A prospective phase 2 study of expeditious EGFR genotyping and immediate therapeutic initiation through extracellular vesicles (EV)-based bronchoalveolar lavage fluid (BALF) liquid biopsy in advanced NSCLC patients

Introduction

Epidermal growth factor receptor (EGFR) mutation testing is an essential step for the therapeutic decision in newly diagnosed advanced non-small cell lung cancer (NSCLC) patients (1,2) and it is usually performed using tumor tissue DNA after histologic confirmation. Obtaining tumor tissue for EGFR mutation testing is basically invasive and sometimes challenging in the cases of small-sized tumor, risky location for percutaneous targeting, and radiologic characteristics, such as cavity, consolidation, or ground glass type lesions. Surgical biopsy is sometimes adopted for adequate tissue biopsy. Turn-around-time (TAT) is another important unmet need for both the patients and the physicians on the decision of adequate treatment, because it is usually 2–3 weeks. Recently, plasma liquid biopsy using circulating tumor DNA has been introduced, but it has an intrinsic limitation of low sensitivity to be used in real clinical routine practice (3,4). This low sensitivity issue could be overcome with the use of molecular barcoding and deep sequencing; however, they are still too pricy for routine practice. On this background, we have developed a novel platform for high-speed EGFR genotyping using extracellular vesicles (EV)-derived DNA from bronchoalveolar lavage fluid (BALF) and obtained the results almost equally concordant with tissue-based genotyping. EV are ideal carriers of cancer biomarkers, as cancer cells secret an abundance of EV and the contents of tumor cell originated EV reflect the molecular and genetic composition (double-stranded DNA and various subtypes of RNA, proteins and lipids) of parental cells (5,6). EV are potential sources of tumor genetic material for EGFR mutation tests. Furthermore, the lipid bilayer of EV enables EV in the body fluid to exist for stable cargoes from enzymatic degradation (7-10).

In a previous study, an EV-based BALF liquid biopsy for EGFR genotyping showed a sensitivity of 97.8%, specificity of 96.9%, and concordance rate of 97.7% when compared to tissue genotyping in 224 advanced stage III–IV NSCLC patients (11). In addition, its TAT of 1–2 days was significantly shorter than the conventional tissue-based EGFR testing. Thus, therapeutic decision process for EGFR-TKIs or even other therapeutic modalities can be accelerated with the knowledge of the EGFR genotype, especially in the patients with symptomatic disease. In addition, EV-based BALF liquid biopsy can be highly effective when tissue biopsy is risky or impractical to access. In this study, we had prospectively validated the diagnostic and therapeutic performance of EV-based BALF EGFR genotyping in the aspects of both the speed and accuracy in advanced NSCLC patients. Gefitinib in previous research has shown to be beneficial as a first line treatment in patients with EGFR mutations (1). However, there is no prospective clinical trial reporting the efficacy of first line gefitinib treatment based on EV-based BALF liquid biopsy. This study aimed to prove the diagnostic performance of EV-based BALF liquid biopsy and show the possibility of EGFR-TKI treatment without tissue biopsy in real clinical practice. This article is presented in accordance with the TREND reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-892/rc).

Methods

Study design and patient population

This study was single arm, phase II, single center and prospective clinical trial to investigate the clinical performance of EV-based BALF liquid biopsy for EGFR genotyping. From January 2018 to August 2020, 120 treatment-naive and newly diagnosed advanced stage III–IV NSCLC patients were screened for EV-based BALF liquid biopsy for EGFR genotyping after obtaining BALF from tumor site through bronchoscopic examination in the referred patients with suspected lung cancer. We preferentially screened patients of the cohort in the previous study (11) using EV-based BALF liquid biopsy with favorable factors for EGFR mutation: female, never smoker or minimally exposed smoker. Heavy smokers and the patients with central-type lung cancer were not included because likelihood of harboring EGFR mutations is significantly low (12).

This study was planned to provide gefitinib to the selected patients before histologic confirmation to reduce the risk of misdiagnosis. Routine diagnostic work-up for histologic confirmation and staging was simultaneously done in all patients. After confirming EGFR mutation positivity by EV-based BALF liquid biopsy, oral gefitinib 250 mg/day (Iretinib^®, Chong Kun Dang Pharm.) treatment was immediately initiated by investigator after acquisition of patients’ consent for clinical trial even before the acquisition of pathologic report. Inclusion criteria to select patients for gefitinib treatment were: age ≥19 years; histopathologic confirmed and treatment-naive NSCLC patients with stage IIIB or IV advanced NSCLC; active EGFR mutation (E21L858R, E19DEL, E21L861Q, G719X, S768I) or combination with rare EGFR mutation in BALF; ECOG performance status of 0–2; treatment-naive; measurable target lesion according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; life expectancy of ≥12 weeks; normal organ function [absolute nucleophile counts >1,500, platelet ≥100,000/mm³, hemoglobin ≥9.0 g/dL, AST/ALT/ALP ≤3 times of the upper limit of normal (ULN), total bilirubin ≤2.0 mg/dL, serum creatinine ≤ ULN]. Patients were excluded, if they had been treated with cytochrome P450 inhibitor within 1 week; had other active malignancy; had pre-existing interstitial lung disease/pulmonary fibrosis; were pregnant or lactating women; had symptomatic or uncontrolled brain metastasis; had NYHA ≥3 congestive heart failure, uncontrolled hypertension, unstable angina, myocardial infarction within 6 months. Disease stages were based on the 8^th TNM classification criteria (13). Clinical and demographic data of the enrolled patients were reviewed. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study protocol was approved by the institutional review board of Konkuk University Medical Center (No. KUH1010868). Written informed consent was obtained from all patients. This prospective clinical trial was also approved by Ministry of Food and Drug Safety, South Korea (KFDA Registration No. 201700442, Approval No. 31413).

BALF sample collection, EV-based EGFR genotyping

Patients with suspected lung cancer based on chest tomography (CT) underwent bronchoscopy at initial lung cancer work-up for biopsy. BALF was retrieved in a trap by gentle aspiration through operating channel after instilling approximately 70–100 mL of sterile isotonic saline by wedging the bronchoscope at the segment or sub-segment where the tumor lesion was located. At least, more than 10–15 mL of BALF were collected from each patient. After sending for the routine cytological examination, 1 mL of residual BALF sample was used for the isolation of EV. Cells and debris were removed using centrifugation at 1,000 g for 10 min at 4 ℃. Cells and debris free BALF were spun in ultracentrifuge tube at 200,000 g for 1 h at 4 ℃ using a Beckman rotor (Beckman Coulter, Brea, CA, USA). The supernatant was carefully removed, and the pellet was suspended in 200 µL of phosphate-buffered saline. The EV were lysed by mixing cell lysis buffer and detergent, and the EV-derived DNA was purified using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany). The concentration and purity of DNA samples were measured using the NanoDrop (Thermo Scientific, Waltham, MA, USA). The length of the purified DNA was analyzed using a 4200 Tapestion and Genomic DNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA). For detecting EGFR mutations, PANAMutyper™ R EGFR kit (Panagene, Daejeon, Korea) with the peptide nucleic acid (PNA)-mediated PCR clamping method (14) and CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) were used. PCR and the melting curve step were performed according to the manufacturer’s protocol (Panagene, Korea). All reactions had a total volume of 25 µL containing 70 ng of template DNA. Fluorescence was measured on all four channels (FAM, ROX, Cy5, and HEX). With the use of a mutant-type DNA specific PNA detection probe that had a fluorescent dye and quencher, EGFR mutations could be genotyped by melting peak analysis (15,16). To prevent false positive or negative results, the same test was performed at least twice times on each sample.

EGFR genotyping of tissue DNA

The tumor samples were prepared as formaline-fixed, paraffin-embedded (FFPE) tissues and tumor DNAs were purified using the TANBead OptiPure FFPE DNA Tube (Taiwan Advanced Nanotech, Taoyuan, Taiwan) according to the manufacturer’s protocol. Then, EGFR genotyping was done through PANAMutyper™ R EGFR kit (Panagene, Daejeon, Korea) according to the manufacturer’s protocol. To prevent the bias, two pathologists read tissue and BALF samples separately in a blinded manner.

Treatment protocol and follow-up assessment

At screening, demographic data and medical history were recorded. All the assessments were made at screening/baseline period (week −2 to week 0). For the patients who were proven to have sensitive EGFR mutation by EV-based BALF liquid biopsy even before histopathologic result, 250 mg gefitinib (Iretinib^®, Chong Kun Dang Pharm.) treatment was immediately initiated after receiving the consent for trial. All the patients received oral gefitinib at a dose of 250 mg/day until tumor progression or death or occurrence of intolerable adverse event (AE) or adverse drug reaction. Dose reductions or temporarily interruption of gefitinib treatment were permitted, if patients encountered any grade ≥3 drug-related AEs [assessed according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0]. Tumor assessment by computed tomography scan was performed every 8 weeks by masked independent radiologist. The primary endpoint was to assess the objective response rate (ORR) at best response according to RECIST criteria version 1.1. ORR was defined as the proportion of patients with complete response (CR) or partial response (PR) to gefitinib. The secondary endpoints were the assessment of progression-free survival (PFS), the concordance rate of EGFR mutation between BALF and tissue, disease control rate (DCR), TAT, and time-to-treatment initiation (TTI). TTI was determined with the time from the date of bronchoscopy or tissue biopsy to the date of starting treatment. All patients with EGFR mutations were treated with Gefitinib based on their BALF genotyping, therefore, the TTI for BALF was determined. TTI for conventional-tissue biopsy was determined with the time from the date of tissue biopsy to the starting date of cytotoxic chemotherapy after the confirmation of no EGFR mutation in tissue. PFS was calculated from the start of treatment to PD or death from any cause, or it was censored on the date of the last follow-up. ORR was defined as the proportion of patents with CR and PR in response to the treatment. DCR was defined as the proportion of patients with CR, PR, or stable disease (SD) in response to the treatment. TAT was determined by the time from the day of bronchoscopy for obtaining BALF to the day of reporting of EGFR mutation result. Drug safety evaluation was performed according to the NCI-CTCAE version 3.0.

Statistical analysis

Sample size calculation

The sample size was determined by the exact single-stage phase II design. The response rate of gefitinib was 70% (55–84%) in previous study (2). On the hypothesis that the EGFR mutation detection rate using BALF liquid biopsy is comparable (non-inferiority) to that using tissue biopsy (d=0.1, non-inferiority difference 0.07, α=5%, 1−β=0.9), the expected actual number was 32 patients. Assuming that the concordance rate of the tissue and BALF test was 90%, mismatched results occur in 3 patients. Therefore, 35 patients are required, and 40 patients were recruited considering a drop-out rate of 10%.

Statistical method

Thirty-eight patients with sensitive EGFR mutation were analyzed, and efficacy of EGFR-TKI was assessed one year after the initiation of gefitinib treatment of the last entered patient. The survival curves, median value of PFS and corresponding 95% confidence interval (CI) were calculated using the Kaplan-Meier method. EGFR status assessment of histological tumor samples was considered as a standard reference for the calculation of concordance. Categorical variables were summarized by calculating frequencies and percentages. The means and standard deviations were used to determine numerical variables. We used t-tests, Fisher’s exact tests, and χ² statistics. All statistical analyses were carried out using SPSS Statistics version 25.0 (IBM Corp, Chicago, IL, USA) and R software version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria), and a P value <0.05 was regarded as the indicator of statistical significance.

Results

Patient characteristics

The mean age of the enrolled patients was 70.3±11.6 years. Females were 49.2% and non-smokers were 55.0%. Most of the patients were histologically classified as adenocarcinoma (80.8%). Stage IIIB were 7.5%, stage IVA were 55.8% and stage IVB were 36.7% (Table 1).

Among the 120 screened patients, 51 cases were detected to harbor EGFR mutations through EV-based BALF liquid biopsy. Eleven patients among them were excluded because 4 were transferred, 2 had symptomatic brain metastasis, 2 had other organ cancers, 3 did not consent the trial. Among 40 cases that were enrolled for immediate initiation of gefitinib treatment, two were dropped out early due to 1 transfer, 1 small cell lung cancer histology, and finally 38 patients were included to be analyzed. The data cutoff for this analysis was September, 2021 (Figure 1). The median follow-up period at the time of analysis was 18.7 months (range, 1–38 months). Two were lost to follow-up due to withdrawal and 36 patients were completed the follow-up.

Figure 1 The study flow diagram of rapid diagnosis and EGFR-TKI initiation by EV-based BALF liquid biopsy in advanced NSCLC patients. EV, extracellular vesicle; BALF, bronchoalveolar lavage fluid; EGFR, epidermal growth factor receptor; SCLC, small cell lung cancer; TKI, tyrosine kinase inhibitor; NSCLC, non-small cell lung cancer.

Performance of EV-based BALF liquid biopsy for EGFR genotyping

Figure 2 displays the result of the concordance between tissue biopsy and EV-based BALF liquid biopsy for EGFR genotyping in 51 EGFR-mutated patients. There was only one mismatched case of E21L858R mutation which was proven to be false positive. The concordance rate in EGFR mutation positive group detected by EV-based BALF liquid biopsy was 98.0% (50/51). There was no false negative case in the wild-type EGFR patients by EV-based BALF liquid biopsy. Overall concordance rate in 120 screened patients was 99.2% (119/120). Tissue samples were unobtainable in two patients out of total 120 patients due to the complication at the time of diagnosis. Both of two patients were confirmed to have EGFR mutation later, one in pleural effusion and another in BALF cytology performed at progression time. The EGFR mutation results of these two patients obtained from BALF were consistent with the EGFR mutation results confirmed through other samples other than lung tissue.

Figure 2 Concordance rate of EV-based BALF liquid biopsy for EGFR genotyping to tissue genotyping and plasma liquid biopsy (n=51). EV, extracellular vesicle; BALF, bronchoalveolar lavage fluid; EGFR, epidermal growth factor receptor.

The proportions of EGFR mutation subtypes were the same as reported in the previous research (4,12,17); the proportion of each sensitive mutation was 56.9% (29 cases) for E19del, 41.2% (21 cases) for E21L858R, and 2.0% (one case) for G719C/S769I compound mutation depending on BALF liquid biopsy. These data suggest that EV-based BALF liquid biopsy has almost the equivalent performance with conventional tissue biopsy for EGFR mutation testing except one case of mismatch.

TAT and TTI of EV-based BALF liquid biopsy

Turn-around time was defined by the time from the biopsy or bronchoscopy to the time of knowing the EGFR mutation result. Conventional TAT of tissue-based EGFR mutation testing took about 12.1±7.2 days in our center. In comparison, the BALF EGFR mutation testing only took about 1.9±1.1 days (P<0.001). Therefore, the time for initiating gefitinib treatment after the day of bronchoscopy was just 7.8±6.5 days and it was 7–10 days earlier than time of conventional tissue genotyping (13.8±12.9 days) (Table 2). It significantly saved the waiting time till initiating treatment and served to reduce the anxiety and suffering of the patients. These means that EV-based BALF liquid biopsy could be a novel platform for EGFR testing enough to replace the conventional tissue-based genotyping in all aspects of speed, accuracy, and less invasiveness. Looking into the surprising sensitivity and specificity, it might be highly feasible and valuable to investigate the therapeutic decision based on EV-based BALF liquid biopsy without tissue biopsy in EGFR-mutated NSCLC patients.

Therapeutic efficacy of gefitinib initiation based on EV-based BALF liquid biopsy

Therapeutic efficacy was evaluated in 38 patients who received gefitinib for more than 4 weeks. The median follow-up period at the time of analysis was 18.7 months (range, 1–38 months). The ORR as a primary endpoint was described in Table 3. During the observation period, one patient exhibited CR, 28 met the criteria for PR, 6 exhibited SD, and 3 exhibited PD. Thus, ORR (CR + PR) were 76.3%. The DCR as a secondary endpoint was 92.1% and the median PFS was 14.6 months (95% CI: 8.8–21.9) (Figure 3). A proportion of 76.3% ORR was better than 69.8–73.7% ORR of previous researches. Median PFS 14.6 months was longer than other previous studies (9.7–10.8 months) (2,18-21). The estimated one-sided confidence interval for gefitinib response rate, using BALF liquid biopsy was 0.635–1.0. The non-inferiority of the EGFR-TKI response rate of BALF liquid biopsy was proven, as the lower margin 0.635 of the calculated confidence interval was better than the pre-assumption lower margin 0.63 (0.7–0.07) before the study. In the case of BALF, the ORR 76.3% was not less than 70% (P=0.045).

Figure 3 Kaplan-Meier curve of PFS in patients treated by gefitinib based on BALF liquid biopsy (n=38). CI, confidence interval; mPFS, median progression-free survival; PFS, progression-free survival; BALF, bronchoalveolar lavage fluid.

The therapeutic outcomes of response and PFS were not inferior to the previous results. They are similar or better than the tissue-based results (2,17-20). Our efficacy endpoints, ORR 76.3% and PFS 14.6 months were numerically better than previous Gefitinib efficacy based by tissue biopsy. The early initiation of gefitinib treatment by BALF EGFR mutation before disease progression can improve the clinical outcomes such as PFS and tumor response.

Safety analysis

All patients who had received at least one dose of Gefitinib were included in the safety analysis. Of the 40 patients who underwent gefitinib treatment, 34 (85%) reported the occurrence of AEs. Severe AEs were reported in 12 (30%) patients among the study population. Five (12.5%) patients discontinued the treatment due to drug-related AEs. The most common AEs were the skin eruption (57.5%), diarrhea (27.5%), and increasing liver enzyme (15%) (Table S1).

Discussion

In previous research, we demonstrated the accuracy and speed of BALF EGFR testing for advanced-stage lung cancer patients (11). To prove the performance of this novel platform in real clinical practice, we prospectively validated the diagnostic performance of EV-based BALF liquid biopsy and the efficacy of EGFR-TKI treatment based on the results of this BALF liquid biopsy. In this study, EGFR-mutated NSCLC patients were detected by EV-based BALF liquid biopsy usually within around two days after bronchoscopy, and gefitinib treatment was promptly initiated before tissue confirmation, while tissue-based EGFR genotyping was confirmed in 10–14 days after bronchoscopic examination. The gefitinib treatments were continued by the time of progression, withdrawal of trial, or death. To the best of our knowledge, this work is the first prospective study reporting the efficacy of first line gefitinib by treating the patients with advanced EGFR-mutated NSCLC detected by EV-based BALF liquid biopsy even before conventional tissue-based genotyping. This study provides clinical evidence for the utility of EV-based EGFR mutation status check to ascertain eligibility for EGFR-TKI treatment with no EGFR result from tissue biopsy needed. The earlier initiation of treatment with the help of EV-based EGFR mutation detection improves the treatment efficacy such as PFS and tumor response.

Among the 120 screened patients, 51 patients were detected as harboring EGFR mutation by EV-based BALF liquid biopsy. A proportion of 42.5% of EGFR mutation positivity was relatively higher than expected and it is because we preferentially screened the patients with favorable factors for EGFR mutations, such as female, never smoker or minimally exposed smoker and peripheral-type tumor (12). Patient with central type tumor and current heavy smokers were not included. As a result, the greater part of EGFR mutant patients detected was never smoker (66.7%) or ex-smoker (25.8%). The concordance rate of EGFR genotype between tissue biopsy and EV-based BALF liquid biopsy was 98% and there was only one case of mismatch which was a false positive case. In one false positive case, we retested the EGFR genotyping in remained BALF and tissue and confirmed that the EGFR mutation was negative. The PANAmutyper EGFR PCR method had 0.1–1% error rate. After the case, we performed the EGFR mutation genotyping testing twice to reduce false positive cases. There was no false negative case in the wild-type EGFR patients by EV-based BALF liquid biopsy. Overall concordance rate in 120 screened patients was 99.2%. This finding suggests that EV-based BALF liquid biopsy has almost identical performance with conventional tissue biopsy for EGFR genotyping. Especially in the cases of difficult tissue biopsy due to small tumor size, location, and the nature of radiologic findings such as ground glass type, consolidation-like tumor and cavitary tumor, EV-based BALF liquid biopsy provides great advantages to avoid invasive or risky biopsy or even surgical biopsy.

In routine practice, EGFR mutation testing is ordered after confirming pathologic report after biopsy. It takes usually 2–3 weeks, and it is an indispensable test item before therapeutic decision. There is a great unmet need to shorten its TAT, especially in the symptomatic patients. Patient’s anxiety and doctor’s waiting for the result to make a therapeutic decision are considerable factors. Even though plasma liquid biopsy using cell-free DNA (cfDNA) has been adopted, its role is only supplementary due to its low sensitivity (22). In our study, TAT for EV-based BALF liquid biopsy was only 1.9±1.1 days, while 12.1±7.2 days in tissue genotyping. In other words, the time to get EGFR genotyping result can be reduced to only 1–2 days after bronchoscopy and that is around 10 days shorter than tissue genotyping. Also, this expeditious EGFR genotyping with high accuracy obtained by EV-based BALF liquid biopsy has been proven to provide a great advantage to initiate early therapeutic intervention in both EGFR-TKI treatment in mutant cases and chemotherapy in wild-type cases after histologic diagnosis. Gefitinib treatment was initiated in only 7.8±6.5 days after bronchoscopic examination.

The response of early gefitinib treatment based on EV-based BALF EGFR genotyping was not different from the usual data of tissue-based treatment. The proportion of achieving objective responses was 76.3% (32/38) and the disease control rate was 92.1% (35/38). The response rates of BALF EGFR genotyping were better than previous trials of tissue EGFR genotyping (ORR, 69.8–73.7%; DCR, 89–95%) (17,19,21). The median PFS was 14.6 months (95% CI: 8.8–21.9), which was longer than the values of previous research (20,23) (median PFS, 6–15 months). It might be because most patients in our study were non-smokers or minimally exposed smokers who had good prognosis and good response for EGFR-TKIs in general (24,25). The AEs from gefitinib (Iretinib^®) in this study were not different from the common AEs of other EGFR-TKIs (26). The most common AEs were the skin eruption, diarrhea, and increasing liver enzyme (Table S1).

In this study, we demonstrated for the first time that EV-based BALF liquid biopsy shows almost identical performance with conventional tissue biopsy for EGFR genotyping. We propose that the decision for EGFR-TKI treatment could be made through EV-based BALF liquid biopsy, even without tissue biopsy. If tissue evaluation is necessary in later time for such as detecting T790M mutation or next-generation sequencing (NGS) analysis, it can be performed with adequate time. At present, we are investigating the role of EV-based BALF liquid biopsy for detecting T790M mutation in the patients with acquired EGFR-TKI resistance. And also, we are developing optimal liquid NGS panel using BALF EV DNA (27). It is worthwhile to investigate prospective clinical trial for the 1^st-line treatment of the 3^rd generation EGFR-TKIs such as osimertinib or lazertinib through EV-based BALF liquid biopsy without tissue biopsy. Considering the risk and invasiveness of tissue lung biopsy, the paradigm shift from tissue-based diagnosis to liquid biopsy will be made in lung cancer soon. In this aspect, plasma liquid biopsy is extensively investigated at present, but the expectation is not optimistic when considering the complexity of blood samples. Furthermore, EV-based BALF liquid biopsy is much more promising, as this study reveals the concordance with tissue biopsy for EGFR genotyping.

Molecular testing of sensitizing EGFR mutations, BRAF V600E, as well as ALK, ROS1, and NTRK fusions, is now standard-of-care for patients with advanced NSCLC. Routine testing of RET fusions and MET exon 14 skipping mutations is also considered standard-of-care based on the recent guidelines. Thus, a comprehensive biomarker testing is recommended for all patients diagnosed with non-squamous NSCLC. Currently, our technique using BALF is limited for EGFR mutation not including other targetable mutations, and further development for detecting other mutations will be required. A safe, sensitive and accurate detection of EGFR mutation in BALF is, nevertheless, beneficial for specific sub-population with high mutation frequency, such as Asian non-smoker whose frequency is 40–50% (28,29). We used PANAMutyper for EV-based BALF liquid biopsy approved by Korean Ministry of food and Drug Safety, but other methods such as droplet digital PCR and other FDA-approved companion diagnostics kits such as therascreen^®EGFR RGQ PCR Kit and cobas^®EGFR Mutation Test can be used for EV-based BALF liquid biopsy.

Bronchoalveolar lavage (BAL) through the bronchoscope is a conventional and safe diagnostic technique for patients with a variety of pulmonary diseases or lung cancer (30). BAL makes it possible to obtain the cellular and non-cellular contents from the disease-located site such as distal airways and peripheral alveoli (31). Recently smoking-related central type lung cancers with visible endobronchial lesions decreases, while peripheral-type lung cancer, especially adenocarcinoma are steadily increasing (32). The matter of lung biopsy becomes a major task to overcome in the era of precision medicine because tissue is the issue. BALF cytology based diagnostic yield is quite low and that is the reason why BAL is not routinely performed in diagnostic work up for lung cancer. We would like to recommend taking supernatants rather than cellular components from BALF in which abundant amount of EVs released by tumor cells or tumor microenvironment. In our study, we demonstrated the usefulness of EVs isolated from BALF of lung cancer patients in special case of EGFR mutation testing (11). We propose that the application of EV-based BALF liquid biopsy will be extended to whole field of genetic, genomic or molecular diagnosis.

Conclusions

We have demonstrated for the time that EV-based BALF liquid biopsy is a novel platform for EGFR mutation testing that can lead to early therapeutic intervention with EGFR-TKI in advanced NSCLC patients. Compared to conventional tissue genotyping, EV-based BALF liquid biopsy in the patients with suspicious advanced lung cancer revealed almost identical concordance with TAT of 1–2 days and relatively less invasiveness. Its performance showed that it has the potential to replace tissue genotyping, overcoming the low sensitivity of plasma liquid biopsy using cfDNA. Large-scaled and multicenter clinical trials should be promptly initiated to obtain approvals for real-world application.

Acknowledgments

We would like to thank Iretinib^®, Chong Kun Dang Pharm for kindly providing gefitinib for use in our study.

Funding: This work was supported by Chong Kun Dang Pharm (to Kye Young Lee).

Footnote

Reporting Checklist: The authors have completed the TREND reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-892/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-892/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-892/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-892/coif). KYL reports that this study received funding from Chong Kun Dang Pharm. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study protocol was approved by the institutional review board of Konkuk University Medical Center (No. KUH1010868) and written informed consent was obtained from all patients. This prospective clinical trial was also approved by Ministry of Food and Drug Safety, Republic of Korea (KFDA Registration No. 201700442, No. 31413).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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