Clinical Utility of ctDNA Analysis in Lung Cancer—A Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a review article aiming to discuss the utility of circulating DNA in lung cancer.
The authors talk about lund cancer in general in their introduction and they attempt to enter the topic of circulating DNA and explain its usefulness in diagnosing and treating lung cancer.

There are a few issues in the manuscript:
- Language is quite simplistic and the topic is not fully covered.
- There are references implying that lung cancer "biopses" may not provide adequate samples. However, techniques susc as endobronchial ultrasound (EBUS) and endooesophageal ultrasound (EUS-B) can provide samples at almost all cases of suspected lung cancer and even other metastatic cancers.
- Although the manuscript aims to talk about lung cancer, there are general general comments that are not really relevant (lines 99-110). It seems unreasonable to talk about urine and cerebrospinal fluid to diagnose lung cancer!
- At another point you refer to "ongoing studies with >10.000 patients tested for lung cancer with circulating DNA. The reference do not seem to support your statements.
- The last paragraph in section 5 seems to follow current literature.

Author Response

Dear Reviewer,

Thank you very much for your valuable comments and suggestions. We have carefully considered your feedback and revised the manuscript accordingly. We provide answers for your comments below:

1. Language is quite simplistic and the topic is not fully covered.
   We tried to improve language in the manuscript, however our goal was to make the article understandable both to a specialized audience and to readers outside this narrow field. We tried to avoid overusing technical jargon and aimed instead to describe the phenomena as precisely as possible while maintaining clarity and simplicity of language.
2. There are references implying that lung cancer "biopses" may not provide adequate samples. However, techniques susc as endobronchial ultrasound (EBUS) and endooesophageal ultrasound (EUS-B) can provide samples at almost all cases of suspected lung cancer and even other metastatic cancers.
   The diagnostic evaluation of thoracic lesions involves procedures with different level of invasiveness, including bronchoscopy, endobronchial ultrasound (EBUS), endoscopic ultrasound (EUS), transthoracic fine-needle or core-needle biopsy, and thoracic surgical interventions. The choice of diagnostic technique primarily depends on the location of the lesion suspected of malignancy. With the advancement of therapeutic options for lung cancer, there is an increasing need to obtain larger and more representative diagnostic samples that allow not only for histopathological subtyping but also for molecular and genetic testing, including next-generation sequencing (NGS). The quality of specimens obtained via EBUS-TBNA or combined EBUS and EUS-B-FNA procedures is influenced by multiple factors, such as needle gauge, the number of needle passes, quality of patient sedation, operator experience etc.. Although the diagnostic yield of EBUS for lung cancer staging reaches up to 93%, it does not guarantee that the obtained sample will be sufficient for additional molecular analyses. https://pubmed.ncbi.nlm.nih.gov/19124238/
   Noteworthy findings were reported in a meta-analysis that assessed the overall adequacy of EBUS-derived samples for next-generation sequencing (NGS), which was estimated at 86.5%. The study highlighted that the proportion of adequate samples increases with the number of needle passes through the target lesion. Adequacy rates were reported as 77.3%, 86.2%, 91.6%, and 94.9% for an average of 3, 4, 5, and 6 needle passes, respectively. https://pubmed.ncbi.nlm.nih.gov/35151114/
   As mentioned in sections 2 and 5, tissue biopsy remains the standard diagnostic method. However, due to the high heterogeneity within the tumor and between the primary lesion and metastatic sites tissue biopsy may fail to capture relevant genetic drivers. Therefore, liquid biopsy serves as a complementary tool to standard diagnostics, enabling faster and more comprehensive identification of genetic alterations associated with the malignancy, particularly in cases where initial tissue biopsy attempts were unsuccessful.
3. Although the manuscript aims to talk about lung cancer, there are general general comments that are not really relevant (lines 99-110). It seems unreasonable to talk about urine and cerebrospinal fluid to diagnose lung cancer!
   Thank you very much for this comment. We simplified lines 99–110 by omitting unnecessary details to improve readability. The key facts regarding ctDNA collection for liquid biopsy were preserved.
4. At another point you refer to "ongoing studies with >10.000 patients tested for lung cancer with circulating DNA. The reference do not seem to support your statements.
   We are grateful for this comment. It allowed us to re-evaluate the manuscript and make improvements. We revised section 4, so that it compares two differing viewpoints presented in the literature concerning the application of ctDNA in cancer screening. The reference to an ongoing study was removed.
5. The last paragraph in section 5 seems to follow current literature.

We appreciate your observation that the last paragraph of section 5 reflects the current literature. We aimed to both highlight the novel aspects of ctDNA and to shortly described existing knowledge for readers who are not specialized in this field. 

 

 

We greatly appreciate your time and effort in reviewing our work.

Sincerely,

Agata Wróbel
on behalf of all co-authors

Reviewer 2 Report

Comments and Suggestions for Authors

Kamil Makar, Agata Wróbel, Adam Antczak, and Damian Tworek analyzed the areas of application of circulating tumor DNA detection in lung cancer. First, the authors defined cell free circulating DNA and circulating tumor DNA, then briefly described the methods for their detection. Finally, the authors described the cases in which ctDNA analysis is already used and in which cases it can be used.

In many countries around the world, the use of ctDNA for determining the molecular subtype of lung cancer has already become common practice, an approximate scheme of which the authors provided in Figure 2. Determination of the molecular subtype of cancer is used to prescribe optimal targeted therapy and has a number of advantages over standard biopsy, such as low invasiveness and the ability to track tumor heterogeneity.

Then the authors described other potential areas of application of ctDNA detection, the most promising of which seems to be monitoring minimal residual disease. The authors provide descriptions of numerous studies, both original articles and meta-analyses and multicenter clinical trials, which have shown with high reliability the association of the presence of ctDNA with the duration of relapse-free survival and overall survival in patients. In addition, the authors discussed the possibility of using ctDNA as a dynamic marker for monitoring the response to therapy. Although the authors mainly considered the response to drug therapy, one can find publications describing the use of ctDNA for monitoring the response to radiotherapy (e.g. doi: 10.1016/j.semradonc.2015.05.001). In Table 1, the authors provide a list of studies devoted to the potential clinical use of ctDNA and conducted from 2020 to 2025.

The authors also point out the need to harmonize preanalytical stages and methods for detecting ctDNA in laboratories to obtain standardized results that will allow more accurate clinical recommendations for treating patients with lung cancer.

 

Author Response

Dear Reviewer,

Thank you very much for your valuable comments and suggestions. We have carefully considered your feedback and revised the manuscript to better reflect what we intended to express.

Our goal in preparing this manuscript was to make the article understandable both to a specialized audience and to readers outside this narrow field. We aimed to cover as many relevant studies as possible to present the current state of knowledge and research in the best possible way. We also made an effort to avoid overusing technical jargon, focusing instead on describing the phenomena as precisely as possible while maintaining clarity and simplicity of language.

We greatly appreciate your time and effort in reviewing our work.

Sincerely,
Agata Wróbel on behalf of all co-authors

Reviewer 3 Report

Comments and Suggestions for Authors

First of all, I would like to congratulate the authors on the quality of the manuscript. The paper is well written, clear, and does not require any English language editing. While the topic is not entirely novel, it remains highly relevant in the field of lung cancer, and further clinical trials are needed to clarify the role of liquid biopsy in this context.

The authors provide a comprehensive overview of the various applications of liquid biopsy. However, it is important to note that, at present, its only approved clinical indication is in cases where tissue samples are inadequate or unavailable.

I would like to highlight three key points that should be addressed in the discussion:

1. Distinction Between cfDNA and ctDNA
   The manuscript would benefit from a clearer explanation of the difference between circulating free DNA (cfDNA) and circulating tumor DNA (ctDNA). This distinction is essential, as ctDNA is typically detected by identifying tumor-specific mutations using next-generation sequencing (NGS). However, in cases where no mutations are present, ctDNA detection becomes more challenging. In this context, the use of broader NGS panels at baseline may increase the chance of identifying mutations and facilitate subsequent monitoring of ctDNA clearance.
2. Complementary NGS Analysis Using Tissue and Liquid Biopsy
   Several studies have investigated the utility of performing NGS on both tissue and plasma samples (orthogonal analysis). This approach can improve mutation detection, as certain alterations may be missed by one method but identified by the other. For example, it is well recognized that ctDNA-based NGS has limitations in detecting gene fusions such as ALK or ROS1. The authors should consider discussing this limitation and referring to the following studies:
• DOI: 10.1016/j.jtocrr.2024.100778
• DOI: 10.1016/j.cllc.2021.10.001
3. Correlation Between Clinical Characteristics and Liquid Biopsy Positivity
   The authors should also cite studies that have explored the association between clinical characteristics—such as tumor burden, disease stage, or histology—and the likelihood of liquid biopsy positivity. These data are important for understanding which patients are most likely to benefit from liquid biopsy testing.

 

Author Response

Dear Reviewer,

We sincerely appreciate your recognition of the content and scientific value of our manuscript. The insightful and constructive comments provided in your review have allowed us to refine and enrich the article, making it more comprehensive and coherent.

We have revised the manuscript thoroughly, addressing all of your comments (1–3). A new section entitled “Discussion” has been added, in which we respond in detail to the points raised in the review. The issues we were asked to address have been discussed both in the main body of the text and in the Discussion section, in order to further highlight aspects that we believe readers should pay particular attention to.
Furthermore, we have included a concise summary of the manuscript in the newly added “Conclusions” section. We have also added over ten new references, including those you suggested.

We firmly believe that your valuable feedback has significantly improved the quality and clarity of our manuscript, and we are truly grateful for your contribution to this process.

Sincerely,
Agata Wróbel