuracy compared to plasma EV DNA genotyping [79]. These results suggest that this might also apply to ncRNA analysis of proximal fluids compared to EVs obtained from plasma or serum.…”

“…Current studies focus on the extracellular vesicles mainly isolated from human blood, or, respectively, serum or plasma [ 71 , 72 , 73 , 74 ]. However, extracellular vesicles can be isolated from a broad variety of human body fluids such as urine [ 75 , 76 ], saliva [ 77 , 78 ], bronchoalveolar liquid [ 79 ], pleural lavage [ 80 ], and cerebrospinal fluid [ 81 ] and even more uncommon ones like tears [ 82 ], semen [ 83 ], menstrual blood [ 84 ], peritoneal lavage [ 85 , 86 ], bile [ 87 ], and pancreatic juice [ 88 ]. An overview of non-coding RNAs that were analyzed in extracellular vesicles isolated from different liquid biopsies of different kinds is presented in Table 1 .…”