Novel Bronchoscopy Method for Molecular Profiling of Lung Cancer: Targeted Washing Technique

Mi-Hyun Kim; Hayoung Seong; Hyojin Jang; Saerom Kim; Wanho Yoo; Soo Han Kim; Jeongha Mok; Kwangha Lee; Ki Uk Kim; Min Ki Lee; Jung Seop Eom

doi:10.4143/crt.2024.1128

Articles

Page Path: HOME > Cancer Res Treat > Volume 58(1); 2026 > Article

Original Article Lung and Thoracic cancer Novel Bronchoscopy Method for Molecular Profiling of Lung Cancer: Targeted Washing Technique: Mi-Hyun Kim^1,2,3, Hayoung Seong^1,2, Hyojin Jang^1,2, Saerom Kim^1,2, Wanho Yoo^1,2, Soo Han Kim^1,2, Jeongha Mok^1,2, Kwangha Lee^1,2, Ki Uk Kim^1,2, Min Ki Lee^1,2, Jung Seop Eom^1,2,3; Cancer Research and Treatment : Official Journal of Korean Cancer Association 2026;58(1):107-114.
DOI: https://doi.org/10.4143/crt.2024.1128
Published online: February 26, 2025

¹Department of Internal Medicine, Pusan National University School of Medicine, Busan, Korea

²Department of Internal Medicine, Pusan National University Hospital, Busan, Korea

³Biomedical Research Institute, Pusan National University Hospital, Busan, Korea

Correspondence: Jung Seop Eom, Department of Internal Medicine, Pusan National University School of Medicine, 179 Gudeok-ro, Seo-gu, Busan 49241, Korea
Tel: 82-51-240-7889 E-mail: ejspulm@pusan.ac.kr

• Received: November 25, 2024 • Accepted: February 25, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2,335 Views
114 Download
2 Web of Science
1 Crossref
2 Scopus

prev next

Full Article

Download PDF

Abstract

Purpose
There have been efforts to find alternative samples other than standard samples of tissue or plasma for mutational analyses for lung cancer patients. However, no other sample or technique has replaced the mutational analyses using standard samples. In this prospective study, we assessed a novel bronchoscopy method, named as targeted washing technique, for detecting the epidermal growth factor receptor (EGFR) mutation.
Materials and Methods
A 3.0-mm ultrathin bronchoscope was precisely navigated to the target lung lesion with the assistance of virtual bronchoscopic navigation and fluoroscopy. Once the bronchoscope is placed in front of target lung lesion, 0.9% normal saline was instilled for targeted washing. EGFR testing using targeted washing fluid (TWF) was compared to standard methods using plasma or tumor tissue.
Results
In 41 TWF samples, the T790M mutation was detected in tissue, plasma, and TWF samples at rates of 22.0%, 9.8%, and 29.3%, respectively. The overall EGFR T790M detection rate using tissue, plasma, or TWF samples was 36.6%, with TWF samples increasing the T790M mutation detection rate by up to 10%. The accuracy of T790M mutation detection using TWF sample was 82.9% compared with standard samples. Four patients were found to have the EGFR T790M mutation solely through EGFR testing using TWF, which repeated rebiopsies using either plasma or tissue finally confirmed to have the T790M mutation.
Conclusion
We demonstrated the clinical potential of targeted washing technique for molecular testing, which can be a good option to overcome spatial heterogeneity, low sensitivity of plasma sample or technical limitations in collecting tumor tissues.
Key words: Bronchoscopy, Pathology, Molecular, Precision Medicine, Lung neoplasms

Introduction

The dawn of precision medicine has ushered in a new era, marked by a thorough understanding of oncogene addiction and the consequent emergence of targeted therapeutic agents [1,2]. Notably, there has been a quantum leap in the treatment landscape of non–small-cell lung cancer (NSCLC), particularly concerning genetic aberrations such as epidermal growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase rearrangements, ROS1 rearrangements, and beyond [3]. The introduction of novel targeted agents has also given rise to concurrent challenges in companion diagnostics and the selection of appropriate samples [4].

The introduction of liquid biopsy techniques using plasma samples has streamlined the process of obtaining mutational information, providing a more accessible alternative compared to analyses conducted using tissue biopsy [5]. Specifically, liquid biopsy has been employed for detecting EGFR mutations in NSCLC patients, which was widely utilized for identifying T790M mutations after the first-line treatment with first or second-generation EGFR–tyrosine kinase inhibitors (EGFR-TKIs) [6]. However, there have been concerns about relatively high false-negative results in liquid biopsy using plasma samples [7]. In contrast, tissue biopsy has raised concerns regarding the invasiveness of procedures and limitations in overcoming spatial heterogeneity [8,9].

To overcome the limitations or drawbacks of mutational analyses using standard samples of plasma or malignant tumor tissue, there have been efforts to find alternative samples for mutational analyses for NSCLC, such as sputum or pleural fluid [10-12]. However, no other sample or technique has replaced the mutational analyses using plasma or tissue. In this study, we assessed a novel bronchoscopy method of targeted washing technique for detecting the EGFR mutation.

Materials and Methods

1. Study subjects

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 Response Evaluation Criteria in Solid Tumor (RECIST) criteria after treatment with EGFR-TKIs, and (4) absence of contraindications for bronchoscopy.

2. Targeted washing technique

A representative case of targeted washing technique is shown in Fig. 1. Before bronchoscopy, local anesthesia was applied to the oropharynx using 4% lidocaine with an atomizer. Subsequently, all study participants received intravenous midazolam and fentanyl to achieve conscious sedation. Initially, a conventional bronchoscopy was conducted using a 4-mm flexible bronchoscope (BF-P260F, Olympus) 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 computed tomography (CT) images and with the assistance of virtual bronchoscopic navigation (LungPoint, Broncus Medical). 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 0.9% normal saline was instilled for targeted washing. After a 3-second interval, the washing fluid was retrieved by gently pulling the bronchoscopy proximally. Targeted washing procedures were repeated until more than 5 mL of washing fluid had been collected. The scheme of targeted washing technique is presented in Fig. 2.

Following the collection of targeted washing fluid (TWF), bronchoscopic biopsies of the primary lung tumor were performed using either forceps biopsy or cryobiopsy based on the physicians’ discretion, aiming for histologic diagnosis and tissue next generation sequencing (NGS). In cases where the target lung lesion could not be identified, EBUS-guided transbronchial needle aspiration was employed to sample mediastinal lymph nodes suspected of metastasis based on axial CT scans.

3. DNA isolation from TWF and droplet digital polymerase chain reaction

Cell-free DNA (cfDNA) was extracted from TWF using QIAamp DSP Circulating NA Kit (Qiagen) 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) and an EGFR mutation analysis kit (Droplex EGFR Mutation Test v2, Gencurix) following the manufacturer’s recommended protocol. Detailed information of EGFR testing using TWF samples was provided in Supplementary Materials.

4. Conventional EGFR mutation analysis

Using tissue specimen, EGFR mutation tests were performed using an EGFR Mutation Detection Kit (PNA clamp, Panagene). Cobas EGFR Mutation Test v2 (cobas, Roche Molecular System) was used on the plasma sample.

5. Statistical analysis

Categorical variables were presented as number (%) and continuous variables were described as mean (range). Detection rate of molecular test was calculated as the number of cases with the presence of oncogenic alteration divided by the total number of cases. The detection of T790M mutation through standard EGFR testing is defined as any identification of EGFR T790M mutation utilizing tissue or plasma specimens. Statistical analyses were conducted using R software for Windows ver. 4.2.3 (R Foundation for Statistical Computing).

Results

1. Study patients

During the study period, a total of 41 TWF samples were collected from 34 patients. Among them, seven patients underwent repeated re-biopsy after the progression of subsequent therapy. The clinical characteristics of the 34 patients are summarized in Table 1. The median age was 72 years (range, 50 to 86 years), and 79.4% were female. The mean longest diameter of primary lung cancer was 41 mm (range, 9 to 100 mm). Second-generation EGFR-TKIs were used in 67.7% of the study subjects, while 29.4% received first-generation EGFR-TKIs. One patient was treated with lazertinib, a third-generation EGFR-TKI, as part of a clinical trial.

2. Bronchoscopy results

For conscious sedation, midazolam (mean, 3.7 mg; range, 2 to 7 mg) and fentanyl (mean, 53 μg; range, 25 to 125 μg) were administered in 40 cases of bronchoscopy, while one received 90 mg of propofol. The mean volumes of instilled and retrieved normal saline were 15.4 mL (range, 10 to 20 mL) and 8.2 mL (range, 5 to 15 mL), respectively. After the collection of TWF, forceps biopsy was performed for the primary lung tumor in 21 cases (51.2%), cryobiopsy in five cases (12.2%), and a combination of forceps biopsy and cryobiopsy in 10 cases (24.4%) out of a total of 41 bronchoscopies. Using radial probe EBUS, primary lung tumor could not be detected in five patients (12.2%); three received EBUS-guided transbronchial needle aspiration for mediastinal lymph node and only targeted washings were done without tissue sampling in two patients. As results, malignant tumor cells were successfully collected in 35 patients (85.4%), whereas tumor tissue could not be collected in six patients (14.6%).

Mean duration of bronchoscopy was 22.5 minutes (range, 6.3 to 44.6 minutes). There was no immediate complication after targeted washing of primary tumor. Balloon hemostasis was performed in two patients who received transbronchial cryobiopsy for peripheral lung lesion. None of patients suffered iatrogenic pneumothorax, procedure-related infection, or life-threatening complication.

3. EGFR testing results

Table 2 presents the results of EGFR testing across 41 cases. Using tissue samples, EGFR mutation was identified in 80.5% (33 out of 41) of cases, while the T790M mutation was detected in 22.0% (9 out of 41). In plasma samples, EGFR mutation positivity was observed in 56.1% (23 out of 41) of cases, with T790M mutations detected in 9.8% (4 out of 41). Standard EGFR testing results, combining tissue and plasma EGFR testing, indicated that 26.8% showed positive T790M mutation (11 out of 41). Out of the total 41 TWF samples, EGFR mutations were detected in 70.7% (29 out of 41), while the T790M mutation was identified in 29.3% (12 out of 41) of cases. The overall EGFR T790M detection rate using tissue, plasma, or TWF samples was 36.6% (15 out of 41), with EGFR testing using TWF samples increasing the T790M mutation detection rate by up to 10%. Detailed yield of each bronchoscopic procedure is presented in S1 Table.

Table 3 shows an agreement analysis of the detection of EGFR T790M mutation between the TWF and standard specimens. The sensitivity and specificity in the T790M mutation detection using the TWF sample were 72.7% (95% CI, 43.4 to 90.3) and 86.7% (95% CI, 70.3 to 94.7), respectively. The accuracy of T790M mutation detection of the TWF sample was 82.9% (95% CI, 68.7 to 91.5) compared with standard samples.

4. Early detection of T790M mutation in TWF sample

Four patients were found to have the EGFR T790M mutation solely through EGFR testing using TWF samples. Subsequently, all of them underwent cytotoxic chemotherapy, and repeated rebiopsies using either plasma or tissue were performed after progression from chemotherapy. As a result, all four patients were confirmed to have the T790M mutation in tissue or plasma samples. Consequently, their treatment was changed to third-generation EGFR-TKIs (osimertinib or lazertinib).

Discussion

As personalized medicine in the oncology field develops, re-biopsy is essential in the treatment process [13-15]. However, tissue re-biopsy is often difficult due to various situations, such as fibrosis of cancer due to previous treatment or deterioration of patients’ performance [7,16]. Uozu et al. [8] reported that 45% of patients were found to encounter difficulty in collecting tumor tissue when the tumor had progressed after first-line EGFR-TKI treatment based on RECIST criteria. Similarly, 14.6% of cases (6 out of 41) were unable to collect appropriate tissue samples for EGFR testing in the present study. Moreover, molecular testing using plasma samples is simple and least invasive; however, its low sensitivity has been regarded as a significant limitation [15,17]. Therefore, there has been a great need for a more sensitive and less invasive method for molecular testing, including EGFR testing, comparable to tissue or plasma samples. Our findings indicated that TWF samples for EGFR testing are less invasive and yield higher detection rates compared to standard specimens for the detection of the T790M mutation (T790M detection rate: tissue, 22.0%; plasma, 9.8%; and TWF, 29.3%).

We previously reported that the diagnostic yield of extracellular vesicles (EVs) from bronchial washing fluid samples [18]. In 55 bronchial washing fluid-derived samples, the overall detection sensitivity of EGFR mutations was found to be 90% with 100% specificity. Based on these findings, we conclude that EVs from bronchial washing fluid represent a promising source for liquid biopsy. However, the lack of standardized protocols and the variability between different isolation techniques make it difficult to apply EVs in clinical practices yet [19-22]. Furthermore, the process involves the cumbersome steps of isolating EVs from samples and subsequently extracting DNA. Therefore, we isolated cfDNA directly from TWF and employed ddPCR for the detection of EGFR mutations in this study, which was found to be an effective method for detecting the EGFR mutation.

There have been previous reports in conventional bronchial washing for detecting EGFR mutation. Lee et al. [23] showed that ROC for sensitivity was 0.895 for bronchial washing fluid detecting sensitive EGFR mutations (19del or L858R). Zhang et al. [24] also demonstrated the sensitivity of EGFR mutation detected in bronchial washing fluid was 92.5%. Additionally, Roncarati et al. [25] performed NGS analysis using bronchial washing fluid and showed a concordance rate of over 90% when compared to tissue [25]. However, little is known about the utility of bronchial washing fluid samples for re-biopsy, such as detecting the EGFR T790M mutation. Target agents or immunochemotherapy has been demonstrated to effectively shrink tumor volume compared to the conventional chemotherapy [26-28]; therefore, even after progression from first-line treatment, the overall tumor volume is often much less compared to the initial presentation. This could potentially affect the sensitivity and specificity of molecular tests using conventional re-biopsy specimens, such as plasma or tumor tissue. Our results demonstrate that targeted washing techniques are a promising method for collecting cfDNA for molecular testing, even in situations of lower tumor burden during re-biopsy.

Additionally, we noticed the early detection of T790M from TWF in the present study. Strictly speaking, it corresponds to a false positive when assessed based on results from tissue or plasma samples. However, given the subsequent confirmation of the T790M mutation in tissue or plasma specimens, it is deemed highly probable that early detection occurred in the TWF, in which case the aforementioned results align with the mutation’s presence. Many studies reported that tumors can secrete circulating tumor DNA (ctDNA) into the bloodstream before they are visible on imaging, and symptoms of disease are detected [29,30]. Our results suggest that mutational testing using TWF can be a reasonable option for overcoming spatial heterogeneity, low sensitivity of plasma samples, or technical limitations in collecting tissue samples.

Traditionally, deep washing has been utilized for various lung diseases, such as tuberculosis; however, the bronchoscope used for deep washing has typically been a conventional bronchoscope with a diameter of 5 to 6 mm. We developed a targeted technique for acquiring ctDNA from areas surrounding lung tumors, in which the distal tip of the bronchoscope must be positioned directly in front of the tumor, as shown in Fig. 2. To achieve precise localization of the lung lesion, we employed an ultrathin bronchoscope with a 3 mm diameter in combination with radial probe EBUS and fluoroscopy. Regarding the volume of saline instillation, we repeatedly instilled 3 mL of 0.9% normal saline, as the bronchial washing was performed in peripheral lesions where the bronchoscope was positioned just before the lung tumor, leaving very limited space within the bronchus. In our previous experience, instilling a large volume of saline led to endobronchial bleeding, likely due to an abrupt increase in pressure within the peritumoral space and bronchial tree. To mitigate this risk, we opted for repeated instillations of 3 mL of 0.9% normal saline.

This study had several limitations. First, this was a single-center study with available samples from only 41 cases. EGFR T790M was detected exclusively in TWF but not in tissue samples in six cases (14.6%). Due to the small number of study subjects, determining the specific candidates who could benefit from TWF was not feasible. Further studies are required to identify patients who may derive the most benefit from this procedure. Second, the methods of EGFR detection varied depending on the tissue, plasma, and TWF sample. Third, our data showed a lower rate of T790M mutation than other previous studies. It may be explained that the L858R mutation accounts higher portion of our data. Finally, the utility of TWF samples was solely assessed in EGFR testing; hence, further studies are warranted to validate TWF samples for various oncogenic drivers, including next-generation sequencing.

In conclusion, our distinctive technology, named as targeted washing technique, for molecular testing in NSCLC patients showed less invasive, high yield of EGFR T790M mutation detection compared with standard methods. We anticipated that our technique can be readily applied in various kinds of molecular testing in NSCLC, not just EGFR mutation.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

crt-2024-1128_S1_Table.pdf

crt-2024-1128_Supplementary-Materials.pdf

NOTES

Ethical Statement

The study and protocol were approved by the Institutional Review Board of Pusan National University Hospital (No. 2204-006-113) and registered as the Clinical Trials.Gov (NCT05517083). The study was conducted in accordance with the principles of the Declaration of Helsinki and written informed consent was obtained from all study participants.

Author Contributions

Conceived and designed the analysis: Kim MH, Eom JS.

Collected the data: Kim MH, Seong H, Jang H, Kim S, Yoo W, Kim SH, Mok J, Lee K, Kim KU, Lee MK.

Contributed data or analysis tools: Kim MH, Seong H, Kim SH.

Performed the analysis: Kim MH, Seong H, Kim SH, Eom JS.

Wrote the paper: Kim MH, Eom JS.

Conflicts of Interest

Conflict of interest relevant to this article was not reported. Yuhan Cooperation provided research funding, but had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funding

The present study was supported by Yuhan Cooperation.

Acknowledgments

We would like to thank Editage (www.editage.co.kr) for English language editing.

Fig. 1.

Representative case of targeted washing in patients who progressed from the treatment with afatinib. (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 epidermal growth factor receptor (EGFR) testing using a 3.0-mm-sized ultrathin bronchoscope under 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.

Fig. 2.

Schema 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 computed tomography images and with the assistance of virtual bronchoscopic navigation and fluoroscopy. (B) Subsequently, a radial probe endobronchial ultrasound (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 0.9% normal saline (blue lines) was instilled. After a 3-second interval, the washing fluid was retrieved for collecting cell-free DNA derived from the lung tumor. (D) Fluoroscopy image during targeted washing; an ultrathin bronchoscope is placed in front of the target lung lesion (black arrows).

Table 1.

Patient characteristics

Characteristic	No. (%)
Age (yr), median (range)	72 (50-86)
Female sex	27 (79.4)
Never smoker	29 (85.3)
Histology
Adenocarcinoma	30 (88.2)
Squamous cell carcinoma	2 (5.9)
Non-small cell lung cancer, not otherwise specified	2 (5.9)
Stage
III	7 (20.6)
IV	27 (79.4)
*Initial EGFR* mutation type**
19del	16 (47.1)
L858R	18 (52.9)
First-line EGFR-TKI
Gefitinib/Erlotinib	10 (29.4)
Afatinib	23 (67.7)
Lazertinib	1 (2.9)
Longest diameter of lung lesion (mm)	41 (9-100)
Lobar location of lung lesion
Right upper lobe	3 (7.3)
Right middle lobe	3 (7.3)
Right lower lobe	16 (39.0)
Left upper lobe	13 (31.7)
Left lower lobe	6 (14.7)

EGFR-TKI, epidermal growth factor receptor-tyrosine kinase inhibitors.

Table 2.

EGFR testing results

No.	Sex	Age (yr)	Initial mutation (biopsy methods)	Re-biopsy for T790M
No.	Sex	Age (yr)	Initial mutation (biopsy methods)	Tissue results (biopsy methods)	Plasma results	TWF results
1^a)	M	58	19del (TBFB)	19del (TBFB)	19del	19del/T790M^b)
2	F	72	19del (TBFB)	19del/T790M (TBC)	19del/T790M	19del/T790M
3	F	75	19del (TBFB)	19del (TBFB)	19del	Wild type
4^a)	F	67	19del (TBFB)	No cancer cell (TBFB)	19del	Wild type
5	M	50	L858R (TBC+TBFB)	L858R/T790M (TBC+TBFB)	Wild type	L858R/T790M
6	F	55	L858R (TBFB)	L858R (TBC+TBFB)	L858R	L858R
7	F	67	19del (EBUS-TBNA)	No cancer cell (TBFB)	19del	19del
8^a)	F	79	L858R (TBFB)	L858R (TBC)	L858R	L858R
9	F	80	L858R (EBUS-TBNA)	L858R/T790M (TBFB)	Wild type	L858R
10	F	80	L858R (TBFB)	L858R/T790M (TBC+TBFB)	Not detected	L858R/T790M
11	F	65	L858R (TBFB)	L858R (TBC+TBFB)	L858R	L858R
12^a)	M	68	L858R (EBUS-TBNA)	L858R (TBFB)	L858R	L858R/T790M^b)
13^a)	F	67	19del (TBFB)	No cancer cell (TBC)	Wild type	Wild type
14^a)	F	80	L858R (TBFB)	L858R/T790M (TBC+TBFB)	L858R	L858R/S768I/L718X
15^a)	M	59	19del (TBFB)	19del/T790M (TBFB)	19del	19del/T790M
16	F	55	19del (TBFB)	19del/T790M (TBC+TBFB)	19del/T790M	19del/T790M
17	F	75	L858R (TBFB)	L858R (TBFB)	Wild type	L858R
18^a)	F	69	19del (TBFB)	19del (TBFB)	Wild type	19del
19	F	72	L858R (TBFB)	L858R (TBFB)	L858R	L858R/G724S/C797X
20	F	70	19del (TBFB)	19del (TBFB)	Wild type	19del
21	F	86	19del (TBFB)	19del (TBFB)	Wild type	Wild type
22	M	73	L858R (TBFB)	No cancer cell (TBFB)	L858R	L858R
23	F	74	19del (TBFB)	Wild type (TBFB)	Wild type	Wild type
24	F	68	L858R (EBUS-TBNA)	L858R (TBC+TBFB)	L858R	L858R
25^a)	M	68	L858R (EBUS-TBNA)	L858R (TBFB)	L858R	L858R/T790M^b)
26	F	66	19del (TBFB)	19del (TBC+TBFB)	19del	19del
27	F	74	19del (TBFB)	Wild type (TBC+TBFB)	Wild type	Wild type
28	F	70	19del (TBFB)	No cancer cell (not performed)	Wild type	19del/T790M^b)
29^a)	F	64	19del (TBC)	19del (TBFB)	19del	19del
30^a)	F	69	19del (TBFB)	19del (TBFB)	19del/T790M	19del/T790M
31	F	72	19del (TBC+TBFB)	19del (TBC)	Wild type	Wild type
32	F	78	19del (TBFB)	19del/T790M (TBFB)	19del	19del/T790M
33	M	70	L858R (TBFB)	L858R (EBUS-TBNA)	Wild type	L858R/C797X
34	M	76	L858R (TBFB)	L858R (TBFB)	Wild type	Wild type
35^a)	M	77	L858R (TBFB)	L858R (EBUS-TBNA)	Wild type	Wild type
36	F	80	L858R (TBFB)	L858R (TBC+TBFB)	L858R/T790M	L858R/T790M
37	F	71	19del (thoracoscopy)	19del (TBFB)	19del	Wild type
38	F	67	L858R (TBFB)	No cancer cell (not performed)	Wild type	Wild type
39^a)	F	64	19del (TBFB)	19del (TBFB)	19del	19del
40^a)	M	77	L858R (TBFB)	L858R/T790M (EBUS-TBNA)	Wild type	Wild type
41	F	75	L858R (TBC)	L858R (TBC)	Wild type	L858R

EBUS-TBNA, endobronchial ultrasound-guided transbronchial needle aspiration; EGFR, epidermal growth factor receptor; TBC, transbronchial cryobiopsy; TBFB, transbronchial forceps biopsy; TWF, targeted washing fluid.

^a) No. 1 and No. 15, No. 4 and No. 13, No. 8 and No. 14, No. 12 and No. 25, No. 18 and No. 30, No. 29 and No. 39, and No. 35 and No. 40 are the same patients with different dates of bronchoscopy procedures,

^b) T790M mutations were additionally found in four patients using TWF samples.

Table 3.

Agreement analysis of the detection of EGFR T790M mutation between the TWF and standard specimens

T790M detection using standard specimen	T790M detection using TWF
T790M detection using standard specimen	Positive	Negative
Tissue specimen
Positive	6 (14.6)	3 (7.3)
Negative	6 (14.6)	26 (63.4)
Plasma specimen
Positive	4 (9.8)	0
Negative	8 (19.5)	29 (70.7)
Overall standard specimen
Positive^a)	8 (19.5)	3 (7.3)
Negative	4 (9.8)	26 (63.4)

Values are presented as number (%). EGFR, epidermal growth factor receptor; TWF, targeted washing fluid.

^a) Patients with EGFR T790M detection using either tissue or plasma.

REFERENCES

1. Tan AC, Tan DS. Targeted therapies for lung cancer patients with oncogenic driver molecular alterations. J Clin Oncol. 2022;40:611–25. Article PubMed
2. Singh N, Jaiyesimi IA, Ismaila N, Leighl NB, Mamdani H, Phillips T, et al. Therapy for stage IV non-small-cell lung cancer with driver alterations: ASCO living guideline, version 2023.1. J Clin Oncol. 2023;41:e42–50. Article PubMed
3. Lindeman NI, Cagle PT, Aisner DL, Arcila ME, Beasley MB, Bernicker EH, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Thorac Oncol. 2018;13:323–58. PubMed
4. Jorgensen JT. The current landscape of the FDA approved companion diagnostics. Transl Oncol. 2021;14:101063.Article PubMed PMC
5. Malapelle U, Pisapia P, Pepe F, Russo G, Buono M, Russo A, et al. The evolving role of liquid biopsy in lung cancer. Lung Cancer. 2022;172:53–64. Article PubMed
6. Herbst RS, Wu YL, John T, Grohe C, Majem M, Wang J, et al. Adjuvant osimertinib for resected EGFR-mutated stage IB-IIIA non-small-cell lung cancer: updated results from the phase III randomized ADAURA trial. J Clin Oncol. 2023;41:1830–40. Article PubMed PMC
7. Kim S, Kim SH, Kim J, Kim MH, Lee MK, Eom JS. Should we perform repeated re-biopsy for the detection of T790M mutation? Cancer Res Treat. 2023;55:1190–7. Article PubMed PMC PDF
8. Uozu S, Imaizumi K, Yamaguchi T, Goto Y, Kawada K, Minezawa T, et al. Feasibility of tissue re-biopsy in non-small cell lung cancers resistant to previous epidermal growth factor receptor tyrosine kinase inhibitor therapies. BMC Pulm Med. 2017;17:175.Article PubMed PMC PDF
9. Dagogo-Jack I, Shaw AT. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol. 2018;15:81–94. Article PubMed PDF
10. Lee JS, Hur JY, Kim IA, Kim HJ, Choi CM, Lee JC, et al. 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. BMC Cancer. 2018;18:1236.Article PubMed PMC PDF
11. Wang S, Chen H, Zhong J, Qin H, Bai H, Zhao J, et al. Comparative study of EGFR mutations detected in malignant pleural effusion, plasma and tumor tissue in patients with adenocarcinoma of the lung. Lung Cancer. 2019;135:116–22. Article PubMed
12. Hubers AJ, Heideman DA, Yatabe Y, Wood MD, Tull J, Taron M, et al. EGFR mutation analysis in sputum of lung cancer patients: a multitechnique study. Lung Cancer. 2013;82:38–43. Article PubMed
13. Arcila ME, Oxnard GR, Nafa K, Riely GJ, Solomon SB, Zakowski MF, et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clin Cancer Res. 2011;17:1169–80. Article PubMed PMC PDF
14. Hong KS, Cho J, Jang JG, Jang MH, Ahn JH. Endobronchial ultrasound-guided re-biopsy of non-small cell lung cancer with acquired resistance after EGFR tyrosine kinase inhibitor treatment. Thorac Cancer. 2023;14:363–70. PubMed
15. Kim I, Seol HY, Kim SH, Kim MH, Lee MK, Eom JS. Favorable conditions for the detection of EGFR T790M mutation using plasma sample in patients with non-small-cell lung cancer. Cancers (Basel). 2023;15:1445.Article PubMed PMC
16. Yoon HJ, Lee HY, Lee KS, Choi YL, Ahn MJ, Park K, et al. Repeat biopsy for mutational analysis of non-small cell lung cancers resistant to previous chemotherapy: adequacy and complications. Radiology. 2012;265:939–48. Article PubMed
17. Seto T, Nogami N, Yamamoto N, Atagi S, Tashiro N, Yoshimura Y, et al. Real-world EGFR T790M testing in advanced non-small-cell lung cancer: a prospective observational study in Japan. Oncol Ther. 2018;6:203–15. Article PubMed PMC PDF
18. Park J, Lee C, Eom JS, Kim MH, Cho YK. Detection of EGFR mutations using bronchial washing-derived extracellular vesicles in patients with non-small-cell lung carcinoma. Cancers (Basel). 2020;12:2822.Article PubMed PMC
19. Connal S, Cameron JM, Sala A, Brennan PM, Palmer DS, Palmer JD, et al. Liquid biopsies: the future of cancer early detection. J Transl Med. 2023;21:118.Article PubMed PMC PDF
20. De Rubis G, Rajeev Krishnan S, Bebawy M. Liquid biopsies in cancer diagnosis, monitoring, and prognosis. Trends Pharmacol Sci. 2019;40:172–86. Article PubMed
21. Doyle LM, Wang MZ. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. 2019;8:727.Article PubMed PMC
22. Ramirez-Garrastacho M, Bajo-Santos C, Line A, Martens-Uzunova ES, de la Fuente JM, Moros M, et al. Extracellular vesicles as a source of prostate cancer biomarkers in liquid biopsies: a decade of research. Br J Cancer. 2022;126:331–50. Article PubMed PMC PDF
23. Lee SH, Kim EY, Kim T, Chang YS. Compared to plasma, bronchial washing fluid shows higher diagnostic yields for detecting EGFR-TKI sensitizing mutations by ddPCR in lung cancer. Respir Res. 2020;21:142.Article PubMed PMC PDF
24. Zhang X, Li C, Ye M, Hu Q, Hu J, Gong Z, et al. Bronchial washing fluid versus plasma and bronchoscopy biopsy samples for detecting epidermal growth factor receptor mutation status in lung cancer. Front Oncol. 2021;11:602402.Article PubMed PMC
25. Roncarati R, Lupini L, Miotto E, Saccenti E, Mascetti S, Morandi L, et al. Molecular testing on bronchial washings for the diagnosis and predictive assessment of lung cancer. Mol Oncol. 2020;14:2163–75. Article PubMed PMC PDF
26. Cho BC, Ahn MJ, Kang JH, Soo RA, Reungwetwattana T, Yang JC, et al. Lazertinib versus gefitinib as first-line treatment in patients with EGFR-mutated advanced non-small-cell lung cancer: results from LASER301. J Clin Oncol. 2023;41:4208–17. Article PubMed
27. Peters S, Camidge DR, Shaw AT, Gadgeel S, Ahn JS, Kim DW, et al. Alectinib versus crizotinib in untreated ALK-positive non-small-cell lung cancer. N Engl J Med. 2017;377:829–38. Article PubMed
28. Gadgeel S, Rodriguez-Abreu D, Speranza G, Esteban E, Felip E, Domine M, et al. Updated analysis from KEYNOTE-189: pembrolizumab or placebo plus pemetrexed and platinum for previously untreated metastatic nonsquamous non-small-cell lung cancer. J Clin Oncol. 2020;38:1505–17. Article PubMed
29. Fiala C, Kulasingam V, Diamandis EP. Circulating tumor DNA for early cancer detection. J Appl Lab Med. 2018;3:300–13. Article PubMed PDF
30. Campos-Carrillo A, Weitzel JN, Sahoo P, Rockne R, Mokhnatkin JV, Murtaza M, et al. Circulating tumor DNA as an early cancer detection tool. Pharmacol Ther. 2020;207:107458.Article PubMed PMC

Figure & Data

REFERENCES

Citations

Citations to this article as recorded by

Comparison of the safety and efficacy of remimazolam for sedation during bronchoscopy: a meta-analysis of randomized controlled trials
Yupei Yuan, Chunlei Chang, Jing Zhang, Liang Liang
PeerJ.2026; 14: e20552. CrossRef

PubReader
ePub Link

Cite

CITE: export Copy Download Format; Close

Download Citation

Download a citation file in RIS format that can be imported by all major citation management software, including EndNote, ProCite, RefWorks, and Reference Manager.

Format:

RIS — For EndNote, ProCite, RefWorks, and most other reference management software
BibTeX — For JabRef, BibDesk, and other BibTeX-specific software

Include:

Citation for the content below
Citation and abstract for the content below

Novel Bronchoscopy Method for Molecular Profiling of Lung Cancer: Targeted Washing Technique

Cancer Res Treat. 2026;58(1):107-114. Published online February 26, 2025

DOI: https://doi.org/10.4143/crt.2024.1128

XML Download

Abstract
Introduction
Materials and Methods
Results
Discussion
Electronic Supplementary Material
NOTES
REFERENCES

Novel Bronchoscopy Method for Molecular Profiling of Lung Cancer: Targeted Washing Technique

Fig. 1. Representative case of targeted washing in patients who progressed from the treatment with afatinib. (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 epidermal growth factor receptor (EGFR) testing using a 3.0-mm-sized ultrathin bronchoscope under 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.

Fig. 2. Schema 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 computed tomography images and with the assistance of virtual bronchoscopic navigation and fluoroscopy. (B) Subsequently, a radial probe endobronchial ultrasound (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 0.9% normal saline (blue lines) was instilled. After a 3-second interval, the washing fluid was retrieved for collecting cell-free DNA derived from the lung tumor. (D) Fluoroscopy image during targeted washing; an ultrathin bronchoscope is placed in front of the target lung lesion (black arrows).

Fig. 1.

Fig. 2.

Novel Bronchoscopy Method for Molecular Profiling of Lung Cancer: Targeted Washing Technique

Characteristic	No. (%)
Age (yr), median (range)	72 (50-86)
Female sex	27 (79.4)
Never smoker	29 (85.3)
Histology
Adenocarcinoma	30 (88.2)
Squamous cell carcinoma	2 (5.9)
Non-small cell lung cancer, not otherwise specified	2 (5.9)
Stage
III	7 (20.6)
IV	27 (79.4)
*Initial EGFR* mutation type**
19del	16 (47.1)
L858R	18 (52.9)
First-line EGFR-TKI
Gefitinib/Erlotinib	10 (29.4)
Afatinib	23 (67.7)
Lazertinib	1 (2.9)
Longest diameter of lung lesion (mm)	41 (9-100)
Lobar location of lung lesion
Right upper lobe	3 (7.3)
Right middle lobe	3 (7.3)
Right lower lobe	16 (39.0)
Left upper lobe	13 (31.7)
Left lower lobe	6 (14.7)

No.	Sex	Age (yr)	Initial mutation (biopsy methods)	Re-biopsy for T790M
No.	Sex	Age (yr)	Initial mutation (biopsy methods)	Tissue results (biopsy methods)	Plasma results	TWF results
1^a)	M	58	19del (TBFB)	19del (TBFB)	19del	19del/T790M^b)
2	F	72	19del (TBFB)	19del/T790M (TBC)	19del/T790M	19del/T790M
3	F	75	19del (TBFB)	19del (TBFB)	19del	Wild type
4^a)	F	67	19del (TBFB)	No cancer cell (TBFB)	19del	Wild type
5	M	50	L858R (TBC+TBFB)	L858R/T790M (TBC+TBFB)	Wild type	L858R/T790M
6	F	55	L858R (TBFB)	L858R (TBC+TBFB)	L858R	L858R
7	F	67	19del (EBUS-TBNA)	No cancer cell (TBFB)	19del	19del
8^a)	F	79	L858R (TBFB)	L858R (TBC)	L858R	L858R
9	F	80	L858R (EBUS-TBNA)	L858R/T790M (TBFB)	Wild type	L858R
10	F	80	L858R (TBFB)	L858R/T790M (TBC+TBFB)	Not detected	L858R/T790M
11	F	65	L858R (TBFB)	L858R (TBC+TBFB)	L858R	L858R
12^a)	M	68	L858R (EBUS-TBNA)	L858R (TBFB)	L858R	L858R/T790M^b)
13^a)	F	67	19del (TBFB)	No cancer cell (TBC)	Wild type	Wild type
14^a)	F	80	L858R (TBFB)	L858R/T790M (TBC+TBFB)	L858R	L858R/S768I/L718X
15^a)	M	59	19del (TBFB)	19del/T790M (TBFB)	19del	19del/T790M
16	F	55	19del (TBFB)	19del/T790M (TBC+TBFB)	19del/T790M	19del/T790M
17	F	75	L858R (TBFB)	L858R (TBFB)	Wild type	L858R
18^a)	F	69	19del (TBFB)	19del (TBFB)	Wild type	19del
19	F	72	L858R (TBFB)	L858R (TBFB)	L858R	L858R/G724S/C797X
20	F	70	19del (TBFB)	19del (TBFB)	Wild type	19del
21	F	86	19del (TBFB)	19del (TBFB)	Wild type	Wild type
22	M	73	L858R (TBFB)	No cancer cell (TBFB)	L858R	L858R
23	F	74	19del (TBFB)	Wild type (TBFB)	Wild type	Wild type
24	F	68	L858R (EBUS-TBNA)	L858R (TBC+TBFB)	L858R	L858R
25^a)	M	68	L858R (EBUS-TBNA)	L858R (TBFB)	L858R	L858R/T790M^b)
26	F	66	19del (TBFB)	19del (TBC+TBFB)	19del	19del
27	F	74	19del (TBFB)	Wild type (TBC+TBFB)	Wild type	Wild type
28	F	70	19del (TBFB)	No cancer cell (not performed)	Wild type	19del/T790M^b)
29^a)	F	64	19del (TBC)	19del (TBFB)	19del	19del
30^a)	F	69	19del (TBFB)	19del (TBFB)	19del/T790M	19del/T790M
31	F	72	19del (TBC+TBFB)	19del (TBC)	Wild type	Wild type
32	F	78	19del (TBFB)	19del/T790M (TBFB)	19del	19del/T790M
33	M	70	L858R (TBFB)	L858R (EBUS-TBNA)	Wild type	L858R/C797X
34	M	76	L858R (TBFB)	L858R (TBFB)	Wild type	Wild type
35^a)	M	77	L858R (TBFB)	L858R (EBUS-TBNA)	Wild type	Wild type
36	F	80	L858R (TBFB)	L858R (TBC+TBFB)	L858R/T790M	L858R/T790M
37	F	71	19del (thoracoscopy)	19del (TBFB)	19del	Wild type
38	F	67	L858R (TBFB)	No cancer cell (not performed)	Wild type	Wild type
39^a)	F	64	19del (TBFB)	19del (TBFB)	19del	19del
40^a)	M	77	L858R (TBFB)	L858R/T790M (EBUS-TBNA)	Wild type	Wild type
41	F	75	L858R (TBC)	L858R (TBC)	Wild type	L858R

T790M detection using standard specimen	T790M detection using TWF
T790M detection using standard specimen	Positive	Negative
Tissue specimen
Positive	6 (14.6)	3 (7.3)
Negative	6 (14.6)	26 (63.4)
Plasma specimen
Positive	4 (9.8)	0
Negative	8 (19.5)	29 (70.7)
Overall standard specimen
Positive^a)	8 (19.5)	3 (7.3)
Negative	4 (9.8)	26 (63.4)

Table 1. Patient characteristics

EGFR-TKI, epidermal growth factor receptor-tyrosine kinase inhibitors.

Table 2. EGFR testing results

No. 1 and No. 15, No. 4 and No. 13, No. 8 and No. 14, No. 12 and No. 25, No. 18 and No. 30, No. 29 and No. 39, and No. 35 and No. 40 are the same patients with different dates of bronchoscopy procedures,

T790M mutations were additionally found in four patients using TWF samples.

Table 3. Agreement analysis of the detection of EGFR T790M mutation between the TWF and standard specimens

Values are presented as number (%). EGFR, epidermal growth factor receptor; TWF, targeted washing fluid.

Patients with EGFR T790M detection using either tissue or plasma.