Study participants
From April 2022 to September 2023, a prospective observational pilot study was conducted to assess the feasibility of a targeted washing technique for detecting EGFR mutation at Pusan National University Hospital, Busan, a university-affiliated, tertiary referral hospital in Busan, South Korea. The inclusion criteria were as follows: 1) patients aged 20 years or older, 2) individuals with advanced NSCLC with EGFR mutations, 3) confirmation of progression based on RECIST criteria after treatment with EGFR-TKIs, and 4) absence of contraindications for bronchoscopy.
Targeted washing technique
A representative example of the targeted washing technique is shown in Fig. 1.
Fig. 1 Representative case of targeted washing in patients who progressed from afatinib treatment.
A Lung cancer with a size of 31 × 17 mm (arrow) was found in the right middle lobe with multiple bone metastases.
B After 30 months of afatinib treatment, disease progression was identified with the development of numerous hematogenous metastases in both lungs and an increase in the size of bone metastases. At the time of progression, right middle lobe mass was found to be 24 × 11 mm (arrow).
C Three-dimensional images of virtual bronchoscopy navigation were created to precisely locate the target lung lesion.
D Targeted washing was performed at the medial segment of right middle lobe for EGFR testing using a 3.0 mm-sized ultrathin bronchoscope with the assistance of fluoroscopy. The T790M mutation was detected in EGFR testing using targeted washing fluid, whereas neither the tissue nor plasma sample could detect the T790M mutation.
Before bronchoscopy, local anesthesia was administered to the oropharynx using 4% lidocaine via an atomizer. Subsequently, all the participants received intravenous midazolam and fentanyl for conscious sedation. Initially, conventional bronchoscopy was conducted using a 4-mm flexible bronchoscope (BF-P260F; Olympus, Tokyo, Japan) to administer lidocaine for local anesthesia to the tracheobronchial tree. If no endobronchial lesion was identified during conventional bronchoscopy, an ultrathin bronchoscope (BF-MP190F; Olympus) with a diameter of 3.0-mm was navigated precisely to the target lung lesion as close as possible based on guidance from thin-section chest CT images and with the assistance of virtual bronchoscopic navigation (LungPoint; Broncus Medical, Mountain View, CA) [13-15]. To accurately locate the target lung lesion, a radial probe endobronchial ultrasound (EBUS) (UM-S20-17S; Olympus) was advanced through a 1.7-mm-diameter working channel of the ultrathin bronchoscope under X-ray fluoroscopy guidance. Once the target lung lesion was identified, the radial probe EBUS was withdrawn and 3 mL of normal saline was instilled for targeted washing. After a 3-s interval, the washing fluid was retrieved by gently pulling the bronchoscope proximally. The targeted washing procedure was repeated until more than 5cc of the washing fluid was collected. A schematic of the target-washing technique is shown in Fig. 2.
Fig. 2 Scheme of targeted washing technique.
A Initially, an ultrathin bronchoscope with a diameter of 3.0 mm (indicated by the black arrow) was precisely navigated to the target lung lesion as closely as possible, based on thin-section chest CT images and with the assistance of virtual bronchoscopic navigation and fluoroscopy.
B Subsequently, a radial probe EBUS (red line) was advanced through the working channel of the ultrathin bronchoscope to accurately locate the target lung lesion.
C Finally, the radial probe EBUS was withdrawn, and 3 mL of normal saline (blue lines) was instilled. After a 3-s interval, the washing fluid was retrieved for collecting cfDNA derived from the lung tumor.
D Fluoroscopy image during targeted washing; an ultrathin bronchoscope is placed in front of the target lung lesion (black arrow).
Abbreviation: EBUS, endobronchial ultrasound
Following the collection of the targeted washing fluid, bronchoscopic biopsies of the primary lung tumor were performed using either forceps biopsy or cryobiopsy at the physician’s discretion, with the aim of histologic diagnosis and tissue NGS. In cases where the target lung lesion could not be identified, endobronchial ultrasound-guided transbronchial needle aspiration was employed to sample the mediastinal lymph nodes suspected of metastasis based on axial CT scans.
DNA isolation from targeted washing fluid (TWF) and droplet digital polymerase chain reaction
Cell-free DNA (cfDNA) was extracted from the TWF using the QIAamp DSP Circulating NA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Using cfDNA from TWF, droplet digital polymerase chain reaction (ddPCR) was conducted using a ddPCR system (QX200 Droplet Digital PCR System; Bio-Rad, Hercules, CA, USA) and an EGFR mutation analysis kit (Droplex EGFR Mutation Test v2; Gencurix, Seoul, South Korea), following the manufacturer’s recommended protocol. Detailed information on EGFR testing using TWF samples is provided in the Supplementary Appendix.
Conventional EGFR mutation analysis
EGFR mutation tests were performed on tissue specimens using an EGFR Mutation Detection Kit (PNA clampTM, Panagene, Daejeon, Republic of Korea). Cobas EGFR Mutation Test v2 (cobas®; Roche Molecular System, Pleasanton, CA, USA) was used on the plasma sample.
Statistical analysis
Categorical variables are presented as numbers (percentages), and continuous variables are described as means (ranges). The detection rates of the molecular tests were calculated as the number of cases with oncogenic alterations divided by the total number of cases. Detection of the T790M mutation through standard EGFR testing was defined as any identification of the EGFR T790M mutation using tissue or plasma specimens. Statistical analyses were conducted using the R software for Windows (ver. 4.2.3, R Foundation for Statistical Computing, Vienna, Austria).