Review Reports - The Dynamic Role of Liquid Biopsy in Patients with Non-Small Cell Lung Cancer Receiving Immune Checkpoint Inhibitors: Emerging Opportunities and Clinical Challenges

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the study entitled “The Dynamic Role of Liquid Biopsy in Patients with Non-Small Cell Lung Cancer Receiving Immune Checkpoint Inhibitors: Emerging Opportunities and Clinical Challenges.” Authors discuss Immune checkpoint inhibitors (ICIs) have transformed the treatment of non-small-cell lung cancer (NSCLC), but primary and acquired resistance, heterogeneous benefit, and atypical response patterns continue to limit precision treatment. Tissue biomarkers, including PD-L1 expression and tumor mutational burden, provide clinically useful yet static information and incompletely capture spatial heterogeneity and treatment-driven tumor–immune evolution. Liquid biopsy enables minimally invasive, longitudinal assessment of circulating tumor DNA (ctDNA) and other blood-based analytes, offering a dynamic view of tumor burden, molecular response, residual disease, and emerging resistance. Across respectable, locally advanced, and metastatic NSCLC, liquid biopsy is emerging as a complementary strategy for immunotherapy stratification and longitudinal disease monitoring. ctDNA clearance consistently correlates with pathological response and favorable survival, whereas postoperative molecular residual disease and persistent or rising ctDNA identify patients at high risk of recurrence or progression, often before radiological detection. Beyond mutation-based assays, methylation, fragmentomics, blood-based tumor mutational burden, plasma proteomics, extracellular vesicles, and immune-cell profiling may improve sensitivity and characterize tumor-host interactions. However, low tumor shedding, clonal hematopoiesis, pre-analytical variability, assay heterogeneity, and the absence of validated intervention thresholds constrain routine implementation. Prospective trials must determine whether biomarker-guided escalation, de-escalation, or treatment discontinuation improves clinical outcomes and enables patient-centered decision-making.. However, there are certain pitfalls/comments which need to be addressed before acceptance of this manuscript.

One of the major things that come over and over again in this review is poor references/poor citations. Authors have not cited appropriately information put in the review.

To ensure transparency and accountability I would like each of its authors to provide Author Contributions and Contribution statements (per author)
Please specify each author's topical and intellectual contributions to the paper (avoid generic wording).
Include a statement of the professional relationship between authors.
Lead Author:
What did their conceptual leadership role entail?
In addition, could you ensure your abstract highlights the specific perspective, assessment, or research synthesis your work offers? It must include a statement that outlines the contribution your article makes to the field. This summary should focus on the review's timeliness and impact, communicating its importance to the general audience.
Appropriate references are missing like end of paragraphs (line 210, 220, 230 etc)…
Some more figures could be added in the review to make it more attractive by summarizing the roadmap of each setting.
Table needs to be revised. Instead of classifying it as per authors. Authors should classify it as per Trials data and should include more data in it.
Topic 5 should also be explained in the form of figure.

Author Response

Reviewer 1

In the study entitled “The Dynamic Role of Liquid Biopsy in Patients with Non-Small Cell Lung Cancer Receiving Immune Checkpoint Inhibitors: Emerging Opportunities and Clinical Challenges.” Authors discuss Immune checkpoint inhibitors (ICIs) have transformed the treatment of non-small-cell lung cancer (NSCLC), but primary and acquired resistance, heterogeneous benefit, and atypical response patterns continue to limit precision treatment. Tissue biomarkers, including PD-L1 expression and tumor mutational burden, provide clinically useful yet static information and incompletely capture spatial heterogeneity and treatment-driven tumor–immune evolution. Liquid biopsy enables minimally invasive, longitudinal assessment of circulating tumor DNA (ctDNA) and other blood-based analytes, offering a dynamic view of tumor burden, molecular response, residual disease, and emerging resistance. Across respectable, locally advanced, and metastatic NSCLC, liquid biopsy is emerging as a complementary strategy for immunotherapy stratification and longitudinal disease monitoring. ctDNA clearance consistently correlates with pathological response and favorable survival, whereas postoperative molecular residual disease and persistent or rising ctDNA identify patients at high risk of recurrence or progression, often before radiological detection. Beyond mutation-based assays, methylation, fragmentomics, blood-based tumor mutational burden, plasma proteomics, extracellular vesicles, and immune-cell profiling may improve sensitivity and characterize tumor-host interactions. However, low tumor shedding, clonal hematopoiesis, pre-analytical variability, assay heterogeneity, and the absence of validated intervention thresholds constrain routine implementation. Prospective trials must determine whether biomarker-guided escalation, de-escalation, or treatment discontinuation improves clinical outcomes and enables patient-centered decision-making. However, there are certain pitfalls/comments which need to be addressed before acceptance of this manuscript.

One of the major things that come over and over again in this review is poor references/poor citations. Authors have not cited appropriately information put in the review.

To ensure transparency and accountability I would like each of its authors to provide Author Contributions and Contribution statements (per author)

Response:
Thank you for this comment. We have revised the Authors’ contributions section to ensure that an individual contribution statement is provided for each author, clearly specifying their respective roles in the conception, design, literature acquisition and analysis, interpretation, writing, and critical revision of the manuscript.

Please specify each author's topical and intellectual contributions to the paper (avoid generic wording).

Response:
Thank you for this suggestion. We have further expanded the Authors’ contributions section by specifying the main topical and intellectual areas to which each author contributed, including the scientific conception of the review, liquid biopsy and ctDNA-based monitoring, molecular diagnostics, emerging multi-omics biomarkers, and the clinical and translational implications of immunotherapy biomarkers in NSCLC. Generic descriptions were avoided wherever possible.

Include a statement of the professional relationship between authors.

Response:
Thank you for this comment. We have added a statement clarifying the professional relationship among the authors. The authors are clinicians and researchers working in the fields of medical oncology, molecular pathology, and translational oncology at the University of Palermo and the University Federico II of Naples. Their collaboration on this review reflects complementary clinical, molecular, pathological, and translational expertise in NSCLC, immunotherapy, and liquid biopsy.

Lead Author:

Response:
Tancredi Didier Bazan Russo (T.D.B.R.) served as the lead author of the manuscript, with Valerio Gristina (V.G.) contributing equally, as indicated by the equal-contribution designation in the manuscript.

What did their conceptual leadership role entail?

Response:
T.D.B.R.’s conceptual leadership included defining the overall scope and scientific objectives of the review, developing its conceptual framework and structure, coordinating the integration of the different sections, synthesizing the evidence on liquid biopsy and immunotherapy across the different stages of NSCLC, and leading the drafting and subsequent revision of the manuscript. V.G., as co-first author, contributed to the conceptual development, study design, interpretation of the literature, and critical refinement of the manuscript.

In addition, could you ensure your abstract highlights the specific perspective, assessment, or research synthesis your work offers? It must include a statement that outlines the contribution your article makes to the field. This summary should focus on the review's timeliness and impact, communicating its importance to the general audience.

Response:

We thank the reviewer for this helpful suggestion. We have revised the abstract to explicitly state the review’s perspective and contribution: integrating evidence on longitudinal ctDNA monitoring and emerging multi-omics approaches across NSCLC disease stages, while distinguishing prognostic associations from evidence that biomarker-guided treatment improves patient outcomes. We also emphasize the review’s timeliness in the context of expanding perioperative immunotherapy and growing interest in biomarker-guided treatment adaptation. Finally, we have clarified its relevance to a broader audience by highlighting the potential to identify ineffective treatment earlier and reduce unnecessary treatment exposure, while emphasizing that these benefits require prospective validation.

Appropriate references are missing like end of paragraphs (line 210, 220, 230 etc)…

Response:
Thank you for this comment. Appropriate references are already provided at the end of the corresponding paragraphs, specifically at lines 204, 213, and 223. We have carefully rechecked these sections to ensure that the cited references appropriately support the statements made.

Some more figures could be added in the review to make it more attractive by summarizing the roadmap of each setting.

Response:

We thank the reviewer for this helpful suggestion. We have included figures summarizing complementary aspects of liquid biopsy in NSCLC immunotherapy. Figure 1 illustrates longitudinal ctDNA monitoring alongside conventional clinicopathological assessment in the perioperative setting. The newly added Figure 2 depicts the integration of blood-based molecular profiling with imaging, tissue biomarkers, and clinical data, providing a visual overview of the approaches discussed in the review.

Table needs to be revised. Instead of classifying it as per authors. Authors should classify it as per Trials data and should include more data in it.

Response

We thank the reviewer for this suggestion. We have revised the tables according to your suggestions

Topic 5 should also be explained in the form of figure.

Response:

We agree and have added Figure 2 to illustrate Section 5. The figure summarizes plasma proteomics, cfDNA methylation, fragmentomics, and blood-based tumor mutational burden, alongside ctDNA genotyping and kinetics. It shows how these complementary measurements may be integrated with other clinical modalities to support patient stratification, response monitoring, and assessment of emerging resistance.

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks to the authors for this well written and comprehensive review. I have some minor suggestions as follows:

I find that ‘ctDNA’ is used throughout as a uniform nomenclature, despite it encompassing many different potential applications such as tumour-informed MRD assays, tumour agnostic fixed panel sequencing, assays, single-variant approaches, broad genomic profiling etc. I think the review would be improved by a section at the beginning- maybe an additional section 2.3- describing the differing assays, their main application, strengths and weaknesses. Perhaps a table showing the same. This would help the reader interpret what is then later presented by defining the exact type of ‘ctDNA’ test being applied in a particular context and discussed. This would improve the review as assay heterogeneity is one of the main things which people struggle with in terms of their understanding of the area and often the reason ctDNA findings cannot be generalised across studies.

Page 8, line 340. Presence of KRAS G12C along with abnormalities in STK11/ KEAP3 presented with significantly higher sensitivity to ICIs.

I think this is factually incorrect, and the references seem truncated or not supportive. My understanding is that this is a negative cluster of prognostic markers, including with ICI. Please check this and check references.

Table 1 is a fairly impenetrable list at the moment. It could be improved by splitting into sections and to try and make clearer the intended use of the assay. For example split it into disease setting (perioperative, adjuvant, advanced etc). Then have type of assay used (as per suggested additional section 2.3 above), study aim, main findings. It needs to be presented in a way which makes it more accessible.

Goes from section 5.2 to 5.4

Conclusion should state that no ctDNA assay is currently validated for routine selection, escalation or discontinuation of ICI therapy in lung cancer as yet.

Reference 74 seems incorrect, and some have truncated titles eg 31, 36. Need checked

Author Response

Thanks to the authors for this well written and comprehensive review. I have some minor suggestions as follows:

Response:

We would really thank the reviewer for the king suggestion. We have modified the latest version of the manuscript accordingly:

“Liquid biopsy became cornerstone guiding treatment decision making procedures dynamically tracking longitudinal evolution of molecular profile of tumor patients. Cap-turing traces of ctDNA across time points LBx can early prevent recurrence in surgically resected patients or identify high tumor burden in perioperative setting electing patients to adjuvant therapy. In this scenario, ctDNA detection poses basis for minimal residual dis-ease (MRD) evaluation supporting dynamic assessment of LBx samples. NGS platforms play a key role in detecting ctDNA in bloodstream. Heterogeneous NGS strategies are cur-rently available to identify ctDNA traces across clinical points. It has been seen that NGS systems investigating MRD can be classified in tumor informed and tumor agnostic de-pending on the need of tumor genotype. In detail, tumor informed strategies are built tar-geting tumor private alterations previously detected on paired tissue samples whereas tumor agnostic approaches include comprehensive genome profile assays (CGP), mainly WES or WGS, track tumor signatures. Of note, tumor informed approaches demonstrated higher analytical sensitivity than agnostic procedures but are affected by scant reference range, longer turnaround time (TAT) and consistent technical costs (requiring paired tis-sue testing). Conversely, tumor agnostic procedures showed lower sensitivity, TAT and technical costs than informed strategies. “

Page 8, line 340. Presence of KRAS G12C along with abnormalities in STK11/ KEAP3 presented with significantly higher sensitivity to ICIs.

Response:

We thank the reviewer for identifying this error. We agree that the original statement suggesting increased sensitivity to ICIs in KRAS G12C tumors with STK11/KEAP1 co-alterations was inaccurate. We have removed this statement and revised the paragraph to describe the association of STK11/LKB1 co-mutations with reduced response and shorter survival following PD-1 blockade in KRAS-mutant lung adenocarcinoma. We also explicitly distinguish the adverse prognostic significance of STK11 and KEAP1 alterations across treatment classes from an ICI-specific predictive effect.

Response:

We thank the reviewer for this constructive suggestion. We have reorganized the original table into Table 1A and Table 1B, distinguishing tissue-based and liquid biopsy studies, and moved studies of emerging multi-omic biomarkers to Table 2. Dedicated columns now specify the disease setting and study population, investigated biomarkers, study aim, assay, and technical parameters. This structure clarifies the intended application of each assay and facilitates comparison across studies, complementing the technical overview added in Section 2.3.

Goes from section 5.2 to 5.4

Response:

We have also corrected the subsection numbering: the former Section 5.4 is now Section 5.3.

Conclusion should state that no ctDNA assay is currently validated for routine selection, escalation or discontinuation of ICI therapy in lung cancer as yet.

Response:

We would really thank the reviewer for the king suggestion. We have modified the latest version of the manuscript accordingly:

“Despite recent advances, ctDNA analysis guiding ICIs schemes in perioperative NSCLC patients is not codified in clinical setting”

Reference 74 seems incorrect, and some have truncated titles eg 31, 36. Need checked

Response:

We thank the reviewer for identifying these issues. We have corrected the reference previously numbered 74 and restored the truncated titles, including those of references 31 and 36. We have also checked the reference list for accuracy and completeness and updated the corresponding in-text citations.

Reviewer 3 Report

Comments and Suggestions for Authors

Please split Table 1 to show tissue biopsies and liquid biopsies separately

Please compare tissue and liquid biopsies in terms of prediction accuracy.

Please present the contents of sections 5.1, 5.2, and 5.4 (there is no section 5.3) in a Table.

The definition of liquid biopsy is provided at the beginning of section 4. Please move to the beginning of the Introduction.

Please provide a reference for the paragraph: “Notably, approximately 62% of patients who were ctDNA-positive after surgery achieved molecular clearance following adjuvant chemotherapy, which was associated with substantially improved outcomes, while integration of ctDNA status with PD-L1 expression further refined postoperative prognostic stratification.”

The abbreviations should be presented in full the first time they appear in the text (KEAP1 and KMT2C).

Please use bullet points to indicate the challenges and future directions.

Please avoid repetitions.

Author Response

Please split Table 1 to show tissue biopsies and liquid biopsies separately

Response:

We thank the reviewer for this suggestion. We have divided Table 1 into two parts, presenting studies involving tissue biopsies in Table 1A and those involving liquid biopsies in Table 1B, to clarify the distinction between the two approaches.

Please compare tissue and liquid biopsies in terms of prediction accuracy.

Response:

We would really thank the reviewer for the king suggestion. We have modified the latest version of the manuscript accordingly:

“Tissue samples remain gold standard for molecular profiling of predictive biomarkers, but spatial heterogeneity and preanalytical issues significantly impact the successful rate of molecular in advanced NSCLC patients. LBx samples have proven consistent results detecting actionable alterations when tissue samples are unavailable for genomic profiling. In detail, LBx highlighted positive predictive value of 99.0% identifying molecular alterations in torrent blood but analytical negative predictive value spans from 30.0 to 90.0% depending on tumor burden, clinical framework, heterogeneity in ctDNA shedding.”

Please present the contents of sections 5.1, 5.2, and 5.4 (there is no section 5.3) in a Table.

Response:

We thank the reviewer for this suggestion. We have added Table 2 to summarize the studies discussed in Sections 5.1, 5.2, and the former Section 5.4, covering plasma proteomics, methylation and fragmentomics, and blood-based tumor mutational burden. The table outlines the study populations, biomarkers, objectives, assays, and technical parameters. We have also corrected the subsection numbering, renumbering Section 5.4 as Section 5.3.

The definition of liquid biopsy is provided at the beginning of section 4. Please move to the beginning of the Introduction.

Response:

We would really thank the reviewer for the king suggestion. We have modified the latest version of the manuscript accordingly:

Response

We thank the reviewer for highlighting this citation discrepancy. We have corrected the citation and specified the evaluable population and denominator. We have corrected the citation and specified the evaluable population and denominator.

The abbreviations should be presented in full the first time they appear in the text (KEAP1 and KMT2C).

Response

We thank the reviewer for this comment. We have revised the manuscript to define KEAP1 (Kelch-like ECH-associated protein 1) and KMT2C (lysine methyltransferase 2C) in full at their first occurrence in the text.

Please use bullet points to indicate the challenges and future directions.

Response

We thank the reviewer for this helpful suggestion. To improve readability, we have reorganized Section 6, “Current Challenges and Future Directions for Clinical Integration,” into four thematic subsections addressing biological and analytical limitations, standardization and clinical implementation, clinical interpretation, and future directions and prospective validation. We have used short narrative subsections rather than bullet points to preserve the flow of the discussion and maintain the connections between the supporting evidence, its limitations, and the implications for clinical practice.

Please avoid repetitions.

Response

We thank the reviewer for this suggestion. We have revised the manuscript to reduce repetition

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accepted