Assessing Reporting Quality and Pre-Analytical Standards in Extrachromosomal Circular DNA Studies in Cancer: A Systematic Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an important and timely issue: the incomplete reporting of pre-analytical variables in circulating eccDNA studies in cancer. The topic is relevant for the reproducibility and future clinical translation of liquid biopsy biomarkers, particularly given the low abundance and methodological complexity of eccDNA analysis.

However, in its current form, I do not consider the manuscript sufficiently aligned with the scope and expectations of Cancers. Rather than providing a cancer-focused systematic review with substantial biological, clinical, or translational conclusions, the study mainly assesses whether previously published eccDNA studies report methodological variables included in the NCI guideline “Cell-free DNA: Biospecimen Collection and Processing”. This makes the manuscript closer to a methodological/reporting-quality assessment than to a cancer biology or clinical oncology review. Therefore, I believe it would be more suitable for a journal more specifically focused on methodological standardization, reporting quality, diagnostics, or protocols, such as Methods and Protocols or, if appropriately reframed toward molecular diagnostic implementation, Diagnostics.

The study is potentially useful, but several conceptual and methodological aspects should be clarified before it can be considered for publication.

Major comments

1. Scope and suitability for Cancers

The manuscript focuses primarily on adherence to pre-analytical reporting items derived from the NCI cfDNA biospecimen collection and processing guideline. Although this is relevant for cancer liquid biopsy research, the manuscript does not provide sufficient cancer-specific insight across tumour types, nor does it substantially discuss how the observed reporting gaps affect eccDNA biomarker development in specific oncological contexts.

Given the methodological nature of the work, the authors should consider whether the manuscript would be better suited to a more methods-oriented journal. If the manuscript is to remain under consideration for Cancers, the authors should strengthen the cancer-specific discussion, including how pre-analytical underreporting may affect biomarker discovery, diagnostic performance, clinical translation, and comparability across different tumour types.

2. Clarify the conceptual use of the NCI guideline

The authors have adapted the NCI Cell-free DNA: Biospecimen Collection and Processing guideline into a 22-item checklist and use it as the central framework for the review. The manuscript states that the checklist was derived from the NCI guideline and that items were coded as “reported”, “not reported”, or “deviated from recommendation”.

However, the NCI guideline is primarily intended to provide evidence-based guidance on biospecimen collection and processing for cfDNA analyses. It is not necessarily a formal reporting guideline or a standardized reporting system for pre-analytical variables. The authors should make this distinction clearer. In particular, they should avoid presenting the NCI guideline as if it were a validated reporting checklist for eccDNA studies. A more accurate framing would be that the NCI document provides a useful reference framework to identify relevant pre-analytical variables that should be transparently reported.

3. Discuss other reporting and pre-analytical standardization initiatives

The discussion would benefit from a broader consideration of existing initiatives in biospecimen reporting and pre-analytical standardization. In particular, the authors should discuss the Standard PREanalytical Code (SPREC), promoted by ISBER, which is specifically designed to identify and record key pre-analytical factors that may affect the integrity of clinical fluids and solid biospecimens.

The authors should also refer to the EQUATOR Network, which provides a searchable repository of reporting guidelines for health research and could help position their proposed checklist within the broader ecosystem of reporting standards.

This is particularly important because the manuscript proposes a reporting checklist. The authors should explain whether their checklist is intended as a practical extension of the NCI recommendations, a provisional eccDNA-specific reporting tool, or a first step toward a future consensus-based reporting guideline.

4. Heterogeneity of included studies

The studies included in the review are highly heterogeneous. They differ in tumour type, clinical setting, sample size, biospecimen type, analytical workflow, and study design. The manuscript itself reports that the 14 studies included multiple malignancies, with sample sizes ranging from 3 to 98 participants, using plasma, serum, tissue, bile, or urine in different combinations.

This heterogeneity substantially limits comparability across studies. While the authors acknowledge that no meta-analysis was performed because of methodological heterogeneity, this issue should be discussed more explicitly as a central limitation of the review.

The authors should avoid overgeneralizing their conclusions and should clarify that the review mainly evaluates reporting completeness, not necessarily true methodological adherence or analytical quality.

5. Risk of bias and QUADAS-2

The authors state that formal bias assessment tools such as QUADAS-2 were not applied because the objective was to assess methodological reporting quality rather than clinical outcomes or effect sizes.

This is reasonable, but the manuscript should still include an appropriate reference for QUADAS-2 when mentioning it. QUADAS-2 is a widely used tool for assessing risk of bias and applicability in diagnostic accuracy studies, structured around domains such as patient selection, index test, reference standard, and flow/timing.

The authors should also clarify why QUADAS-2 was not suitable for their specific objective, while acknowledging that some included studies may have diagnostic biomarker aims.

6. Data synthesis and software

In the Data Synthesis section, the authors state that descriptive statistics were used to summarize reporting, missing, and deviation rates, and that horizontal bar graphs were generated. However, the software used for the descriptive analyses and figure generation is not specified. The authors should indicate whether analyses were performed using R, Python, Excel, GraphPad Prism, or another tool, including software version and relevant packages where applicable.

7. Practical implementation of the proposed checklist

The conclusion recommends adoption of a concise eccDNA reporting checklist, together with protocol preregistration and mandatory checklist submission. This is a potentially useful recommendation, but the authors should discuss more clearly where and how they expect this checklist to be used. For example, should it be required by journals as supplementary material? Should it be incorporated into liquid biopsy study protocols? Should it be used by biobanks when releasing biospecimens? Should it be promoted by scientific societies or networks working on cfDNA/eccDNA standardization?

A more practical discussion would substantially increase the value of the manuscript.

Minor comments

1. The manuscript title contains typographical errors: “Systemmatical Review” should be corrected to “Systematic Review”.
2. The authors should ensure consistency in terminology, particularly between eccDNA, cfDNA, circulating eccDNA, and cell-free eccDNA.
3. The authors should specify more clearly whether “deviation” refers to deviation from an explicit NCI recommendation or from the authors’ interpretation of best practice.
4. The limitations section should more strongly emphasize that absence of reporting does not necessarily mean absence of compliance.
5. The authors should avoid implying that reported practices generally aligned with guidance, since most variables were not reported and therefore true adherence cannot be reliably assessed.

Author Response

Response to REVIEWER #1

 

The manuscript addresses an important and timely issue: the incomplete reporting of pre-analytical variables in circulating eccDNA studies in cancer. The topic is relevant for the reproducibility and future clinical translation of liquid biopsy biomarkers, particularly given the low abundance and methodological complexity of eccDNA analysis.

However, in its current form, I do not consider the manuscript sufficiently aligned with the scope and expectations of Cancers. Rather than providing a cancer-focused systematic review with substantial biological, clinical, or translational conclusions, the study mainly assesses whether previously published eccDNA studies report methodological variables included in the NCI guideline “Cell-free DNA: Biospecimen Collection and Processing”. This makes the manuscript closer to a methodological/reporting-quality assessment than to a cancer biology or clinical oncology review. Therefore, I believe it would be more suitable for a journal more specifically focused on methodological standardization, reporting quality, diagnostics, or protocols, such as Methods and Protocols or, if appropriately reframed toward molecular diagnostic implementation, Diagnostics.

The study is potentially useful, but several conceptual and methodological aspects should be clarified before it can be considered for publication.

Response: We appreciate the reviewer’s time and the feedback they provided to help us improve our manuscript. Following the reviewer’s comments, we made the changes to the manuscript shown below.

 

Major comments

1. Scope and suitability for Cancers

The manuscript focuses primarily on adherence to pre-analytical reporting items derived from the NCI cfDNA biospecimen collection and processing guideline. Although this is relevant for cancer liquid biopsy research, the manuscript does not provide sufficient cancer-specific insight across tumour types, nor does it substantially discuss how the observed reporting gaps affect eccDNA biomarker development in specific oncological contexts. Given the methodological nature of the work, the authors should consider whether the manuscript would be better suited to a more methods-oriented journal. If the manuscript is to remain under consideration for Cancers, the authors should strengthen the cancer-specific discussion, including how pre-analytical underreporting may affect biomarker discovery, diagnostic performance, clinical translation, and comparability across different tumour types.

Response: We thank the reviewer for this thoughtful comment. We respectfully note that the central aim of our review—appraising the pre-analytical and reporting quality of eccDNA as an emerging liquid-biopsy biomarker in cancer patients—falls squarely within the oncology liquid-biopsy scope of Cancers, and all included studies are cancer studies spanning multiple malignancies (Table 1). At the same time, we fully agree that the cancer-specific implications should be strengthened. We have therefore added a dedicated paragraph to the Discussion explaining how pre-analytical underreporting affects eccDNA biomarker discovery, diagnostic performance, clinical translation, and comparability across tumour types.

Discussion (p. 11, line 333): Beyond general reproducibility, incomplete pre-analytical reporting has tumour-type-specific consequences for eccDNA biomarker development. The studies reviewed here span malignancies with markedly different circulating tumour burdens and eccDNA yields—from haematological cancers such as multiple myeloma to solid tumours including lung, colorectal, prostate, renal, and thyroid carcinomas. Because eccDNA abundance and fragment characteristics differ across tumour types and disease stages, undocumented variation in collection tubes, processing delay, centrifugation, and freeze–thaw history makes it difficult to determine whether between-study differences reflect genuine tumour biology or divergent specimen handling. This ambiguity directly affects biomarker discovery (identifying tumour-specific eccDNA signatures), diagnostic performance (establishing reproducible thresholds and limits of detection), and cross-tumour comparability (benchmarking eccDNA against established liquid-biopsy analytes such as ctDNA). For clinical translation, regulatory and laboratory-accreditation pathways for liquid-biopsy assays require fully specified pre-analytical conditions; the gaps identified here therefore represent a concrete barrier to advancing eccDNA from exploratory studies toward validated, tumour-context-specific clinical applications.

 

2. Clarify the conceptual use of the NCI guideline

The authors have adapted the NCI Cell-free DNA: Biospecimen Collection and Processing guideline into a 22-item checklist and use it as the central framework for the review. The manuscript states that the checklist was derived from the NCI guideline and that items were coded as “reported”, “not reported”, or “deviated from recommendation”.

However, the NCI guideline is primarily intended to provide evidence-based guidance on biospecimen collection and processing for cfDNA analyses. It is not necessarily a formal reporting guideline or a standardized reporting system for pre-analytical variables. The authors should make this distinction clearer. In particular, they should avoid presenting the NCI guideline as if it were a validated reporting checklist for eccDNA studies. A more accurate framing would be that the NCI document provides a useful reference framework to identify relevant pre-analytical variables that should be transparently reported.

Response: Thank you for bringing this issue to our attention. According to the reviewer’s feedback, we have clarified the use of the NCI guideline as a guiding framework rather than a validated reporting checklist.

Introduction (p. 2, line 90): In the absence of such guidelines, existing evidence-based recommendations for biospecimen collection and processing may provide a helpful guiding framework for identifying key pre-analytical details that should be transparently reported in eccDNA studies.

Introduction (p. 3, line 107): For the purposes of this review, key pre-analytical variables described in the NCI guideline were adapted into a reporting assessment checklist; the NCI guideline itself is not a formal methodological reporting guideline.

Materials and Methods (p. 3, line 117): In the absence of a standardized methodological reporting checklist for eccDNA studies, included articles were evaluated using recommendations described in NCI Cell-free DNA: Biospecimen Collection and Processing guideline as a reference framework [7].

 

3. Discuss other reporting and pre-analytical standardization initiatives

The discussion would benefit from a broader consideration of existing initiatives in biospecimen reporting and pre-analytical standardization. In particular, the authors should discuss the Standard PREanalytical Code (SPREC), promoted by ISBER, which is specifically designed to identify and record key pre-analytical factors that may affect the integrity of clinical fluids and solid biospecimens.

The authors should also refer to the EQUATOR Network, which provides a searchable repository of reporting guidelines for health research and could help position their proposed checklist within the broader ecosystem of reporting standards.

This is particularly important because the manuscript proposes a reporting checklist. The authors should explain whether their checklist is intended as a practical extension of the NCI recommendations, a provisional eccDNA-specific reporting tool, or a first step toward a future consensus-based reporting guideline.

Response: As suggested by the reviewer, we have added a discussion of SPREC and EQUATOR in our revised manuscript. We have also added comments on how we intend our proposed checklist to be used.

 

Discussion (page 11, line 399)

Existing reporting and standardization initiatives

The need for better reporting methods in eccDNA research aligns with efforts to standardize biospecimen documentation and research reporting across biomedical fields. One important initiative is the Standard PREanalytical Code (SPREC), created by the International Society for Biological and Environmental Repositories (ISBER) in 2009 [26]. This code helps researchers document pre-analytical information that can affect the integrity of fluid and solid biospecimens [27]. SPREC aims to improve quality management and method harmonization by systematically coding variables related to biospecimen collection, processing, and storage that influence downstream analyses into a short string of letters [27]. After its release, SPREC has been integrated into biobank databases and quality management systems throughout the world, making it easier to standardize the annotation of pre-analytical biospecimens across different institutions [26]. Notably, many variables that this review found to be frequently underreported, such as blood collection procedures and freeze-thaw history, are factors that SPREC was designed to capture [26,27]. The continuous development of new versions of SPREC underscores the need to standardize comprehensive and concise documentation of pre-analytical variables in biospecimen-based research [28].

Another important effort comes from the EQUATOR (Enhancing the QUAlity and Transparency Of Health Research) Network. This network was established to improve the reliability of health research through more complete and clear reporting [29]. The EQUATOR Network has a searchable repository of reporting guidelines and encourages the development as well as use of reporting standards across a wide range of study designs and research fields [30]. While frameworks like CONSORT, STROBE, and PRISMA are widely used in clinical and observational studies, there is currently no reporting guideline specific to plasma or serum-based eccDNA research. As a result, methodological reporting in eccDNA studies heavily relies on the practices of individual investigators, leading to the inconsistencies noted in this review.

 

Recommendations

The lack of detailed methodological reporting in eccDNA research highlights the urgent need for standardized reporting practices. To address this, we recommend the incorporation of a reporting checklist using the key pre-analytical variables described in the NCI guideline as a reference, encompassing 22 key items across biospecimen collection, blood processing, and eccDNA processing (Supplementary Table 3). The checklist includes most crucial and frequently underreported pre-analytical procedural details for eccDNA studies, and it provides researchers with a succinct and easy-to-read way of reporting methodology. Examples of included items are whole blood collection considerations (tube type, blood volume) as well as plasma/serum centrifugation information (speed, temperature, number of spins). Journals could encourage or require the submission of such checklist as supplementary material, similar to existing reporting guidelines used for systematic reviews. By ensuring that key pre-analytical details are transparently documented in publications, experiment reproducibility will be enhanced, and batch effects across studies would be reduced. This checklist may also be useful for biobanks that handle plasma or serum samples for eccDNA studies as a record for researchers to assess potential upstream sources of variability. Ultimately, improving methodological rigor and reporting standards will be essential for advancing eccDNA research toward clinical application in cancer diagnostics and monitoring. Our proposed checklist may provide a starting point for developing standardized reporting recommendations that are tailored specifically to eccDNA studies.

 

4. Heterogeneity of included studies

The studies included in the review are highly heterogeneous. They differ in tumour type, clinical setting, sample size, biospecimen type, analytical workflow, and study design. The manuscript itself reports that the 14 studies included multiple malignancies, with sample sizes ranging from 3 to 98 participants, using plasma, serum, tissue, bile, or urine in different combinations.

This heterogeneity substantially limits comparability across studies. While the authors acknowledge that no meta-analysis was performed because of methodological heterogeneity, this issue should be discussed more explicitly as a central limitation of the review.

The authors should avoid overgeneralizing their conclusions and should clarify that the review mainly evaluates reporting completeness, not necessarily true methodological adherence or analytical quality.

Response: We appreciate the reviewer’s comment on addressing heterogeneity of included studies as a central limitation. We have added the following passages according to the reviewer’s suggestions.

 

Discussion (p. 11, line 366)

Limitations

This review also has several limitations. Firstly, the included studies were highly heterogeneous in the context of cancer type, sample size, biospecimen type, and study design. The studies investigated diverse malignancies, ranging from multiple myeloma to nodular thyroid goitre, with varying sample sizes from 6 to 252 participants. Some study designs involved healthy controls, but not all. While plasma and serum were the predominant biospecimen types being investigated, some studies also used tissue or other types of bodily fluids such as bile or urine. Such heterogeneity limits the comparability across studies and presents a barrier to assess reporting practices according to specific cancer types, biospecimen types, or study designs.

Secondly, because eccDNA remains an emerging biomarker in liquid biopsy research, only a small number of studies using plasma or serum are currently available. The limited sample size (n = 14) reduces statistical power to detect consistent patterns across cancer types and hinders robust evaluation of guideline adherence. Publication bias may also contribute to the limited range of eccDNA literature as studies reporting negative or statistically insignificant findings are less likely to be published than studies with conclusive and positive findings.

Thirdly, while most studies (13/14) included in our analysis were published after the release of the NCI guideline in 2020, the study by Kumar et al. [8] predates the guideline. As a result, it is important to be aware that the pre-analytical practices in that study should not be expected to reflect the guideline recommendations, which were not available at that time.

Finally, our analysis necessarily relied on published information; when studies did not report specific procedures, it remains unclear whether these steps were not performed or simply omitted in the description. As a result, true adherence and deviation rates may differ from those captured in our review, raising additional concerns regarding transparency and reproducibility. Consequently, the findings of this review should be interpreted as an evaluation of reporting completeness in the recent clinical eccDNA literature rather than evidence of a lack of methodological consistency or quality. Poor adherence to reporting recommendations does not directly correlate to poor experimental conduct or analysis. However, it does hinder the transparency, reproducibility, and comparability across eccDNA studies.

 

5. Risk of bias and QUADAS-2

The authors state that formal bias assessment tools such as QUADAS-2 were not applied because the objective was to assess methodological reporting quality rather than clinical outcomes or effect sizes.

This is reasonable, but the manuscript should still include an appropriate reference for QUADAS-2 when mentioning it. QUADAS-2 is a widely used tool for assessing risk of bias and applicability in diagnostic accuracy studies, structured around domains such as patient selection, index test, reference standard, and flow/timing.

The authors should also clarify why QUADAS-2 was not suitable for their specific objective, while acknowledging that some included studies may have diagnostic biomarker aims.

Response: We appreciate the reviewer's comment. We have added a citation for QUADAS-2 and expanded the Materials and Methods to clarify why it was not applied, while explicitly acknowledging that several included studies had diagnostic biomarker aims.

 

Materials and Methods (p. 6, line 192)

Because the objective of this review was to assess the completeness of pre-analytical methodological reporting rather than diagnostic accuracy or effect estimates, formal risk-of-bias tools such as QUADAS-2 [22] were not applied. QUADAS-2 appraises risk of bias and applicability in diagnostic accuracy studies across four domains—patient selection, index test, reference standard, and flow and timing—which are designed to evaluate the validity of diagnostic performance estimates. Although several included studies pursued diagnostic biomarker aims, our unit of assessment was the reporting of pre-analytical variables (collection, processing, storage, and extraction) rather than diagnostic test accuracy; consequently, the QUADAS-2 domains do not map onto the reporting items evaluated here. A checklist-based reporting assessment was therefore better suited to the aims of this review.

 

6. Data synthesis and software

In the Data Synthesis section, the authors state that descriptive statistics were used to summarize reporting, missing, and deviation rates, and that horizontal bar graphs were generated. However, the software used for the descriptive analyses and figure generation is not specified. The authors should indicate whether analyses were performed using R, Python, Excel, GraphPad Prism, or another tool, including software version and relevant packages where applicable.

Response: As the reviewer pointed out, it is important to explicitly state the software used for data synthesis. We have added a statement clarifying our use of Excel in the Data Synthesis section.

Data Synthesis (p. 6, line 209): Descriptive statistics and graph generation were performed through Excel.

 

7. Practical implementation of the proposed checklist

The conclusion recommends adoption of a concise eccDNA reporting checklist, together with protocol preregistration and mandatory checklist submission. This is a potentially useful recommendation, but the authors should discuss more clearly where and how they expect this checklist to be used. For example, should it be required by journals as supplementary material? Should it be incorporated into liquid biopsy study protocols? Should it be used by biobanks when releasing biospecimens? Should it be promoted by scientific societies or networks working on cfDNA/eccDNA standardization?

A more practical discussion would substantially increase the value of the manuscript.

Response: We appreciate the reviewer for bringing up this significant point. We have added more discussion around implementing the checklist in journal submission and biobank biospecimen handling.

Discussion (p. 12, line 437): Journals could encourage or require the submission of such checklist as supplementary material, similar to existing reporting guidelines used for systematic reviews. By ensuring that key pre-analytical details are transparently documented in publications, experiment reproducibility will be enhanced, and batch effects across studies would be reduced. This checklist may also be useful for biobanks that handle plasma or serum samples for eccDNA studies as a record for researchers to assess potential upstream sources of variability.

 

Minor comments

1. The manuscript title contains typographical errors: “Systemmatical Review” should be corrected to “Systematic Review”.

Response: Thank you for your comment. We have verified that the manuscript title is “Systematic Review.” (p. 1, line 1)

 

2. The authors should ensure consistency in terminology, particularly between eccDNA, cfDNA, circulating eccDNA, and cell-free eccDNA.

Response: Thank you for bringing this to our attention. We have gone through the manuscript and clarified our terminology by using “eccDNA”.

 

3. The authors should specify more clearly whether “deviation” refers to deviation from an explicit NCI recommendation or from the authors’ interpretation of best practice.

Response: We appreciate the Reviewer’s comment on specifying our definition of “deviation.”  We have added the clarification in several locations.

Introduction (p. 3, line 102): Each study was assessed for deviations from the NCI guideline recommendations and the lack of reporting of key pre-analytical methodological variables identified in the guideline.

Materials and methods (p. 6, lines 188): Each item was coded as “reported,” “not reported,” or “deviated from NCI guideline recommendation.” Missing and deviation rates were calculated for each item across all studies: missing rate = (# of studies that have not reported an item) ¸ (total # of studies); deviation rate = (# of studies that have deviated from NCI recommendation) ¸ (total # of studies).

 

4. The limitations section should more strongly emphasize that absence of reporting does not necessarily mean absence of compliance.

Response: As noted by the reviewer, poor reporting does not directly correspond to poor compliance. We have included the following paragraph in the limitations section, per the reviewer’s comment.

Discussion (p. 11, line 388): Finally, our analysis necessarily relied on published information; when studies did not report specific procedures, it remains unclear whether these steps were not performed or simply omitted in the description. As a result, true adherence and deviation rates may differ from those captured in our review, raising additional concerns regarding transparency and reproducibility. Consequently, the findings of this review should be interpreted as an evaluation of reporting completeness in the recent clinical eccDNA literature rather than evidence of a lack of methodological consistency or quality. Poor adherence to reporting recommendations does not directly correlate to poor experimental conduct or analysis. However, it does hinder the transparency, reproducibility, and comparability across eccDNA studies.

 

5. The authors should avoid implying that reported practices generally aligned with guidance, since most variables were not reported and therefore true adherence cannot be reliably assessed.

Response: Thank you for pointing this out. This is a very crucial point for us to clarify, which we have added in several places throughout the manuscript.

 

Results (p. 9, line 265): Since deviation cannot be determined for unreported items, the actual frequency of non-conformant practice also may not be reliably assessed.

 

Discussion (p. 9, line 284): However, it is not possible to determine the true frequency of deviations in these studies due to the large percentage of omitted pre-analytical details, further emphasizing the problem of underreporting as the primary limitation in the current literature.

 

Conclusions (p. 12, line 450): While deviations from the NCI guidelines, despite being rare, cannot be fairly assessed due to the frequency of underreporting. This issue indicates that the principal barrier lies in incomplete reporting.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

First of all, congratulations for your interesting work. I hope that my hints will help you in the next steps of improvement and the final manuscript will be really valuable for the readers. Your systematic review has clear scientific value because it addresses an important but underexplored methodological problem in eccDNA research: the lack of standardized pre-analytical and reporting procedures. The paper highlights major reproducibility gaps in liquid biopsy studies and proposes a practical reporting checklist aligned with NCI recommendations. Its conclusions are consistent with current knowledge regarding the importance of biospecimen handling and methodological transparency in cfDNA-based oncology research.

The first weakness is the relatively limited methodological depth of the systematic review itself. Although the study successfully identifies reporting deficiencies, it does not critically evaluate how these deficiencies may biologically influence eccDNA detection, quantification, or downstream interpretation. For example, the manuscript discusses freeze–thaw cycles and centrifugation variability but does not connect these variables to specific eccDNA fragmentation patterns, sequencing artifacts, or analytical bias. The authors could improve the manuscript by adding a mechanistic discussion summarizing how pre-analytical variability may alter eccDNA topology, abundance, or sequencing fidelity. This would strengthen the translational impact without requiring additional experiments.

A second weakness is the limited integration of the review into the broader landscape of modern precision oncology and multi-omics medicine. eccDNA is presented primarily as an isolated liquid biopsy biomarker, whereas its greatest future value may lie in integration with genomics, methylomics, transcriptomics, chromatin accessibility profiling, and AI-assisted biomarker modeling. The manuscript would benefit from a dedicated section discussing how eccDNA could complement circulating tumour DNA, exosomal RNA, epigenetic signatures, or single-cell analyses in personalized cancer monitoring. Even a theoretical framework or schematic model (yes, pictures!) of multi-omics integration would substantially modernize the review and increase its relevance to contemporary oncology.

The third weakness concerns the lack of quantitative quality assessment tools commonly used in systematic reviews and biomarker studies. The authors intentionally avoided formal bias tools such as QUADAS-2, explaining that the focus was methodological reporting quality rather than diagnostic performance. However, the absence of a structured risk-of-bias framework weakens the rigor of evidence interpretation. The manuscript could be improved by incorporating a simplified adapted quality-scoring system or evidence-grading approach specifically designed for pre-analytical reporting studies. Even a qualitative classification of studies into high-, intermediate-, and low-reporting-quality categories would enhance interpretability without additional data collection.

Finally, there is no figures, even if modern AI-based era allows to create entire plethora of wonderful graphs and schemes. Would it be possible to add some  figures and graphs too? It will definitely increase the value of your already well-written manuscript. There are several mechanisms and many molecules described in the paper, it may not be easy to digest for younger readers, and good pictures showing these pathways and molecules, also aberrant places, will definitely be of great value. 

Author Response

Response to REVIEWER #2

Dear Authors,

First of all, congratulations for your interesting work. I hope that my hints will help you in the next steps of improvement and the final manuscript will be really valuable for the readers. Your systematic review has clear scientific value because it addresses an important but underexplored methodological problem in eccDNA research: the lack of standardized pre-analytical and reporting procedures. The paper highlights major reproducibility gaps in liquid biopsy studies and proposes a practical reporting checklist aligned with NCI recommendations. Its conclusions are consistent with current knowledge regarding the importance of biospecimen handling and methodological transparency in cfDNA-based oncology research.

1. The first weakness is the relatively limited methodological depth of the systematic review itself. Although the study successfully identifies reporting deficiencies, it does not critically evaluate how these deficiencies may biologically influence eccDNA detection, quantification, or downstream interpretation. For example, the manuscript discusses freeze–thaw cycles and centrifugation variability but does not connect these variables to specific eccDNA fragmentation patterns, sequencing artifacts, or analytical bias. The authors could improve the manuscript by adding a mechanistic discussion summarizing how pre-analytical variability may alter eccDNA topology, abundance, or sequencing fidelity. This would strengthen the translational impact without requiring additional experiments.

Response: We thank the reviewer for this valuable suggestion. We have added a mechanistic paragraph to the Discussion linking the underreported pre-analytical variables to their potential effects on eccDNA topology (supercoiled versus nicked/relaxed forms and exonuclease susceptibility), abundance (genomic-DNA contamination and dilution of low-abundance signal), and sequencing fidelity (junction-read recovery and background artefacts).

 

Discussion (p. 10, line 315):

Mechanistically, the pre-analytical variables identified as poorly reported are precisely those most likely to distort eccDNA topology, abundance, and sequencing fidelity. eccDNA enrichment typically relies on exonuclease-based digestion of linear DNA within a multi-step purification of closed-circular molecules [5], a workflow that depends on the structural integrity of the circles. Repeated freeze–thaw cycles and prolonged storage can introduce single-strand nicks that convert supercoiled circles into relaxed or linearized forms, rendering them susceptible to exonuclease digestion and biasing recovery toward shorter, more stable species; the same processes shift cfDNA fragment-size distributions and can therefore alter the apparent eccDNA size profile [6,7]. Delayed processing and the absence of stabilizing collection tubes promote leukocyte lysis and genomic-DNA contamination, which dilutes the low-abundance eccDNA signal, lowers the proportion of informative junction-spanning reads, and inflates background during sequencing [7]. Suboptimal or unreported centrifugation can leave residual cells and platelets that contribute further contaminating linear DNA [23]. Because eccDNA is present at very low abundance and is identified bioinformatically through split/junction reads, even modest, undocumented variation in these steps can translate into systematic differences in measured eccDNA quantity, fragment topology, and false-positive junction calls, undermining quantitative interpretation and cross-study comparability.

 

2. A second weakness is the limited integration of the review into the broader landscape of modern precision oncology and multi-omics medicine. eccDNA is presented primarily as an isolated liquid biopsy biomarker, whereas its greatest future value may lie in integration with genomics, methylomics, transcriptomics, chromatin accessibility profiling, and AI-assisted biomarker modeling. The manuscript would benefit from a dedicated section discussing how eccDNA could complement circulating tumour DNA, exosomal RNA, epigenetic signatures, or single-cell analyses in personalized cancer monitoring. Even a theoretical framework or schematic model of multi-omics integration would substantially modernize the review and increase its relevance to contemporary oncology.

Response: We thank the Reviewer for this insightful suggestion. We have added a dedicated Discussion subsection, “eccDNA integration within multi-omics precision oncology,” to place eccDNA within the broader context of modern precision oncology. This new section discusses how eccDNA could complement ctDNA, fragmentomics, DNA methylation and chromatin-accessibility profiling, exosomal RNA/transcriptomics, single-cell analyses, and AI-assisted biomarker modeling for personalized cancer monitoring. We also provide a theoretical framework for multi-omics integration and emphasize that harmonized pre-analytical reporting is essential for enabling such integrative applications.

 

Discussion (p. 10, line 349):

The translational value of eccDNA is likely to be greatest not in isolation but as one layer within an integrated, multi-omics liquid-biopsy framework. Circulating tumour DNA (ctDNA) and fragmentomic profiles capture point mutations, copy-number changes, and fragment-length signatures; DNA methylation and chromatin-accessibility profiling add epigenetic and regulatory context; and exosomal RNA and circulating transcriptomic data report on active gene expression. eccDNA is mechanistically complementary to these layers because it frequently carries amplified oncogenes and can act as a mobile regulatory element, linking copy-number amplification to transcriptional output. Combining eccDNA with ctDNA, methylomic, transcriptomic, and single-cell readouts could therefore improve early detection, help resolve tumour heterogeneity, and enable longitudinal monitoring of treatment response and resistance [24]. Machine-learning and AI-assisted models are increasingly used to integrate such high-dimensional, multimodal data into composite biomarkers, and eccDNA junction and abundance features are well suited as inputs to these models [25]. Realizing this potential, however, depends on harmonized and transparently reported pre-analytical workflows, because batch effects introduced upstream propagate and confound across data layers.

 

3. The third weakness concerns the lack of quantitative quality assessment tools commonly used in systematic reviews and biomarker studies. The authors intentionally avoided formal bias tools such as QUADAS-2, explaining that the focus was methodological reporting quality rather than diagnostic performance. However, the absence of a structured risk-of-bias framework weakens the rigor of evidence interpretation. The manuscript could be improved by incorporating a simplified adapted quality-scoring system or evidence-grading approach specifically designed for pre-analytical reporting studies. Even a qualitative classification of studies into high-, intermediate-, and low-reporting-quality categories would enhance interpretability without additional data collection.

Response: We thank the reviewer for this constructive suggestion. We have added a simplified reporting-quality classification grouping studies into high (≥50% of the 22 items reported), intermediate (30–49%), and low (<30%) reporting-completeness categories. Applying this scheme, 5 studies were classified as high, 7 as intermediate, and 2 as low; the corresponding per-study percentages and category assignments are provided in Supplementary Table 1. We define the categories in the Materials and Methods and report the distribution in the Results.

 

Materials and Methods (p. 6, line 205)

To provide an overall summary of reporting quality, each study was additionally classified by the proportion of the 22 checklist items reported into high (≥50% reported), intermediate (30–49%), and low (<30%) reporting-completeness categories.

 

Results (p. 7, line 250)

Overall, the average percentage of items reported per study was 9/22 (41%). Across the 14 studies, the percentage ranged from 14/22 (64%) to 4/22 (18%). Using this classification, 5/14 studies were categorized as high (≥50% of items reported), 7/14 as intermediate (30–49%), and 2/14 as low (<30%) reporting completeness (Supplementary Table 1). Notably, no study reported more than 64% of items, indicating that even the most complete reports omitted a substantial share of recommended pre-analytical variables.

 

4. Finally, there is no figures, even if modern AI-based era allows to create entire plethora of wonderful graphs and schemes. Would it be possible to add some figures and graphs too? It will definitely increase the value of your already well-written manuscript. There are several mechanisms and many molecules described in the paper, it may not be easy to digest for younger readers, and good pictures showing these pathways and molecules, also aberrant places, will definitely be of great value. 

 

Response: We thank the Reviewer for this constructive suggestion. We fully agree that figures and graphical summaries can substantially improve the readability and accessibility of the manuscript, particularly for younger readers and those less familiar with this field. In response, we have revised the existing PRISMA flow diagram (Figure 1) and the missing/deviation-rate graphs (Figure 2 and Supplementary Figure 1) to improve visual clarity. We hope that these revisions make the manuscript easier to follow and enhance its educational value for a broad readership.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Major Comments:

 

1. Abstract - The abstract is generally good but has some redundancy and awkward phrasing.

- In the Simple Summary and Background, you repeat the importance of standardization - merge these ideas. 

- Results section: Clearly state the total number of checklist items (22) and categories early. 

- Conclusions: Strengthen the impact statement. Instead of “a concise checklist is recommended,” briefly mention what makes your proposed checklist valuable (e.g., brevity, direct alignment with NCI, focus on eccDNA-specific challenges). 

Suggestion: Reduce Simple Summary to 4 - 5 sentences max and ensure the structured abstract flows logically without repetition.

 

2. Methods - Add Essential Details for Reproducibility

As this is a systematic review, readers will expect stricter methodological transparency. 

- Mention compliance with PRISMA 2020 guidelines and registration (e.g., PROSPERO). 

- Provide the exact search strategy (databases used: PubMed, Scopus, Web of Science?), keywords, and date of last search. 

- Clarify how you adapted the 22 NCI items specifically for eccDNA (what was added/removed and why?). 

- Describe the screening process (who performed it? dual independent reviewers? kappa statistic for agreement?). 

- Explain how you calculated “missing/deviation rates” include a clear formula or reference.  These additions are critical for a methods-focused review paper.

 

3. Results - Enhance Presentation and Visual Impact

The results are informative but text-heavy. 

- Add a Table 1 summarizing the 14 included studies (author, year, cancer type, sample size, key findings). 

- Add a Figure 1 (PRISMA flow diagram).  

- Create a Table 2 or heatmap showing compliance/missing rates for all 22 checklist items (grouped by category: collection, processing, eccDNA-specific). Highlight the 100% missing items visually. 

- Quantify overall reporting quality (e.g., mean percentage of items reported per study). This would make the gaps more compelling.

 

4. Discussion - Strengthen Novelty, Limitations, and Implications

- Expand on why these specific gaps (e.g., freeze-thaw cycles, storage duration, venipuncture site) matter uniquely for eccDNA compared to general cfDNA. 

- Discuss potential bias introduced by poor reporting (e.g., how it affects biomarker validation or liquid biopsy standardization). 

- Address limitations clearly: small number of studies (n = 14), possible publication bias, and the challenge that some studies pre-date the NCI guideline. 

- Elaborate on the proposed checklist: Provide 1-2 example items in the main text and include the full concise checklist as a supplementary table or Box. Explain how it improves upon the original NCI guideline for eccDNA context.

 

5. Minor Comments (Language & Structure)

- Use consistent terminology: “eccDNA” after first use; avoid switching between “extrachromosomal circular DNA” repeatedly. 

- Improve sentence structure in several places (e.g., “This limits the reproducibility…” → make subject clearer). 

- Ensure all percentages in Results match the numbers (e.g., double-check 13/14 = 93%). 

- Add a dedicated “Recommendations” subsection at the end of Discussion with your proposed checklist. 

- Consider expanding the conclusion to discuss broader impact on clinical translation of eccDNA as a liquid biopsy biomarker.

 

Author Response

Response to REVIEWER #3

Major Comments:

1. Abstract - The abstract is generally good but has some redundancy and awkward phrasing. In the Simple Summary and Background, you repeat the importance of standardization. Merge these ideas. 

Response: We appreciate your comment on repetition in the Abstract. We have moved the emphasis on standardization to the structured abstract.

 

Abstract (p. 1, line 27):

Background/Objectives: eccDNA is a promising cancer biomarker in liquid biopsy. However, the reliability and reproducibility of eccDNA studies rely on the standardization of pre-analytical handling and processing of eccDNA as well as transparent methodological reporting. Although evidence-based guidelines for cell-free DNA handling provide clear recommendations for plasma/serum processing, the extent to which eccDNA studies report and adhere to these key procedures remains uncertain.

 

2. Results section: Clearly state the total number of checklist items (22) and categories early. 

Response: Thank you for your comment. The total number of checklist items and item categories were stated in the Methods section of the structured abstract.

 

Abstract (p. 1, line 32):

Methods: We systematically reviewed 14 studies (2017–2025) assessing eccDNA in plasma or serum from cancer patients. Each study was evaluated against 22 checklist items summarized from the NCI Biospecimen Collection and Processing Guideline, categorized into biospecimen collection, blood processing, and eccDNA processing. Items were classified as “reported,” “not reported,” or “deviated from NCI guideline,” and missing/deviation rates were calculated.

 

3. Conclusions: Strengthen the impact statement. Instead of “a concise checklist is recommended,” briefly mention what makes your proposed checklist valuable (e.g., brevity, direct alignment with NCI, focus on eccDNA-specific challenges). 

Response: As suggested by the reviewer, we have incorporated an impact statement in the Abstract.

 

Abstract (p. 2, line 49):

We propose a concise reporting checklist informed by NCI guideline. By focusing on the most frequently underreported aspects of key pre-analytical eccDNA procedures, the checklist provides researchers with an efficient and succinct methodological reporting framework that will enhance transparency and promote standardization in future eccDNA studies.

 

4. Suggestion: Reduce Simple Summary to 4 - 5 sentences max and ensure the structured abstract flows logically without repetition.

Response: According to the reviewer’s suggestion, we have shortened the Simple Summary and merged repetitive ideas into the Abstract.

 

Simple Summary (p. 1, line 17)

Extrachromosomal circular DNA (eccDNA) shows potential as a cancer detection biomarker in liquid biopsy. We systematically reviewed 14 studies between 2017 to 2025 that used eccDNA in plasma or serum from cancer patients, evaluating their methods against 22 checklist items summarized from the United States National Cancer Institute (NCI) “Cell-free DNA: Biospecimen Collection and Processing” guideline. Reporting gaps were significant, particularly in procedural details for eccDNA isolation, which limits the reproducibility and validity of eccDNA research. Based on our findings, we propose a concise reporting checklist for eccDNA isolation methods aligned with NCI guidelines to improve transparency and method standardization in future eccDNA studies.

 

5. Methods - Add Essential Details for Reproducibility. As this is a systematic review, readers will expect stricter methodological transparency. Mention compliance with PRISMA 2020 guidelines and registration (e.g., PROSPERO). Provide the exact search strategy (databases used: PubMed, Scopus, Web of Science?), keywords, and date of last search. Describe the screening process (who performed it? dual independent reviewers? kappa statistic for agreement?). 

Response: Thank you for your comment. We have mentioned compliance with PRISMA and provided the search process in the Study Selection subsection. The review was not registered on PROSPERO, and a separate protocol was not prepared. This is acknowledged in the Methods section.

 

Materials and Methods (p. 4, line 140)

Study Selection

Two reviewers independently screened all retrieved records by title and abstract, followed by a full-text review of potentially eligible studies. Discrepancies were resolved by consensus. A total of 14 studies met the inclusion criteria: 11 full-length journal articles, two letters, and one preprint. The study selection process is summarized in the PRISMA flow diagram (Figure 1). This review was performed in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 guidelines and has not been registered. A separate review protocol was not prepared. The PRISMA systematic review checklist for this review can be found in Supplementary Table 2. Data extraction and checklist coding were performed by one reviewer (F.T.) and verified by a second reviewer (H.S.K.); disagreements were resolved by consensus.

 

6. Clarify how you adapted the 22 NCI items specifically for eccDNA (what was added/removed and why?). 

Response: We appreciate the reviewer for making this suggestion. We have clarified how the 22 items were derived in the Materials and Methods section. We intended to summarize and reorganize the NCI guideline recommendations into 22 assessable reporting items to facilitate systematic evaluation of reporting completeness in eccDNA studies. Since the NCI guideline already includes collection, processing, storage, extraction, quantification, and quality assessment variables relevant to eccDNA analyses, no major domains were added or removed. We have revised the manuscript to make this distinction clearer.

 

Materials and Methods (p. 6, line 179)

Risk of Bias and Quality Assessment

We developed a 22-item methodological reporting checklist summarized from pre-analytical variables described in the NCI guideline that were applicable to plasma- and serum-based eccDNA workflows. The items were directly organized based on the NCI guideline recommendations because the pre-analytical variables (such as methods of blood sample collection, processing times, centrifugation parameters, storage conditions, DNA isolation processes, and DNA quality evaluation) are thought to impact the integrity of cfDNA, which includes eccDNA. No additional domains for reporting were included or excluded; instead, the NCI guidelines were transformed into an assessment framework that was systematically applied to included studies in this review.

 

7. Explain how you calculated “missing/deviation rates” include a clear formula or reference.  These additions are critical for a methods-focused review paper.

Response: We appreciate the reviewer’s comment on the inclusion of missing/deviation rate formula. According to the reviewer’s suggestion, we have added the formula to the Risk of Bias and Quality Assessment subsection of the manuscript.

 

Materials and Methods (page 6, line 188)

Each item was coded as “reported,” “not reported,” or “deviated from NCI guideline recommendation.” Missing and deviation rates were calculated for each item across all studies: missing rate = (# of studies that have not reported an item) ¸ (total # of studies); deviation rate = (# of studies that have deviated from NCI recommendation) ¸ (total # of studies).

 

8. Results - Enhance Presentation and Visual Impact. The results are informative but text-heavy. Add a Table 1 summarizing the 14 included studies (author, year, cancer type, sample size, key findings). Add a Figure 1 (PRISMA flow diagram).

Response: Thank you for your comment on the visual representation of our results. As suggested by the Reviewer, we added Table 1 summarizing the 14 included studies and Figure 1 PRISMA flow diagram).

 

9. Create a Table 2 or heatmap showing compliance/missing rates for all 22 checklist items (grouped by category: collection, processing, eccDNA-specific). Highlight the 100% missing items visually. 

Response: .We thank the Reviewer for this valuable suggestion. We have revised Figure 2 to present the compliance and missing rates for all 22 checklist items, grouped into sample collection, sample processing, and eccDNA-specific categories. The corresponding detailed information is provided in Supplementary Table 1. We have also visually highlighted the items with a 100% missing rate in both Figure 2 and Supplementary Table 1, as recommended by the Reviewer.

 

10. Quantify overall reporting quality (e.g., mean percentage of items reported per study). This would make the gaps more compelling.

Response: According to the reviewer’s suggestion, we have calculated the percentage of reported items per study as well as the average. The data has also been included in Supplementary Table 1.

 

Results (p. 7, line 250)

Overall, the average percentage of items reported per study was 9/22 (41%). Across the 14 studies, the percentage ranged from 14/22 (64%) to 4/22 (18%). Using this classification, 5/14 studies were categorized as high (≥50% of items reported), 7/14 as intermediate (30–49%), and 2/14 as low (<30%) reporting completeness (Supplementary Table 1). Notably, no study reported more than 64% of items, indicating that even the most complete reports omitted a substantial share of recommended pre-analytical variables.

 

11. Discussion - Strengthen Novelty, Limitations, and Implications

- Expand on why these specific gaps (e.g., freeze-thaw cycles, storage duration, venipuncture site) matter uniquely for eccDNA compared to general cfDNA. 

- Discuss potential bias introduced by poor reporting (e.g., how it affects biomarker validation or liquid biopsy standardization). 

Response: Thank you for bringing up this point. In our revised manuscript, we have added a discussion on poor reporting as a source of bias.

Discussion (p. 9, line 304):

The issue of poor reporting of methodological details can also serve as a source of bias when analyzing eccDNA as a cancer biomarker. If essential pre-analytical variables are poorly described, it is difficult to understand whether any observed differences in eccDNA yield, fragment composition, or biomarker performance arise from biological variance or result from variations in sample handling and preparation procedures. It is also harder to achieve consistency of findings and estimates between different studies due to this issue, which can further hinder attempts at validating study results. Incomplete reporting of methodological details also prevents the process of reproducing research protocols, investigating technical biases, and testing the generalizability of study results. A lack of reporting detail can slow down the process of creating standardized methods and generating evidence to support the wider use of eccDNA in clinical settings.

 

12. Address limitations clearly: small number of studies (n = 14), possible publication bias, and the challenge that some studies pre-date the NCI guideline. 

Response: According to the reviewer’s feedback, we have elaborated further on our limitations subsection.

 

Discussion (p. 11, line 366)

Limitations

This review also has several limitations. Firstly, the included studies were highly heterogeneous in the context of cancer type, sample size, biospecimen type, and study design. The studies investigated diverse malignancies, ranging from multiple myeloma to nodular thyroid goitre, with varying sample sizes from 6 to 252 participants. Some study designs involved healthy controls, but not all. While plasma and serum were the predominant biospecimen types being investigated, some studies also used tissue or other types of bodily fluids such as bile or urine. Such heterogeneity limits the comparability across studies and presents a barrier to assess reporting practices according to specific cancer types, biospecimen types, or study designs.

Secondly, because eccDNA remains an emerging biomarker in liquid biopsy research, only a small number of studies using plasma or serum are currently available. The limited sample size (n = 14) reduces statistical power to detect consistent patterns across cancer types and hinders robust evaluation of guideline adherence. Publication bias may also contribute to the limited range of eccDNA literature as studies reporting negative or statistically insignificant findings are less likely to be published than studies with conclusive and positive findings.

Thirdly, while most studies (13/14) included in our analysis were published after the release of the NCI guideline in 2020, the study by Kumar et al. [8] predates the guideline. As a result, it is important to be aware that the pre-analytical practices in that study should not be expected to reflect the guideline recommendations, which were not available at that time.

Finally, our analysis necessarily relied on published information; when studies did not report specific procedures, it remains unclear whether these steps were not performed or simply omitted in the description. As a result, true adherence and deviation rates may differ from those captured in our review, raising additional concerns regarding transparency and reproducibility. Consequently, the findings of this review should be interpreted as an evaluation of reporting completeness in the recent clinical eccDNA literature rather than evidence of a lack of methodological consistency or quality. Poor adherence to reporting recommendations does not directly correlate to poor experimental conduct or analysis. However, it does hinder the transparency, reproducibility, and comparability across eccDNA studies.

 

13. Elaborate on the proposed checklist: Provide 1-2 example items in the main text and include the full concise checklist as a supplementary table or Box. Explain how it improves upon the original NCI guideline for eccDNA context.

Response: According to the reviewer’s suggestions, we have discussed our proposed checklist in more detail. Since the original NCI guideline was not meant as a reporting checklist, we explained how our checklist includes the most important and frequently underreported details (using the NCI guideline as a reference) for eccDNA studies.

 

Discussion (p. 12, line 432)

The checklist includes most crucial and frequently underreported pre-analytical procedural details for eccDNA studies, and it provides researchers with a succinct and easy-to-read way of reporting methodology. Examples of included items are whole blood collection considerations (tube type, blood volume) as well as plasma/serum centrifugation information (speed, temperature, number of spins).

 

Minor Comments (Language & Structure)

1. Use consistent terminology: “eccDNA” after first use; avoid switching between “extrachromosomal circular DNA” repeatedly. 

Response: Thank you for your comment. We have gone through the manuscript and ensured that consistent terminology for eccDNA has been used.

 

2. Improve sentence structure in several places (e.g., “This limits the reproducibility…” → make subject clearer). 

Response: Per the reviewer’s suggestion, we have edited sentences that seem ambiguous.

 

Abstract (p. 1, line 21)

Reporting gaps were significant, particularly in procedural details for eccDNA isolation, which limits the reproducibility and validity of eccDNA research.

 

3. Ensure all percentages in Results match the numbers (e.g., double-check 13/14 = 93%). 

Response: Thank you for your comment. We have double-checked all deviation and missing rates in our manuscript. The percentages are rounded to their nearest integer.

 

4. Add a dedicated “Recommendations” subsection at the end of Discussion with your proposed checklist. 

Response: According to the reviewer’s suggestion, we have added a Recommendations subsection at the end of Discussion.

 

Discussion (p. 12, line 427)

Recommendations

The lack of detailed methodological reporting in eccDNA research highlights the urgent need for standardized reporting practices. To address this, we recommend the incorporation of a reporting checklist using the key pre-analytical variables described in the NCI guideline as a reference, encompassing 22 key items across biospecimen collection, blood processing, and eccDNA processing (Supplementary Table 3). The checklist includes most crucial and frequently underreported pre-analytical procedural details for eccDNA studies, and it provides researchers with a succinct and easy-to-read way of reporting methodology. Examples of included items are whole blood collection considerations (tube type, blood volume) as well as plasma/serum centrifugation information (speed, temperature, number of spins). Journals could encourage or require the submission of such checklist as supplementary material, similar to existing reporting guidelines used for systematic reviews. By ensuring that key pre-analytical details are transparently documented in publications, experiment reproducibility will be enhanced, and batch effects across studies would be reduced. This checklist may also be useful for biobanks that handle plasma or serum samples for eccDNA studies as a record for researchers to assess potential upstream sources of variability. Ultimately, improving methodological rigor and reporting standards will be essential for advancing eccDNA research toward clinical application in cancer diagnostics and monitoring. Our proposed checklist may provide a starting point for developing standardized reporting recommendations that are tailored specifically to eccDNA studies.

 

5. Consider expanding the conclusion to discuss broader impact on clinical translation of eccDNA as a liquid biopsy biomarker.

Response: Thank you for your suggestion. In our revised manuscript, we discussed briefly the impact on translation of eccDNA as cancer biomarker.

 

Conclusion (p. 13, lines 460)

Ultimately, greater transparency in eccDNA research helps improve study reproducibility and accelerate the clinical translation of eccDNA as a biomarker in cancer detection, prognosis, and monitoring.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

There are significant changes in the manuscript, however it still lack the proper graphical representation. Would it be possible to add some  figures and graphs too? It will definitely increase the value of your already well-written manuscript. There are several mechanisms and many molecules described in the paper, it may not be easy to digest for younger readers, and good pictures showing these pathways and molecules, also aberrant places, will definitely be of great value. 

Author Response

Response to REVIEWER #2

There are significant changes in the manuscript, however it still lacks the proper graphical representation. Would it be possible to add some figures and graphs too? It will definitely increase the value of your already well-written manuscript. There are several mechanisms and many molecules described in the paper, it may not be easy to digest for younger readers, and good pictures showing these pathways and molecules, also aberrant places, will definitely be of great value.

Response: We sincerely thank the Reviewer for this positive and constructive suggestion. We agree that graphical representations improve both the clarity and the accessibility of the manuscript. To address this comment, we have added two new figures (Figure 3 and Figure 4). Each new figure is now cited at the relevant point in the main text, and the corresponding figure legends have been added.

(1) New Figure 3 — study-level reporting completeness. To complement the item-level analysis in Figure 2, Figure 3 summarizes the reporting gaps from a study-level perspective. For each of the 14 included studies it shows the missing rate (100% − percentage of recommended items reported) for all 22 checklist items combined (panel a) and for the three pre-analytical domains separately: biospecimen collection (b), blood processing (c), and eccDNA processing (d). Studies are ordered consistently across panels and colour-coded by missing rate, so that readers can appreciate at a glance both the between-study variability and the domain in which reporting is weakest (blood processing, with a mean missing rate of 72%). Figure 3 is now cited in Section 3.2.
(page 9 lines 258-265)

Figure 3. Study-level reporting gaps, expressed as the missing rate (100% − percentage of recommended items reported) for each of the 14 included studies. Panels show (a) all 22 checklist items combined and the three pre-analytical domains: (b) Category A, biospecimen collection; (c) Category B, blood processing; and (d) Category C, eccDNA processing. Studies are ordered by overall missing rate, and bar color scales with the missing rate (blue, lower; red, higher). Dashed lines denote the mean missing rate within each panel. Reporting was least complete for blood processing (Category B; mean 72% missing), and no study reported more than 64% of all items.

(2) New Figure 4 — pre-analytical workflow and the molecular sources of bias. To directly address the Reviewer’s request for figures that depict the mechanisms, molecules, pathways and “aberrant places” described in the text, we converted the previously text-only mechanistic discussion into a schematic. Figure 4 presents the analytical workflow (A, biospecimen collection → B, blood processing → C, eccDNA three-step purification → detection by sequencing) together with the specific pre-analytical pitfall that arises at each step, annotated at the molecular level: (i) processing delay leads to leukocyte lysis and genomic-DNA contamination, diluting the already low-abundance eccDNA signal; (ii) inadequate centrifugation leaves residual cells and platelets that contribute contaminating linear DNA; (iii) freeze–thaw cycles and prolonged storage introduce single-strand nicks that relax supercoiled circles and ultimately linearize them, rendering them susceptible to the exonuclease step of purification and biasing recovery toward short, stable species; and (iv) because eccDNA is identified bioinformatically from split/junction reads, small undocumented variation propagates into systematic differences in measured abundance, fragment topology, and false-positive junction calls. A dedicated lower panel illustrates this molecular topology cascade (closed-circular and supercoiled → nicked and relaxed → linearized → degraded). The figure therefore conveys, in a single image, why complete pre-analytical reporting is essential for eccDNA studies. Figure 4 is now cited in the Discussion (page 11 lines 340-351).

We are grateful to the Reviewer for prompting these additions, which we believe materially improve the readability and value of the manuscript.

Figure 4. Pre-analytical workflow for plasma/serum eccDNA studies (left) and the mechanistic sources of bias introduced when individual steps are not standardized or reported (right). Across biospecimen collection (A), blood processing (B), and eccDNA processing (C), inadequate or undocumented handling promotes genomic-DNA contamination and alters the structure of circular molecules. The lower panel summarizes the resulting topology cascade: intact, exonuclease-resistant supercoiled eccDNA acquires single-strand nicks during freeze–thaw cycles and prolonged storage, relaxing and ultimately linearizing the circles so that they become susceptible to the exonuclease step of purification. Because eccDNA is quantified from split/junction reads, these undocumented changes propagate into systematic differences in measured abundance, fragment topology, and false-positive junction calls. Blue boxes denote workflow steps; red boxes denote the associated pre-analytical pitfalls.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for addressing the queries raised for this manuscript. The manuscript is in acceptable form.

Author Response

We sincerely thank the Reviewer for the positive evaluation of our revised manuscript. We are grateful for the Reviewer’s constructive comments. Thank you.