Feasibility of next-generation sequencing using bronchoscopic specimens in potentially resectable non-small cell lung cancer

Introduction

Lung cancer is one of the leading causes of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases (1). Although cytotoxic chemotherapy is conventionally used to prolong prognosis in patients with unresectable NSCLC, the median overall survival period remains only 10–14 months (2). Since the advent of molecular targeted drugs, the prognosis in patients with unresectable NSCLC harboring gene aberrations, such as EGFR mutation and ALK fusion, has drastically improved (3-5). Therefore, molecular biomarker testing is crucial for the development of treatment strategies for unresectable NSCLC (6). Thereafter, various driver oncogenes have been discovered other than EGFR and ALK, and molecular targeted drugs for them are already being introduced into clinical practice. However, testing with individual companion diagnostics has resulted in an increase in the number of specimens required and cost of testing.

Next-generation sequencing (NGS) has enabled the simultaneous detection of multiple targetable gene aberrations using smaller specimens than using multiple sequential single-gene assays (7,8). In addition, NGS is associated with higher accuracy and lower cost than whole-genome sequencing in clinical settings (9,10). Programmed cell death 1/programmed cell death-ligand 1 expression should also be examined by analyzing specimens when deciding on a treatment plan for unresectable NSCLC (11,12).

Recent advances in multidisciplinary treatment for potentially resectable NSCLC have led to the development of various therapies by combining molecular-targeted drugs and immune checkpoint inhibitors. Prolonged prognoses have been reported when these drugs are used as adjuvant therapies in selected patients (13-16). In addition, the efficacy of neoadjuvant therapy has also been reported (17-19), and clinical trials for the preoperative molecular-targeted drugs have recently been conducted (20-22). Patients with multiple and/or invasive N2 who were not previously considered eligible for surgery can now be treated successfully with surgery after preoperative therapy (23). Therefore, molecular biomarker testing is being considered even for potentially resectable NSCLC to make preoperative treatment decisions, and it is becoming increasingly important to perform NGS on preoperative biopsy specimens successfully.

Bronchoscopy is one of the most commonly performed biopsy methods for lung cancer and it is reported to be adequate for obtaining specimens for NGS in unresectable cases (24-27). However, bronchoscopic biopsy specimens have not been widely used for NGS analysis in potentially resectable NSCLC, because NGS has been considered for surgical specimens that have a higher analytical success rate than biopsy specimens (24). This study aimed to investigate the question whether NGS-targeted bronchoscopy for potentially resectable NSCLC is feasible. In order to achieve this objective, we compared the success rate of NGS analysis using bronchoscopic biopsy specimens in potentially resectable cases with those in unresectable cases. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-605/rc).

Methods

Participants

Data for consecutive patients diagnosed with NSCLC via bronchoscopy at National Cancer Center Hospital between October 2019 and December 2022, who were ordered for NGS using bronchoscopy specimens, were retrospectively extracted. The Oncomine DX Target Test (ODxTT), which is used as the standard companion diagnostic test for NGS in Japan, was included.

Traditionally, resectable NSCLC with mediastinal lymph node metastasis has been limited to single-station and non-bulky N2 (28,29). However, this delineation has become unclear with the development of induction or preoperative therapies and most N2 cases are now considered potentially resectable (23,30). Therefore, we defined all N0–2 and M0 NSCLC cases as potentially resectable, regardless of the N2 status (i.e., multiple and/or bulky), whereas N3 and/or M1 NSCLC cases were considered unresectable according to the 8^th edition of the tumor-node-metastasis (TNM) classification, which was the standard during the study period. Staging was determined based on systemic imaging studies and pathological confirmation of biopsies. Based on these results, patients were divided into potentially resectable and unresectable groups.

Accordingly, patients who experienced recurrence after curative treatment (i.e., surgery and radiotherapy) and those who underwent rebiopsy after systemic therapy were excluded.

NGS

The NGS used in this study was ODxTT, which detects mutations in 46 genes from tumor specimen-derived DNA and the fusion of 21 genes from tumor specimen-derived ribonucleic acid extracted from formalin-fixed paraffin-embedded (FFPE) samples. In Japan, ODxTT has been approved as a companion diagnostic for EGFR, ALK, ROS1, BRAF, RET, and HER2 aberrations by the end of 2023.

There are several tissue quality and quantity requirements for analysis using ODxTT. Tumor cell content (TCC), which is defined as the percentage of tumor cells to total nucleated cells in the tissue (31), of ≥30%, is required for accurate analysis to prevent false-negative results (26,32). If the content is 10–30%, microdissection may be considered to extract suitable sites for analysis at the discretion of pathologists. NGS was performed on 10 slides of 5-µm-thick sliced tissues from FFPE specimens. If the tissue surface area was ≤4 mm², >15 slides were prepared. In addition, a tissue surface area of ≥1 mm² is required for accurate analysis (32). If tissues ordered for NGS are judged inadequate by pathologists, the reason is commented upon, and NGS is discontinued at National Cancer Center Hospital.

Procedures

Sampling was performed using any of the following three methods corresponding to each of the three types of target lesions: (I) endobronchial lesions under direct visualization; (II) peri-tracheal/bronchial lesions using convex probe endobronchial ultrasound (CP-EBUS); and (III) lung lesions using radial EBUS (R-EBUS). If there were multiple targetable lesions, they were compared at a conference, and the method that appeared to be the most efficient for collecting sufficient tumor cells was selected.

For endobronchial lesions, the target lesion was biopsied under direct visualization using a conventional bronchoscope (BF-P290, BF-1TQ290, or BF-F260; Olympus, Tokyo, Japan). Forceps are usually used as biopsy devices, and a snare and/or cryoprobe (Erbe Elektromedizin GmbH, Tübingen, Germany) is occasionally used.

For peri-tracheal/bronchial lesions, the target was biopsied under real-time CP-EBUS guidance using a dedicated ultrasound bronchoscope (BF-UC260FW or BF-UC290F; Olympus). EBUS-guided transbronchial needle aspiration (EBUS-TBNA) was performed mainly using 22-gauge (G) needles; however, if the case was also intended for systematic nodal staging or if the target was in contact with a tightly angled bronchus, 25-G needles were used. In cases with multiple enlarged lymph nodes, the target deemed suitable for tumor cell collection was determined with reference to CP-EBUS findings, combining the B, Doppler, and elastography modes (33). Since July 2020, EBUS-guided intranodal forceps biopsy (IFB) has been frequently performed to enhance the quality of specimens (34,35).

For lung lesions, the target was biopsied under X-ray fluoroscopy guidance while referring to R-EBUS findings using one of the following three patterns: (I) a thin bronchoscope (BF-P260F or BF-P290; Olympus) with a smaller guide sheath (GS) (K-201; Olympus); (II) a thin bronchoscope without any GS; or (III) a therapeutic bronchoscope (BF-1T260 or BF-1TQ290; Olympus) with a larger GS (K-203; Olympus). Prior to bronchoscopy, virtual bronchoscopic navigation and fluoroscopy were reconstructed using high-resolution computed tomography (HRCT) data on a workstation (Ziostation2; Ziosoft, Tokyo, Japan). Based on the above virtual images, an R-EBUS probe (UM-S20-17S or UM-S20-20R; Olympus) was inserted toward the target, and forceps biopsy, TBNA, and/or cryobiopsy were performed at the location where the lesion was identified.

In most cases, a rapid on-site evaluation was performed. Tissues were immediately fixed in 10% neutral-buffered formalin for 6–48 hours, embedded in paraffin, and processed for histopathological examination.

Study objectives

The main objective was to compare the rates of successful NGS analysis between the potentially resectable and unresectable groups divided into subgroups, and to investigate the feasibility of NGS-targeted bronchoscopy for potentially resectable NSCLC. ODxTT has been approved as a companion diagnostic tool for six different gene aberrations (EGFR, ALK, ROS1, BRAF, RET, and HER2) at the time of analysis; therefore, the analytical outcome of NGS was determined based on the results of this study. Concerning the EGFR mutations, the results were extracted mainly to focus on common mutations because the variants that were analyzed differed depending on the timing. For this study, NGS analysis was considered analytically successful if a definitive result, either positive or negative for each driver oncogene, was reported. Conversely, the analysis was deemed a failure if it was canceled prior to analysis due to an ‘inadequate’ specimen, as judged by pathologists, or if the post-analytical report was ‘no call’ or ‘invalid’.

Patient data, such as age, sex, smoking status, tumor characteristics on HRCT, histology, and NGS results, were extracted from medical records and the respiratory endoscopy database. Morphology was classified as solid or subsolid based on the presence of ground-glass opacity. The location of lung lesions was classified as inner or outer. We defined “outer” lesions as those located in the outer third of the lung parenchyma, a previously established topographical classification (36).

Statistical analyses

Categorical variables were summarized as frequencies (percentages) and continuous variables as medians (ranges). Differences in distribution between the two groups were analyzed using the Fisher exact test or the Pearson Chi-squared test for categorical variables and the Mann-Whitney U test for continuous variables. For the multivariable logistic regression analysis, variables identified as significant (P<0.05) in the univariable analysis were included. All statistical analyses were performed using JMP Pro 16.0.0 software (SAS Institute Inc. Cary, NC, USA), and two-sided P values <0.05 were considered to be statistically significant.

Ethical approval

This study was approved by the National Cancer Center Hospital Institutional Review Board (No. 2018–090). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The requirement for informed consent was waived by the ethics committee because of the retrospective nature of the study. An opt-out option was provided for patients to refuse the use of their data.

Results

Patient characteristics

Among the 644 patients included, 184 were in the potentially resectable group and 460 in unresectable group. Patient characteristics are summarized in Table 1. The median age of the potentially resectable group was higher than that of the unresectable group (72 vs. 66 years, P<0.001). The rates of male sex (70.7% vs. 59.6%, P=0.009) and smoking habit (83.2% vs. 71.7%, P=0.002) were higher in the potentially resectable group. Although the most common histology in both groups was adenocarcinoma, squamous cell carcinomas were more frequent in the potentially resectable group (29.9% vs. 10.9%). Endobronchial lesions were the targeted site of biopsy more frequently in the potentially resectable group (7.6% vs. 2.6%); however, the frequencies for targeting peri-tracheal/bronchial and lung lesions were similar in both groups.

Overall patients

As shown in Figure 1, the overall rate of successful NGS analysis was significantly lower in the potentially resectable group (81.0% vs. 87.8%, P=0.03). Regarding the cause of failure, the proportion of invalid or no calls was similar in both groups (4.3% vs. 3.9%), whereas the proportion of inadequate calls was higher in the potentially resectable group (14.7% vs. 8.3%); a detailed breakdown of the reasons for failure is provided in Table S1. Among successful cases, the detection rates of EGFR mutations and ALK fusions in the potentially resectable group were lower compared with that of those in the unresectable group (10.9% vs. 24.8% and 2.2% vs. 6.1%, respectively). In both groups, KRAS mutations were the second most common after EGFR mutations (8.2% vs. 10.2%, respectively). Additionally, the proportion of cases in which no genetic aberrations were identified was higher in the potentially resectable group (46.7% vs. 32.8%).

Figure 1 Comparison of the success rates of next-generation sequencing analysis between the potentially resectable and unresectable groups. The success rate of next-generation sequencing analysis is significantly lower in the potentially resectable group than in unresectable group (81.0% vs. 87.8%, P=0.03). In successful cases, the detection rates of EGFR mutations and ALK fusions in the potentially resectable group are lower than those in the unresectable group (10.9% vs. 24.8% and 2.2% vs. 6.1%, respectively). In both groups, KRAS mutations are the second most common after EGFR mutations (8.2% vs. 10.2%). Moreover, the proportion of cases in which no gene aberrations are identified is higher in the potentially resectable group than in the unresectable group (46.2% vs. 32.8%).

Subgroup of endobronchial lesions

The endobronchial lesion subgroup included 14 potentially resectable lesions and 12 unresectable lesions. Cryobiopsy was performed for 5 of 14 patients (35.7%) in the potentially resectable group and 6 of 12 patients (50.0%) in the unresectable group, with no significant difference between the groups (P=0.68). Unlike in the unresectable group, there were two failures in the potentially resectable group. Specifically, all 11 patients who underwent cryobiopsy had successful analyses, whereas the two failures occurred among the 15 patients who underwent forceps biopsy alone (success rate, 100% vs. 86.7%; P=0.53). Consequently, the overall NGS analytical success rates were similarly high in both groups (85.7% vs. 100%, P=0.48) (Figure 2).

Figure 2 Stratified comparisons of the rate of successful NGS analysis and causes of failure in each target lesion between the two groups. The NGS analytical success rates for the endobronchial lesions are high and comparable between the two groups (85.8% vs. 100%, P=0.48). The NGS analytical success rates for the peri-tracheal/bronchial lesions are comparable between the two groups (84.9% vs. 88.2%, P=0.44). The success rates of NGS analysis for lung lesions are significantly lower in the potentially resectable group than in the unresectable group (73.4% vs. 86.4%, P=0.03). NGS, next-generation sequencing.

Subgroup of peri-tracheal/bronchial lesions

The peri-tracheal/bronchial lesion subgroup included 106 potentially resectable and 272 unresectable lesions. In this subgroup, mediastinal lymph nodes were targeted for biopsy less frequently in the potentially resectable group than in the unresectable group (56.6% vs. 70.6%). Furthermore, procedures for the potentially resectable group more often involved the use of a 25-G needle (42.5% vs. 6.6%, P<0.001) and included combined IFB less frequently (38.6% vs. 64.7%, P<0.001) compared to those for the unresectable group (Table 2). The NGS analytical success rate was comparable between the two groups (84.9% vs. 88.2%, P=0.44) (Figure 2). In addition, no significant factors associated with the success of NGS analysis were identified in the univariable analysis (Table 3). Therefore, a multivariable analysis was not performed for this subgroup.

Subgroup of lung lesions

The lung lesion subgroup included 64 potentially resectable and 176 unresectable patients. The rate of successful NGS analysis in the potentially resectable group was significantly lower than that in the unresectable group (73.4% vs. 86.4%, P=0.03) (Figure 2). As shown in Table 4, most baseline characteristics of the lung lesions were comparable between the two groups, including morphology, where the proportion of subsolid lesions did not differ significantly (10.9% vs. 6.8%, P=0.30). However, the use of cryobiopsy was significantly more frequent in the unresectable group than in the potentially resectable group (39.8% vs. 15.6%, P<0.001). Additionally, patients in the potentially resectable group were more likely to have target lesions in the outer areas than the unresectable group (64.1% vs. 39.2%, P<0.001). As shown in Table 5, the univariable analysis identified inner-located tumors (P=0.02) and the use of a cryoprobe (P=0.045), and the multivariable analysis identified inner-located tumors (P=0.04) as a significant factor affecting the success of NGS analysis.

Sub-analysis focusing on stage III potentially resectable and unresectable cases

We conducted a sub-analysis limited to stage III potentially resectable cases (n=136) and the unresectable group (n=460). The overall NGS analytical success rate remained significantly lower in the stage III potentially resectable group compared to the unresectable group (80.1% vs. 87.8%, P=0.03). When stratified by lesion type, no significant differences were observed for endobronchial (85.7% vs. 100%, P=0.37) or peri-tracheal/bronchial lesions (84.2% vs. 88.2%, P=0.41). However, for lung lesions, the analytical success rate was still significantly lower in the stage III potentially resectable group (67.6% vs. 86.4%, P=0.02) (Figure S1). A comparison of the characteristics of lung lesions between these two groups revealed that lesions in the stage III potentially resectable group were significantly more likely to be located in the outer third of the lung (58.9% vs. 39.2%, P=0.04) and were biopsied with cryobiopsy less frequently (17.6% vs. 39.8%, P=0.02) (Table S2).

Discussion

The present study focused on the NGS analytical success rate using bronchoscopic specimens in potentially resectable NSCLC cases and compared it to that in unresectable cases. The overall success rate of NGS analysis in potentially resectable cases was significantly lower (81.0% vs. 87.8%, P=0.03). However, when divided into subgroups according to the target lesion, endobronchial lesions (85.7% vs. 100%, P=0.48) and peri-tracheal/bronchial lesions (84.9% vs. 88.2%, P=0.44) showed high success rates of NGS analysis in both groups. In contrast, the subgroup of lung lesions showed significantly lower success rates of NGS analysis in the potentially resectable group (73.4% vs. 86.4%, P=0.03), which likely contributed to the overall success rate difference in NGS analysis. These findings suggest that, when performing NGS-targeted bronchoscopy in a preoperative setting, targeting endobronchial or peri-tracheal/bronchial lesions is preferable. Therefore, when targeting lung lesions, specific strategies should be employed to ensure specimen adequacy, such as the preferential use of cryobiopsy to obtain larger tissue volumes and meticulous targeting with R-EBUS to ensure accurate sampling, especially for peripheral tumors. By implementing these strategies, bronchoscopy in potentially resectable NSCLC becomes feasible and can inform multimodal therapeutic decision-making. A summary of these findings is shown in Figure 3.

Figure 3 Graphical abstract of this study. M, metastasis; N, node; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer.

Significant differences in baseline patient characteristics were noted between the potentially resectable and unresectable groups (Table 1). These disparities are likely reflective of the distinct clinical presentations associated with different disease stages. Such differences in baseline characteristics represent potential confounding factors, warranting caution in the interpretation of our findings. To address confounding by disease stage, we performed a sub-analysis limited to stage III cases. The results confirmed that the success rate for lung lesions remained significantly lower in the potentially resectable group. This suggests that the disparity is not simply due to different stages but reflects the inherent challenges of biopsying surgical candidates. Clinicians may be more cautious in this preoperative setting, underscoring the need for advanced sampling techniques, such as cryobiopsy, to ensure adequate tissue for critical molecular testing.

As perioperative treatment advances, the treatment strategy changes, especially in stage III or N2-positive NSCLC, and combining preoperative treatment with surgery may improve survival rates (37). Genetic information, which has recently been considered for inclusion in the TNM classification (38), is necessary to make treatment decisions. Furthermore, bronchoscopy plays an increasingly important role in treatment choices. Additional clinical trials of preoperative treatments based on NGS results from bronchoscopic biopsies, as reported by Wang et al. (18), will be conducted in the future. In this study, the number of cases in which genetic aberrations were not detected was higher in the potentially resectable group than in the unresectable group (46.7% vs. 32.8%, respectively). For eligible patients, preoperative use of immune checkpoint inhibitors is considered (19). Furthermore, for patients who experience complete or major pathological response to preoperative treatment, the surgical specimen will not be suitable for NGS, and preoperative specimens will have to be relied upon. Therefore, the results of the present study provide valuable insights into preoperative NGS-targeted bronchoscopy.

In the endobronchial lesion subgroup, NGS was successful in 24 of the 26 patients, demonstrating a high success rate of NGS analysis. However, it is important to note that the comparison between the potentially resectable and unresectable groups should be interpreted with caution, as the small sample for this subgroup (n=14 and n=12, respectively) limits the statistical power. Among the 14 patients in the potentially resectable group, two failed to respond. The reasons for each case were excessive granulation to obtain sufficient tumor cells and insufficient de novo DNA amplification. Although not routinely used, cryobiopsy improves the diagnostic yield for endobronchial lesions (39,40) and increases the success rate of NGS analysis by collecting sufficient amount of the specimens (41). In both failure cases, only forceps biopsy was performed, whereas NGS was successful in all 11 patients who underwent cryobiopsy.

In the subgroup of peri-tracheal/bronchial lesions, the rate of successful NGS analysis was comparable between the two groups (84.9% vs. 88.2%). Consistent with previous findings, the overall analytical success rate was relatively high (25,42). We previously reported that the number of core tissues obtained via EBUS-TBNA was associated with a high NGS analytical success rate (43); however, in this study, there were no significant factors. We attribute this change to the routine implementation of EBUS-IFB (35). Traditional TBNA specimens are cell blocks collected using an aspiration needle, and contamination with blood components is inevitable. In contrast, IFB specimens enable accurate tissue sampling, and we encountered cases in which NGS was difficult with TBNA specimens but could be supplemented with IFB specimens. This standardization of specimen quality likely diminished the predictive value of purely quantitative measures of EBUS-TBNA. Recently, there have been reports of improved diagnostic yields by applying EBUS cryobiopsy, and this technique has attracted increasing attention (44). Further studies are required to determine whether these methods can improve the NGS analytical success rates. In potentially resectable cases, specimens are often collected using a 25-G needle for systematic staging purposes. In this study, no difference in the NGS analytical success rates was observed between the 22- and 25-G needles, which was consistent with previous reports (26), and NGS analysis can be considered for specimens using a 25-G needle.

In contrast, in the subgroup of lung lesions, the NGS analytical success rate was significantly lower in the potentially resectable group (73.4% vs. 86.4%, P=0.03). Forceps specimens from lung lesions reportedly contain fewer tumor cells than EBUS-TBNA or surgical specimens (27,32). In this study, location in the inner two-thirds of the lung was a significant factor associated with success in the multivariable analysis. Although lesions located in the outer third have lower diagnostic yields and lesion visualization rates by R-EBUS (45,46), it is also difficult to secure sufficient number of specimens for genetic analysis because approaching them with forceps consistently is difficult, even if the tumor is large enough and the bronchus sign is positive. Specimens from subsolid lesions, which are expected to be more common in potentially resectable cases, tended to have lower NGS analytical success rates, which is consistent with previous reports (47,48). However, no significant differences in the rate of successful NGS analysis by morphology were observed in this study, likely due to the small number of subsolid cases in our cohort (n=19 total).

In this study, the combined use of cryoprobes, which allowed the collection of a larger volume of specimens, was associated with a higher success rate of NGS analysis in the univariable analysis (Table 5). The effectiveness of genomic analysis using cryobiopsy specimens from lung lesions has been reported (41). Notably, the use of cryobiopsy for lung lesions was significantly more frequent in the unresectable group (39.8% vs. 15.6%, P<0.001), representing a key procedural confounder. This suggests that the higher analytical success rate observed in the unresectable group’s lung lesions may be partially attributable to the more frequent application of this advanced sampling technique.

This study has some limitations. First, this was a single-center retrospective study. Although there is a possibility of selection bias, we believe that this study was conducted with a sufficient number of patients. Second, we used ODxTT, which has been approved for companion diagnostics in Japan. The type of NGS used differs depending on the country and facility, and the requirements for the specimens vary, accordingly. However, the general trend is likely to be similar regardless of the types. Third, clinical decision-making likely contributed to the lower analytical success rate in the potentially resectable group. For surgical candidates, clinicians may adopt a more conservative biopsy approach to avoid complications that could delay surgery, often relying on the forthcoming surgical specimen. This cautious sampling can yield smaller specimens and impact NGS feasibility, a critical issue as neoadjuvant therapies requiring preoperative molecular data become more common. Finally, a key limitation is that the assessment of sample adequacy for NGS relied on the qualitative judgment of expert pathologists rather than on standardized, quantitative metrics such as TCC or tissue surface area. As this was a retrospective study of real-world clinical practice, these quantitative data were not uniformly available for analysis, preventing a more objective evaluation of the reasons for analytical failure.

Conclusions

NGS analytical success rates using bronchoscopic specimens in potentially resectable NSCLC cases were significantly lower compared to those in unresectable cases, especially in lung lesions. Harvesting lung lesions adequately and preferentially targeting endobronchial or peri-tracheal/bronchial lesions can enhance the feasibility of NGS and inform multimodal treatment strategies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-605/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-605/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-605/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-605/coif). Y.M. reports consulting fees from INTUITIVE and personal fees from Olympus, AstraZeneca, Erbe Elektromedizin GmbH, NOVARTIS, COOK, AMCO, Thermo Fisher Scientific, Fujifilm, Chugai, Eli Lilly, Merck, Takeda, and ETHICON. H.F. reports grants from Hitachi High-Tech Corporation and DNA Chip Research Inc., and personal fees from Erbe Elektromedizin GmbH, AstraZeneca, and DNA Chip Research Inc. K.U. reports grants from JSPS KAKENHI (Nos. JP22K15698 and JP19K16966) and personal fees from Insmed, Chugai, AstraZeneca, and Novartis. K.A. reports personal fees from AstraZeneca, Chugai, Intuitive Surgical, Covidien, Johnson & Johnson, and MSD. T.T. reports a grant from JSPS KAKENHI, payment from Nippon Medical School Foundation, and participation on a Safety Monitoring Board for Hamamatsu University School of Medicine. 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 approved by the National Cancer Center Hospital Institutional Review Board (No. 2018–090). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The requirement for informed consent was waived by the ethics committee because of the retrospective nature of the study. An opt-out option was provided for patients to refuse the use of their data.

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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