Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.This research focused on Molecular analysis of EBUS-TBNA samples for accurate nodal staging in non-small cell lung cancer, after check in pubmed, not so many references about this topic（PMID: 40345259）, this was mean this manuscript was with some Innovation.

2.This was a review manuscript, although not so many articles about this topic especially review article, but some questions should be deal with.

3.Firstly, the title“Molecular analysis of EBUS-TBNA samples for accurate nodal staging in non-small cell lung cancer”I think can be revised,  I donot know which was main important content of this manuscript.

4. Are there any differences of Molecular analysis between EBUS-TBNA samples and normal samples in NSCLC?. I only found Common Molecular Alterations in NSCLC.

5.Are there any direct relationship between Molecular analysis of EBUS-TBNA samples and accurate nodal staging?. Or compare EBUS-TBNA samples and normal samples for accurate nodal staging？

6. Whole review lack of many proof about the Molecular analysis of EBUS-TBNA samples for accurate nodal staging.
7. No figures and no tables in this manuscript.

Author Response

Reply to the Reviewer (1)

 

Manuscript ID:

Title: cancers-4315591

Molecular analysis of EBUS-TBNA samples for nodal staging in non-small cell lung cancer

 

Dear reviewer,

Firstly, I would to like the thank you to initially review my manuscript and allow me to send my revision. I also apricate the time and effort that dedicated to provide feedback on our manuscript.

Please see below, for a point-by-point response to the comments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.This research focused on Molecular analysis of EBUS-TBNA samples for accurate nodal staging in non-small cell lung cancer, after check in pubmed, not so many references about this topic(PMID: 4034525 ）, this was mean this manuscript was with some Innovation.

• Reply: Many thanks for the valuable comment.

2.This was a review manuscript, although not so many articles about this topic especially review article, but some questions should be deal with.

3.Firstly, the title“Molecular analysis of EBUS-TBNA samples for accurate nodal staging in non-small cell lung cancer”I think can be revised, I donot know which was main important content of this manuscript.

• Reply:
  • Thank you very much for your valuable comments. The primary aim of this review is to discuss the molecular aspects of EBUS-TBNA samples in the context of non-small cell lung cancer (NSCLC). Therefore, we focused on the current molecular alterations in NSCLC and the various factors that may influence the adequacy and diagnostic yield of EBUS-TBNA specimens for molecular analysis. In our opinion, the title appropriately reflects the scope and scientific content of the manuscript.

4. Are there any differences of Molecular analysis between EBUS-TBNA samples and normal samples in NSCLC?. I only found Common Molecular Alterations in NSCLC.

• Reply:
  • Thank you for the thoughtful comment highlighting the differences between molecular analyses performed on EBUS-TBNA samples and those obtained from conventional surgical specimens in NSCLC. In Section 4.2, “Concordance of EBUS-TBNA with Surgical Specimens for Molecular Profiling,” we specifically addressed the agreement between EBUS-TBNA and surgical samples regarding molecular testing outcomes. Yasufuku et al. compared EBUS-TBNA samples with mediastinoscopy specimens in a prospective study and demonstrated substantial concordance between both techniques (κ = 0.8; 95% CI, 0.7–0.9). Both methods achieved 100% specificity and positive predictive value. The sensitivity, negative predictive value, and overall diagnostic accuracy were 81%, 91%, and 93% for EBUS-TBNA, compared with 79%, 90%, and 93% for mediastinoscopy, respectively, with no statistically significant difference in pathological N-stage determination (p = 0.78)[1].
  • Regarding molecular profiling, Tsunoda et al. conducted a prospective study evaluating PD-L1 expression and driver mutation analysis using EBUS-TBNA samples. Using a PD-L1 tumor proportion score (TPS) cutoff of ≥50%, the authors reported a 78% concordance rate between EBUS-TBNA and surgically resected specimens, with moderate agreement (Cohen’s κ = 0.45). Although PD-L1 expression in biopsy samples correlated with surgical specimens, the authors emphasized that the clinical accuracy of PD-L1 assessment from small biopsy samples remains limited and should therefore be interpreted cautiously in therapeutic decision-making[2].
  • For driver mutation analysis, the study further demonstrated that comprehensive molecular profiling can be reliably performed using EBUS-TBNA specimens. EGFR mutations (exons 18–21), KRAS mutations (exons 2–3), and ALK rearrangements were detected in 16.9%, 31.6%, and 3.9% of EBUS-TBNA samples, respectively, compared with 14.8%, 29.0%, and 3.4% in the corresponding surgical specimens. Importantly, no statistically significant differences were observed between the two specimen types, supporting the suitability of EBUS-TBNA samples for comprehensive molecular analysis in advanced NSCLC[3].
  • Collectively, these studies demonstrate that EBUS-TBNA specimens provide high concordance with surgical specimens for molecular profiling and represent a reliable and minimally invasive alternative for genomic testing in advanced non-small cell lung cancer.

5.Are there any direct relationship between Molecular analysis of EBUS-TBNA samples and accurate nodal staging?. Or compare EBUS-TBNA samples and normal samples for accurate nodal staging？

• Reply:
  • We fully appreciate the importance of clearly addressing the relationship between adequate molecular analysis of EBUS-TBNA samples and improved nodal staging accuracy in NSCLC.
  • In our view, the association between molecular analysis of EBUS-TBNA specimens and accurate nodal staging can be understood through two complementary points. First, EBUS-TBNA samples demonstrate high diagnostic performance in nodal staging in patients with NSCLC with a sensitivity of 81%, specificity of 100%, and an overall diagnostic accuracy of 93%, comparable to mediastinoscopy (p = 0.78) [1]. Importantly, the same procedure that confirms or excludes nodal metastasis also provides sufficient tissue for comprehensive molecular profiling, including analyses of EGFR, ALK, KRAS, ROS1, and PD-L1 using next-generation sequencing (NGS). Thus, EBUS-TBNA offers the unique advantage of combining anatomical nodal staging and molecular characterization within a single minimally invasive procedure. Second, from a molecular perspective, conventional nodal staging is primarily based on cytomorphological identification of malignant cells, which may fail to detect occult or micrometastatic disease in cytologically negative or indeterminate samples. Molecular analysis of mediastinal lymph node specimens may identify tumor-specific genetic alterations even in the absence of definitive cytological evidence of malignancy, thereby potentially increasing the sensitivity of nodal staging beyond standard morphological assessment alone.
  • To address this important point more clearly, we have added the following paragraph to Section 4.0 immediately after the introductory section and has been highlighted with yellow: The molecular analysis of EBUS-TBNA–derived lymph node specimens may improve nodal staging accuracy through two complementary mechanisms. First, EBUS-TBNA enables simultaneous pathological confirmation of nodal metastasis and comprehensive molecular profiling within a single minimally invasive procedure, thereby integrating anatomical staging with molecular characterization. Second, the detection of tumor-specific oncogenic driver mutations in mediastinal lymph node samples may enhance the sensitivity of nodal staging beyond conventional cytomorphological assessment, particularly in cases of occult or micrometastatic nodal involvement where standard cytological evaluation may be insufficient.

6. Whole review lack of many proof about the Molecular analysis of EBUS-TBNA samples for accurate nodal staging.

• Reply:
  • This relation was discussed across several sections of the review. For example, under section 3.1 Clinical importance of comprehensive mediastinal lymph node staging in early-stage NSCLC, it was demonstrated that inaccurate nodal staging directly impacts survival outcomes[4]. As well in section 3.2.3. Clinical importance of comprehensive mediastinal lymph node staging in locally advanced NSCLC: EBUS-guided sampling upstages approximately 20% of PET-CT–based N0/N1 patients to pathological N2[5],[6]. Additionally, in section 4.0: Role of EBUS-TBNA samples for immunohistochemistry and NGS confirms NGS success rates of 92.8% with 100% concordance with paired surgical specimens[7], [3].

7. No figures and no tables in this manuscript.

• Reply: 2 figures have been added within the manuscript.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References

1. Yasufuku, K.; Pierre, A.; Darling, G.; de Perrot, M.; Waddell, T.; Johnston, M.; da Cunha Santos, G.; Geddie, W.; Boerner, S.; Le, L.W.; et al. A prospective controlled trial of endobronchial ultrasound-guided transbronchial needle aspiration compared with mediastinoscopy for mediastinal lymph node staging of lung cancer. J. Thorac. Cardiovasc. Surg. 2011, 142, 1393–1400.
2. Tsunoda, A.; Nakamura, A.; Motoi, N.; Kodama, T.; Nakamura, H.; Yoneda, K.; Yamamoto, K.; Saiki, M.; Kikuchi, T.; Tochigi, N.; et al. A prospective observational study to assess PD-L1 expression in small biopsy samples for non-small-cell lung cancer. BMC Cancer 2019, 19, 546.
3. Casadio, C.; Guarize, J.; Donghi, S.; Di Tonno, C.; Fumagalli, C.; Morenghi, E.; Dell'Orto, P.; Testori, A.; Leo, F.; Spaggiari, L.; et al. Molecular testing for targeted therapy in advanced non–small cell lung cancer: Suitability of endobronchial ultrasound transbronchial needle aspiration. Am. J. Clin. Pathol. 2015, 144, 629–634.
4. Gagliasso, M.; Migliaretti, G.; Ardissone, F. Assessing the prognostic impact of the International Association for the Study of Lung Cancer proposed definitions of complete, uncertain, and incomplete resection in non-small cell lung cancer surgery. Lung Cancer 2017, 111, 124–130.
5. Steinfort, D.P.; Lim, A.; Antippa, P.; Conron, M.; Kok, P.S.; Wright, G.M.; Jennings, B.; Smallwood, D.; Jennings, G.; Slavin, M.; et al. Systematic endoscopic staging of mediastinum to guide radiotherapy planning in patients with locally advanced non-small-cell lung cancer (SEISMIC): An international, multicentre, single-arm, clinical trial. Lancet Respir. Med. 2024, 12, 467–475.
6. Vial, M.R.; O'Connell, O.J.; Grosu, H.B.; Eapen, G.A.; Hernandez, M.; Liang, J.; Gonzalez, A.V.; Casal, R.F.; Ost, D.E.; Jimenez, C.A.; et al. Diagnostic performance of endobronchial ultrasound-guided mediastinal lymph node sampling in early stage non-small cell lung cancer: A prospective study. Respirology 2018, 23, 76–81.
7. Martin-Deleon, R.; Teixido, C.; Reguart, N.; Vollmer, I.; Marti-Pages, C.; Viladot, M.; Garcia-Pelaez, B.; Domenech, M.; Molins, L.; Feu-Collell, N.; et al. EBUS-TBNA cytological samples for comprehensive molecular testing in non–small cell lung cancer. Cancers 2021, 13, 2084

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-written and timely review that synthesizes the current state of molecular profiling from EBUS-derived samples. The authors successfully highlight the shift from mere cytological diagnosis to comprehensive molecular characterization. The inclusion of modern techniques like EBUS-guided cryobiopsy and AI-assisted evaluation is particularly commendable.

Specific Points for Revision:

1. Optimization of Sample Processing

The review discusses the importance of sample adequacy but could be strengthened by a more detailed section on the "pre-analytical" phase.

Please elaborate on the specific effects of different fixatives (e.g., formalin vs. alcohol-based) on the success rates of Next-Generation Sequencing (NGS) from EBUS-TBNA aspirates. A brief comparison of cell blocks versus smear preparations for PD-L1 testing would also be highly valuable for the reader.

2. The Role of EBUS-Guided Cryobiopsy

The authors introduce EBUS-guided cryobiopsy as a promising tool for obtaining larger tissue volumes.

Given that this is a relatively new technique, the authors should include a summary table comparing the diagnostic yield and complication rates (e.g., bleeding, pneumothorax) of EBUS-cryobiopsy versus conventional EBUS-TBNA based on the most recent clinical trials mentioned.

3. Liquid Biopsy Integration

The review focuses on tissue/cytology samples.

The authors should briefly discuss the "complementary" role of liquid biopsy (ctDNA) in cases where EBUS-TBNA samples are insufficient for full molecular profiling. How should clinicians prioritize these two modalities in the staging workflow?

4. Artificial Intelligence (AI) and Future Perspectives

The section on AI is forward-thinking.

To make this section more concrete, please specify whether the AI tools discussed are currently used for real-time intraoperative decision-making (e.g., selecting the most suspicious node) or for post-hoc pathological analysis.

5. Figures and Tables

While the text is descriptive, the review would benefit significantly from a central "Workflow Algorithm" figure. This should depict the decision-making process starting from an EBUS-detected node to the selection of sampling tool, rapid on-site evaluation (ROSE), and subsequent molecular testing tiers.

Author Response

Reply to the Reviewer (2)

 

Manuscript ID:

Title: cancers-4315591

Molecular analysis of EBUS-TBNA samples for nodal staging in non-small cell lung cancer

 

Dear reviewer,

Firstly, I would to like the thank you to initially review my manuscript and allow me to send my revision. I also apricate the time and effort that dedicated to provide feedback on our manuscript.

Please see below, for a point-by-point response to the comments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- This is a well-written and timely review that synthesizes the current state of molecular profiling from EBUS-derived samples. The authors successfully highlight the shift from mere cytological diagnosis to comprehensive molecular characterization. The inclusion of modern techniques like EBUS-guided cryobiopsy and AI-assisted evaluation is particularly commendable.

• Reply: Thank you for this positive comments.

- Specific Points for Revision:

1. Optimization of Sample Processing:

- The review discusses the importance of sample adequacy but could be strengthened by a more detailed section on the "pre-analytical" phase.

• Reply:
  • We would like to clarify that the pre-analytical phase is already addressed in Section 4.1.2, which discusses in detail the influence of needle gauge selection, number of passes, suction technique, specimen expulsion methods, collection media, and the role of ROSE on sample adequacy and molecular testing success. Additionally, we would like to note that this review was intentionally focused on the molecular analysis of EBUS-TBNA samples. Other aspects of nodal staging will be comprehensively addressed in companion papers within this special issue dedicated to Contemporary Lung Cancer Nodal Staging.

- Please elaborate on the specific effects of different fixatives (e.g., formalin vs. alcohol-based) on the success rates of Next-Generation Sequencing (NGS) from EBUS-TBNA aspirates. A brief comparison of cell blocks versus smear preparations for PD-L1 testing would also be highly valuable for the reader.

• Reply:
  • The following has been added within the manuscript and marked in yellow: Additionally, alcohol-based fixatives have been investigated as an alternative to formalin and paraffin-based fixation methods, which may impair DNA and RNA quality. Several studies have demonstrated that alcohol-based non-crosslinking fixatives provide higher DNA and RNA yields with improved preservation of nucleic acid integrity. Furthermore, these fixatives may enhance the molecular diagnostics by reducing nucleic acid damage and increasing the amount of recoverable genetic material from biopsy specimens.

2. The Role of EBUS-Guided Cryobiopsy

- The authors introduce EBUS-guided cryobiopsy as a promising tool for obtaining larger tissue volumes.

- Given that this is a relatively new technique, the authors should include a summary table comparing the diagnostic yield and complication rates (e.g., bleeding, pneumothorax) of EBUS-cryobiopsy versus conventional EBUS-TBNA based on the most recent clinical trials mentioned.

• Reply:
  • Many thanks for the valuable comments. It is difficult to integrate a table summarizing the comparison between conventional EBUS-TBNA and EBUS-TMC, as several meta-analyses published in recent years have already confirmed the safety and superior diagnostic yield of EBUS-TMC. However, the following text has been added to the manuscript and marked in yellow: A pooled analysis of seven studies including 555 patients reported an overall diagnostic yield of 92% for EBUS-TMC compared with 80% for EBUS-TBNA, mostly in patients with lymphoma and benign mediastinal lesions. Additionally, in 97% of EBUS-TMC samples, genetic and immunohistochemical analyses, including PD-L1 testing, were feasible, highlighting the particular value of EBUS-TMC for molecular profiling of mediastinal samples[1]. Another meta-analysis including 538 patients across 10 studies demonstrated a pooled overall diagnostic yield of 89.59% for EBUS-TMC versus 77.13% for EBUS-TBNA, with a statistically significant advantage of EBUS-TMC in lymphoma and benign disorders. The safety profile of EBUS-TMC was also confirmed. Clinically significant bleeding occurred in 1.12% and pneumothorax in 0.74% of patients, with no procedure-related mortality reported[2]. A further meta-analysis including 20 studies demonstrated significantly higher diagnostic efficacy for EBUS-TMC compared with EBUS-TBNA (RD 0.30; 95% CI: 0.17–0.44; p<0.001). Regarding the complications, there was no significant differences in pneumothorax or bleeding risk between the two techniques[3].

3. Liquid Biopsy Integration

- The review focuses on tissue/cytology samples.

- The authors should briefly discuss the "complementary" role of liquid biopsy (ctDNA) in cases where EBUS-TBNA samples are insufficient for full molecular profiling. How should clinicians prioritize these two modalities in the staging workflow?

• Reply:
  • Thank you for this highly relevant suggestion. We would like to clarify, however, that this review was prepared as an invited contribution to a special issue dedicated to Contemporary Lung Cancer Nodal Staging. The scope of the manuscript was therefore intentionally focused on tissue-based molecular analysis of EBUS-TBNA samples, and a comprehensive discussion of liquid biopsy and ctDNA — while undoubtedly clinically important — falls beyond the defined scope of this contribution.

4. Artificial Intelligence (AI) and Future Perspectives

- The section on AI is forward-thinking.

- To make this section more concrete, please specify whether the AI tools discussed are currently used for real-time intraoperative decision-making (e.g., selecting the most suspicious node) or for post-hoc pathological analysis.

• Reply
  • Thank you for this valuable observation. To the best of our knowledge, the AI tools discussed in this section — including the deep learning models are currently represent investigational prototypes that have not yet received clinical validation, regulatory approval or achieved widespread commercial availability. Consequently, none of these tools has been formally established for routine real-time intraoperative decision-making during EBUS-TBNA procedures in clinical practice. This is precisely why we have presented this content within the context of future directions and emerging perspectives in the field, rather than as current standard-of-care applications.

5. Figures and Tables

While the text is descriptive, the review would benefit significantly from a central "Workflow Algorithm" figure. This should depict the decision-making process starting from an EBUS-detected node to the selection of sampling tool, rapid on-site evaluation (ROSE), and subsequent molecular testing tiers.

• Reply: 2 figures have been added within the manuscript.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References

1. Botana-Rial, M.; Núñez-Delgado, M.; Leiro-Fernández, V.; Álvarez-Kindelán, A.; Tilve-Gómez, A.; Fernández-Villar, A. Is the diagnostic yield of mediastinal lymph node cryobiopsy (cryoEBUS) better for diagnosing mediastinal node involvement compared to endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)? A systematic review. Respir. Med. 2023, 218, 107389.
2. Zhang, Z.; Li, S.; Bao, Y. Endobronchial ultrasound-guided transbronchial mediastinal cryobiopsy versus endobronchial ultrasound-guided transbronchial needle aspiration for mediastinal disorders: A meta-analysis. Respiration 2024, 103, 359–367.
3. Gherman, C.D.; Mocan, T.; Mocan, L.; Lebovici, A.; Cismaru, G.; Bota, M.; Mitre, R. Comparative efficacy of transbronchial needle aspiration and cryobiopsies in thoracic disorders: A systematic review and meta-analysis. Life 2026, 16, 768.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

• Mismatch between the title and manuscript content: The title emphasizes the role of EBUS-TBNA in improving nodal staging accuracy; however, Section 2 is largely devoted to a broad overview of molecular alterations in NSCLC that substantially overlaps with existing reviews. The authors should either revise the title to reflect the actual scope or condense Section 2 into a brief summary within the Introduction.
• Insufficient comparative analysis of EBUS-TMC versus EBUS-TBNA: Although Section 4.3 introduces EBUS-TMC as a complementary technique, the comparison with EBUS-TBNA regarding NGS success rates, tumor cellularity, and safety profile remains fragmentary. A more systematic and balanced comparison of these two modalities is warranted.
• Lack of discussion on clinical outcomes: Section 4 adequately describes the technical feasibility of molecular analysis using EBUS-TBNA specimens but lacks discussion on how these results influenced actual treatment decisions and patient outcomes. Incorporating evidence on response rates or survival data in patients treated based on EBUS-TBNA-derived molecular findings would substantially strengthen the clinical relevance of this review.
• Insufficient critical appraisal of cited studies: Many cited studies are retrospective and single-center analyses. The manuscript lacks critical discussion of their methodological limitations and potential biases, and the authors should appropriately qualify conclusions where the supporting evidence remains limited or heterogeneous.
• Underdeveloped Future Directions section: The discussion of AI (Section 5.2) remains largely descriptive without adequately exploring how AI could specifically improve the adequacy of EBUS-TBNA samples for molecular analysis. The authors should also consider discussing emerging technologies such as liquid biopsy and ctDNA analysis as potential adjuncts to EBUS-TBNA-based molecular profiling.

Author Response

Reply to the Reviewer (3)

 

Manuscript ID:

Title: cancers-4315591

Molecular analysis of EBUS-TBNA samples for nodal staging in non-small cell lung cancer

 

Dear reviewer,

Firstly, I would to like the thank you to initially review my manuscript and allow me to send my revision. I also apricate the time and effort that dedicated to provide feedback on our manuscript.

Please see below, for a point-by-point response to the comments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- Mismatch between the title and manuscript content: The title emphasizes the role of EBUS-TBNA in improving nodal staging accuracy; however, Section 2 is largely devoted to a broad overview of molecular alterations in NSCLC that substantially overlaps with existing reviews. The authors should either revise the title to reflect the actual scope or condense Section 2 into a brief summary within the Introduction.

• Reply
  • Thank you for this valuable observation. The primary aim of this review is to discuss the importance and current status of molecular testing for oncogenic driver mutations in EBUS-TBNA specimens in NSCLC. The relationship between molecular analysis and nodal staging is addressed through two complementary mechanisms throughout the manuscript: first, EBUS-TBNA simultaneously confirms nodal metastasis and provides sufficient material for comprehensive molecular profiling — including EGFR, ALK, KRAS, ROS1, and PD-L1 via NGS — within a single minimally invasive procedure [1]; and second, molecular analysis of mediastinal lymph node specimens may identify tumor-specific genetic alterations even in cytologically negative samples, potentially enhancing staging sensitivity beyond standard morphological assessment alone (Sections 3.1, 3.2, and 4.2).
  • Regarding Section 2, the overview of molecular alterations in NSCLC was intentionally included to provide the scientific foundation necessary for appreciating the clinical relevance of molecular profiling from EBUS-TBNA specimens discussed in subsequent sections. Nevertheless, in response to this comment, the word "accurate" has been removed from the title to better reflect the manuscript's actual scope.

- Insufficient comparative analysis of EBUS-TMC versus EBUS-TBNA: Although Section 4.3 introduces EBUS-TMC as a complementary technique, the comparison with EBUS-TBNA regarding NGS success rates, tumor cellularity, and safety profile remains fragmentary. A more systematic and balanced comparison of these two modalities is warranted.

• Reply:
  • The following text has been added to the manuscript and marked in yellow: A pooled analysis of seven studies including 555 patients reported an overall diagnostic yield of 92% for EBUS-TMC compared with 80% for EBUS-TBNA, mostly in patients with lymphoma and benign mediastinal lesions. Additionally, in 97% of EBUS-TMC samples, genetic and immunohistochemical analyses, including PD-L1 testing, were feasible, highlighting the particular value of EBUS-TMC for molecular profiling of mediastinal samples[1].Another meta-analysis including 538 patients across 10 studies demonstrated a pooled overall diagnostic yield of 89.59% for EBUS-TMC versus 77.13% for EBUS-TBNA, with a statistically significant advantage of EBUS-TMC in lymphoma and benign disorders. The safety profile of EBUS-TMC was also confirmed. Clinically significant bleeding occurred in 1.12% and pneumothorax in 0.74% of patients, with no procedure-related mortality reported[2]. A further meta-analysis including 20 studies demonstrated significantly higher diagnostic efficacy for EBUS-TMC compared with EBUS-TBNA (RD 0.30; 95% CI: 0.17–0.44; p<0.001). Regarding the complications, there was no significant differences in pneumothorax or bleeding risk between the two techniques[3].

- Lack of discussion on clinical outcomes: Section 4 adequately describes the technical feasibility of molecular analysis using EBUS-TBNA specimens but lacks discussion on how these results influenced actual treatment decisions and patient outcomes. Incorporating evidence on response rates or survival data in patients treated based on EBUS-TBNA-derived molecular findings would substantially strengthen the clinical relevance of this review.

• Reply
  • We fully appreciate the clinical importance of linking EBUS-TBNA-derived molecular findings to treatment decisions and patient outcomes. However, we would like to clarify that this review was prepared as an invited contribution to a special issue of Cancers dedicated to molecular nodal staging in lung cancer. In accordance with the defined scope of our contribution, this manuscript was intentionally focused on the diagnostic and molecular aspects of EBUS-TBNA specimens, specifically addressing the feasibility, adequacy, and factors influencing molecular profiling from nodal samples. The clinical implications of molecularly guided treatment decisions — including response rates, survival outcomes, and therapeutic algorithms are being addressed another reviews within the same special issue. We therefore respectfully maintain the current diagnostic focus of the manuscript to preserve the coherence and complementarity of the special issue as a whole.

- Insufficient critical appraisal of cited studies: Many cited studies are retrospective and single-center analyses. The manuscript lacks critical discussion of their methodological limitations and potential biases, and the authors should appropriately qualify conclusions where the supporting evidence remains limited or heterogeneous.

• Reply
  • We fully acknowledge that a proportion of the cited studies are retrospective or single-center investigations, which may limit the generalizability of the findings. However, the primary aim of this review was to evaluate and discuss the influence and clinical relevance of EBUS-TBNA in current practice. In addition, several prospective studies were also included in the analysis. Following the reviewers’ valuable suggestions, we further revised the manuscript to incorporate and discuss relevant meta-analyses and additional high-quality evidence in order to provide a more comprehensive and balanced overview of the topic.

- Underdeveloped Future Directions section: The discussion of AI (Section 5.2) remains largely descriptive without adequately exploring how AI could specifically improve the adequacy of EBUS-TBNA samples for molecular analysis. The authors should also consider discussing emerging technologies such as liquid biopsy and ctDNA analysis as potential adjuncts to EBUS-TBNA-based molecular profiling.

• Reply:
  • Thanks for the comment and observation. In the section of future direction, we aimed to highlight the current experminatal technology in the EBUS examination of the LNs. It worth to mention that to the best of our knowledge, the AI tools which described in this section are still learning models which are not fully validated or widespread commercially aviable, they should be considered as investigational prototypes that remain in need for further investignal studies.
  • Regarding liquid biopsy and ctDNA analysis, we would like to clarify, however, that this review was prepared as an invited contribution to a special issue dedicated to Contemporary Lung Cancer Nodal Staging. The scope of the manuscript was therefore intentionally focused on tissue-based molecular analysis of EBUS-TBNA samples, and a comprehensive discussion of liquid biopsy and ctDNA — while undoubtedly clinically important — falls beyond the defined scope of this contribution.

References

1. Botana-Rial, M.; Núñez-Delgado, M.; Leiro-Fernández, V.; Álvarez-Kindelán, A.; Tilve-Gómez, A.; Fernández-Villar, A. Is the diagnostic yield of mediastinal lymph node cryobiopsy (cryoEBUS) better for diagnosing mediastinal node involvement compared to endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)? A systematic review. Respir. Med. 2023, 218, 107389.
2. Zhang, Z.; Li, S.; Bao, Y. Endobronchial ultrasound-guided transbronchial mediastinal cryobiopsy versus endobronchial ultrasound-guided transbronchial needle aspiration for mediastinal disorders: A meta-analysis. Respiration 2024, 103, 359–367.
3. Gherman, C.D.; Mocan, T.; Mocan, L.; Lebovici, A.; Cismaru, G.; Bota, M.; Mitre, R. Comparative efficacy of transbronchial needle aspiration and cryobiopsies in thoracic disorders: A systematic review and meta-analysis. Life 2026, 16, 768.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have deal with all my concerns，now I think now is suitable for publication in this journal.