Review Reports - Epigenetic and Liquid Biopsy Biomarkers in Prostate Cancer: Bridging Tumor Heterogeneity and Clinical Implementation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review article titled "Epigenetic and liquid biopsy biomarkers in prostate cancer: Bridging tumor heterogeneity and clinical implementation” presents an overview of recent advances in molecular diagnostics for prostate cancer. The focus on epigenetic regulation and liquid biopsy-based biomarkers is relevant. The authors emphasize prostate cancer heterogeneity and the need for minimally invasive biomarkers in clinical practice.

The manuscript is generally well organized. The tables summarizing molecular assays and commercially available test platforms are of practical value for readers. These elements help connect molecular discoveries with real-world applications.

That said, some sections would benefit from tighter synthesis. In Sections 3.1–3.3, multiple examples of microRNAs and long non-coding RNAs are described individually, which leads to some redundancy. A more comparative or integrative discussion would strengthen these sections. At times, the narrative shifts between descriptive and analytical styles without clearly linking the findings to clinical relevance or strength of evidence.

The authors are encouraged to cite the given prior work demonstrating ligand-directed delivery of chemically modified microRNAs to prostate cancer cells, as it directly aligns with the manuscript’s focus on epigenetic regulation, tumor heterogeneity, and translational biomarker–guided therapeutic strategies. This study provides a relevant example of how small molecules targeting and endosomal escape can improve specificity and functional efficacy, addressing key barriers to clinical implementation discussed in the review.

https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(24)00080-5

The “Materials and Methods” section lacks sufficient quantitative detail. Information such as the number of studies screened, inclusion criteria, and selection metrics would improve transparency and methodological rigor. Figures and tables are informative but could be more standardized. Including key performance metrics, such as sensitivity, specificity, and AUC, would better highlight clinical relevance.

The discussion of liquid biopsy components, including ctDNA, CTCs, and exosomes, is conceptually strong. However, clearer separation between biomarkers that are clinically validated and those still under investigation would improve clarity. The “Clinical Implementation” section is effective overall but could be strengthened by incorporating cost-effectiveness data and evidence from real-world validation studies.

Some statements, particularly those related to emerging lncRNA panels or cuproptosis-associated non-coding RNAs, may overstate translational readiness. These claims would benefit from clearer qualification or additional citation of prospective clinical evidence.

In summary, this review discusses epigenetic and liquid biopsy biomarkers in prostate cancer, highlighting their relevance to tumor heterogeneity and clinical translation. With stronger critical synthesis, and improved methodological detail, the manuscript would be significantly strengthened and better positioned for publication.

Author Response

We would like to thank the Reviewer for the thorough, constructive, and insightful evaluation of our review manuscript entitled “Epigenetic and liquid biopsy biomarkers in prostate cancer: Bridging tumor heterogeneity and clinical implementation”. We appreciate the positive assessment of the manuscript’s relevance, organization, and practical value, particularly regarding the integration of molecular biomarkers with clinical application.
All comments have been carefully considered, and the manuscript has been revised accordingly. All changes are highlighted in the revised version (track changes).

Point-by-Point Response to Comments and Suggestions:

Comment 1

“Some sections would benefit from tighter synthesis. In Sections 3.1-3.3, multiple examples of microRNAs and long non-coding RNAs are described individually, which leads to some redundancy. A more comparative or integrative discussion would strengthen these sections.”

Response 1
We agree with the Reviewer’s comment. Sections 3.1 (MicroRNAs), 3.2 (Long non-coding RNAs), and 3.3 (circRNAs) have been revised to improve synthesis and reduce redundancy. Specifically, we streamlined descriptive lists of individual ncRNAs and added comparative and integrative commentary emphasizing shared biological roles, overlaps in clinical relevance, and common translational limitations.

These revisions strengthen the analytical narrative and more clearly link biomarker classes to tumor heterogeneity and clinical applicability.

Location of changes:

Section 3.1, paragraphs 6-8
Section 3.2, paragraphs 3-5
Section 3.3, entire section (concluding paragraph expanded)

Comment 2

“The authors are encouraged to cite the given prior work demonstrating ligand-directed delivery of chemically modified microRNAs to prostate cancer cells…”

Response 2
We thank the Reviewer for highlighting this highly relevant study. We have now incorporated this reference and expanded the discussion to explicitly link it to epigenetic regulation, tumor heterogeneity, and translational barriers in prostate cancer.

A new paragraph has been added to Section 3.1, illustrating how ligand-directed, small-molecule-mediated delivery of chemically modified miRNAs addresses key challenges such as tumor specificity, endosomal escape, and functional efficacy. This example directly supports the translational perspective of biomarker-guided precision therapeutics emphasized throughout the review.

Location of changes:

Section 3.1 (MicroRNAs), final paragraph
Newly cited as Reference [37]

Comment 3

“The “Materials and Methods” section lacks sufficient quantitative detail…”

Response 3
We agree and have substantially revised the Materials and Methods section to improve transparency and methodological rigor. We now explicitly report:

the total number of records initially identified (n= 335),
inclusion and exclusion criteria,
the stepwise selection process (title/abstract screening and full-text review),
and the prioritization strategy for clinically and translationally relevant studies.

This revision clarifies the narrative review methodology and aligns it with best practices for transparent literature synthesis.

Location of changes:

Section 2 (Materials and Methods), entire section revised

Comment 4

“Figures and tables are informative but could be more standardized. Including key performance metrics, such as sensitivity, specificity, and AUC, would better highlight clinical relevance.”

Response 4
We agree with this recommendation. Where available, we have now explicitly included or clarified key performance metrics (e.g., sensitivity, specificity, prognostic relevance) in the descriptive text accompanying Tables 1-3 and in the Clinical Implementation section.

We also standardized table structure by clearly distinguishing:

biomarkers with guideline acknowledgment or regulatory approval,
biomarkers supported by retrospective or exploratory evidence,
and those still at an early research stage.

Location of changes:

Tables 1-3 (descriptive clarifications)
Section 6 (Practical Clinical Use of Molecular and Epigenetic Biomarkers)

Comment 5

“Clearer separation between clinically validated biomarkers and those still under investigation would improve clarity.”

Response 5
We fully agree. This distinction has now been made explicit throughout the manuscript. We systematically differentiate:

biomarkers with regulatory approval or guideline acknowledgment (e.g., PCA3, ConfirmMDx, ExoDx, ctDNA assays),
from investigational or emerging candidates (e.g., novel lncRNA panels, circRNAs, cuproptosis-related signatures).

This clarification is particularly emphasized in Sections 4 (Liquid Biopsy), 6 (Practical Clinical Use), and Table 6-7.

Location of changes:

Sections 4 and 6
Tables 6 and 7 (column “Level of evidence / guideline inclusion”)

Comment 6

“The “Clinical Implementation” section could be strengthened by incorporating cost-effectiveness data and real-world validation studies.”

Response 6
We agree and have expanded the Clinical Implementation section to include discussion of cost-effectiveness considerations, scalability, and real-world validation data where available (e.g., urine-based tests used for biopsy triage and ctDNA testing in advanced disease).

We also emphasize that cost, reimbursement, and infrastructure remain key determinants of clinical adoption, particularly for multi-analyte panels.

Location of changes:

Section 6, paragraphs 5-7

Comment 7

“Some statements, particularly those related to emerging lncRNA panels or cuproptosis-associated non-coding RNAs, may overstate translational readiness.”

Response 7
We appreciate this important observation and have revised the relevant passages to adopt more cautious language. Claims regarding translational readiness have been explicitly qualified, and we now clearly state that these lncRNA panels are hypothesis-generating and investigational, requiring prospective validation and demonstration of added clinical value.

Location of changes:

Section 3.2 (Long non-coding RNAs), final paragraph
Table 3 (Evidence status & limitations)

Additional Clarifications

We would like to emphasize that the revised manuscript now places stronger focus on:

critical synthesis rather than enumeration of biomarkers,
explicit separation between clinical practice and research-stage evidence,
and realistic assessment of translational barriers, including cost, standardization, and guideline perspectives.

We sincerely thank the Reviewer for the valuable comments, which have significantly strengthened the clarity, rigor, and translational relevance of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Overall excellent review. Very informative and seemed pretty extensive. Minor comments below.

Intro: line 58: how were these tests (PSA, PHI, PCa3, etc) chosen? Is this all inclusive? What disease setting are the authors discussing? Early stage, active surveillance, metastatic? Is this summarizing table 3?

Section 3: how frequently is ConfirmMDx utilized? Do the authors have data on real-world or trial data on usage/outcomes /sensitivity/specificity?

3.1: are there any miRNA tests being developed commercially? Any panels?

3.2: what is clinical uptake of the PCA3 test? Include some clinical trials/real world data on outcomes / sensitivity/specificity

(ref 15-22)

Table 1 is a great addition to the paper. Would add another column to include level of evidence, eg. NCCN/guidelines/FDA approval (if relevant)

Section 4.2: which FDA approved CTC test?

Table 2 is a wonderful addition. Would add some references for readers who want to dive deeper. Some of the categories are also vague, would add in range of cost, more details on standardization, turnaround time, etc

Figure 2: individual components are not labeled/distinguished

Excellent table 3

Author Response

We sincerely thank the Reviewer for the very positive evaluation of our manuscript and for the constructive and thoughtful minor comments. We appreciate the recognition of the review’s scope, clarity, and practical value. All suggestions were carefully considered, and the manuscript has been revised accordingly. All changes are highlighted in the revised version (track changes).

Point-by-Point Response to Comments

Comment 1

“Intro: line 58: how were these tests (PSA, PHI, PCA3, etc.) chosen? Is this all-inclusive? What disease setting are the authors discussing? Early stage, active surveillance, metastatic? Is this summarizing table 3?”

Response 1
Thank you for this important clarification request. We have revised the Introduction to explicitly state the rationale for selecting these tests. The listed biomarkers (PSA, PHI, PCA3, 4Kscore) were chosen as representative, clinically established or guideline-acknowledged tools most commonly used in early detection, biopsy triage, and risk stratification, rather than as an exhaustive list.

We also clarified that the review intentionally spans multiple disease settings, including:

early detection and biopsy decision-making,
active surveillance and localized disease,
advanced and metastatic prostate cancer.

Additionally, we clarified that this introductory paragraph conceptually introduces biomarker categories later detailed in Tables 6 and 7, rather than summarizing Table 3 (lncRNAs).

Location of changes:

Introduction, paragraph 2 (around former line 58)

Comment 2

“Section 3: how frequently is ConfirmMDx utilized? Do the authors have data on real-world or trial data on usage/outcomes /sensitivity/specificity?”

Response 2
We agree that utilization and real-world relevance require clarification. In Section 3, we expanded the discussion of ConfirmMDx to include:

Its role is primarily in repeat-biopsy decision support after a negative biopsy,
reported sensitivity (~74%) and specificity (~60%) values from validation studies,
and the fact that real-world utilization remains limited and variable, particularly in the post-mpMRI era.

We also emphasize that while ConfirmMDx is acknowledged in NCCN guidelines, its use is optional and context-dependent, with declining uptake due to increased reliance on mpMRI.

Location of changes:

Section 3 (Epigenetic Biomarkers), paragraph discussing ConfirmMDx
Table 1 (Evidence status & limitations column expanded)

Comment 3

“3.1: are there any miRNA tests being developed commercially? Any panels?”

Response 3
Thank you for raising this point. We have expanded Section 3.1 to explicitly address the commercial development status of miRNA-based assays. We now note that:

no miRNA- only test is currently guideline-endorsed,
several multi-miRNA panels are in early commercial or pre-commercial stages (e.g., urine exosome–based assays),
and that lack of transparency regarding panel composition and assay standardization remains a limitation.

We emphasize that miRNA biomarkers are currently best viewed as components of multi-analyte panels, rather than standalone diagnostic tools.

Location of changes:

Section 3.1 (MicroRNAs), penultimate paragraph

Comment 4

“3.2: what is clinical uptake of the PCA3 test? Include some clinical trials/real world data on outcomes / sensitivity/specificity (ref 15-22)”

Response 4
We appreciate this suggestion and have strengthened the PCA3 discussion accordingly. Section 3.2 now includes:

real-world and trial-based estimates of PCA3 sensitivity (generally moderate) and specificity (higher than PSA),
clarification that PCA3 uptake has declined in recent years, particularly with the adoption of mpMRI,
and reinforcement that PCA3 is most useful in repeat biopsy decision-making, not as a standalone diagnostic tool.

Key clinical studies and reviews (references 15-22) are now more explicitly integrated into the narrative.

Location of changes:

Section 3.2 (Long non-coding RNAs), PCA3 subsection

Comment 5

“Table 1 is a great addition. Would add another column to include level of evidence, e.g., NCCN/guidelines/FDA approval (if relevant).”

Response 5
We thank the Reviewer for this helpful suggestion. Table 1 has been revised to more explicitly reflect level of evidence and guideline status, including:

NCCN acknowledgment,
investigational status per EAU,
and lack of FDA approval where applicable.

This information is now clearly integrated within the “Routine clinical use?” and “Evidence status & limitations” columns.

Location of changes:

Table 1 (revised column descriptions and content)

Comment 6

“Section 4.2: which FDA approved CTC test?”

Response 6
Thank you for pointing out the need for specificity. We have now explicitly named CellSearch® as the FDA-cleared CTC enumeration system used in metastatic prostate cancer for prognostic purposes.

Location of changes:

Section 4.2 (Circulating Tumor Cells), first paragraph

Comment 7

“Table 2 is a wonderful addition. Would add some references for readers who want to dive deeper. Some of the categories are also vague; would add range of cost, more details on standardization, turnaround time, etc.”

Response 7
We appreciate this detailed feedback. Table 2 has been revised to:

include clearer references within the “Evidence status & limitations” column,
clarify terminology related to clinical use and investigational status,
and expand the accompanying text to discuss cost, turnaround time, and standardization challenges at a conceptual level.

Given the variability across laboratories and countries, we opted to discuss cost and logistics qualitatively in the text rather than assigning fixed numerical ranges that may not generalize.

Location of changes:

Table 2
Section 6 (Practical Clinical Use of Molecular and Epigenetic Biomarkers)

Comment 8

“Figure 2: individual components are not labeled/distinguished”

Response 8
We agree and have revised Figure 2 to clearly label and distinguish individual components, including exosomes, miRNAs, lncRNAs, and circulating tumor cells, improving clarity and interpretability.

Location of changes:

Figure 2 (revised labeling)

Overall, the manuscript has been refined to:

improve transparency regarding clinical uptake and real-world use,
clearly distinguish guideline-supported assays from investigational biomarkers,
and enhance practical relevance for clinicians and translational researchers.

We are grateful for the Reviewer’s constructive input, which has helped further strengthen the clarity and clinical orientation of the review.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides a broad narrative overview of epigenetic and liquid biopsy biomarkers in prostate cancer, covering DNA methylation, non-coding RNAs, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and extracellular vesicles. While the topic is clinically relevant and the review is generally well written, the manuscript does not meet the publication standards for Cancers in its current form, primarily due to limitations in novelty, methodological rigor, and critical technical appraisal.

First, the manuscript lacks a clear novel or original perspective. Most of the topics discussed—such as GSTP1 methylation, PCA3, miRNAs, ctDNA, CTCs, and commercially available urine-based assays—have been extensively reviewed in recent years, including in Cancers and related journals. The current review essentially reiterates established knowledge without offering a new conceptual framework, critical prioritization of biomarkers, or explicit guidance on unresolved clinical decision points. Although tumor heterogeneity is highlighted as a central challenge, the manuscript does not clearly articulate how the reviewed technologies differentially address spatial versus temporal heterogeneity, nor does it convincingly link biomarker detection to meaningful improvements in clinical outcomes.

Second, the review's methodological rigor is insufficient. Although search terms and a general time frame are provided, the manuscript lacks transparency regarding literature selection and evaluation. There is no description of how studies were screened, how evidence quality was assessed, or how conflicting results were handled. As a result, the completeness, reproducibility, and objectivity of the review cannot be adequately evaluated. Even for a narrative review, a more structured approach—such as a scoping review framework or explicit stratification by level of evidence—would be expected for a journal of this scope.

Third, and most critically, the manuscript does not sufficiently address the technical and quantitative limitations that ultimately determine clinical feasibility. For example, CTC-based assays are presented as promising tools, yet the fundamental physical and biological constraints of CTC detection are not discussed. Detection from a few milliliters of blood requires a substantial tumor burden, making CTCs inherently unsuitable for early cancer detection or screening. Moreover, issues related to quantitative reliability—such as counting precision at low CTC numbers, inter-operator and inter-platform variability, and the distinction between biological signal and measurement noise—are not addressed.

Similarly, for RNA-based biomarkers, normalization strategies and reproducibility concerns are largely absent. Urinary PCA3 is normalized to PSA mRNA rather than creatinine, a distinction with essential implications for urine dilution effects and inter-individual variability, yet this is not discussed. Plasma miRNAs, such as miR-16, are reported as biomarkers without clarification of whether they are used as targets or as reference controls, despite well-known problems related to hemolysis, platelet contamination, and normalization instability. For tissue-based biomarkers, increasing analytical sensitivity and decreasing input material may exacerbate the impact of intratumoral heterogeneity—particularly in multifocal prostate cancer—yet this critical trade-off is not considered.

In addition, the manuscript does not sufficiently address pre-analytical and logistical challenges, including blood and urine collection conditions, sample preservation, transport, batch effects, and the stringency of standard operating procedures required for reproducible measurement. These factors are central to real-world clinical implementation, especially in multicenter or routine diagnostic settings, but are largely overlooked. The discussion of multi-marker panels also lacks a balanced assessment of the increased costs, greater complexity in quality control, and reduced long-term reproducibility compared with simpler single-analyte assays.

Finally, the review does not adequately engage with the broader context of population-level screening and risk stratification. The historical limitations of PSA-based screening—including low specificity, overdiagnosis, and lack of mortality benefit in specific populations—are not critically examined as a cautionary framework for evaluating new biomarkers. The potential role of pre-test risk enrichment (e.g., age, family history, lifestyle factors) to improve biomarker performance and clinical utility is also underdeveloped.

In summary, while the manuscript offers a biologically comprehensive overview, it lacks sufficient novelty, methodological transparency, and critical discussion of quantitative, technical, and translational constraints. These limitations substantially reduce its value for clinicians and researchers seeking realistic guidance on biomarker implementation. Addressing these issues would require a fundamental restructuring of the manuscript rather than incremental revision. So, I think it's a good idea to reject the manuscript.

Author Response

We thank the Reviewer for the extensive, detailed, and highly technical critique of our manuscript. We appreciate the recognition of the clinical relevance of the topic and the overall clarity of the manuscript. We respectfully disagree, however, with the conclusion that the manuscript lacks sufficient novelty, methodological rigor, and critical technical appraisal.

Importantly, many of the key concerns raised by the Reviewer-particularly those related to technical, quantitative, and translational limitations-have been explicitly addressed and substantially expanded in the revised version of the manuscript. Below, we respond point-by-point and indicate where the revised manuscript directly engages with the issues raised.

Point-by-Point Response to Major Comments

Comment 1: Lack of novelty and original perspective

“The manuscript reiterates established knowledge without offering a new conceptual framework or clear guidance on unresolved clinical decision points. Tumor heterogeneity is highlighted but not meaningfully operationalized.:

Response 1
We respectfully disagree that the manuscript lacks an original perspective. While we acknowledge that many individual biomarkers discussed have been previously reviewed, the novel contribution of this review lies in its integrative and translational framework, which explicitly connects:

Tumor heterogeneity (spatial and temporal)
Epigenetic and liquid biopsy biomarker classes
Realistic clinical implementation pathways and guideline perspectives

In the revised manuscript, we have strengthened this conceptual framework by:

explicitly differentiating spatial heterogeneity (e.g., multifocal primary tumors, tissue-based assays) from temporal heterogeneity (e.g., clonal evolution monitored via ctDNA and CTCs),
clarifying how individual technologies address (or fail to address) these dimensions,
and emphasizing why no single biomarker can adequately capture prostate cancer biology in isolation.

This integrative, clinically grounded synthesis-rather than the introduction of a new biomarker-is the intended novel perspective of the review.

Location of changes:

Introduction, final paragraph
Section 4 (Liquid Biopsy), introductory paragraphs
Section 5 (Critical Technical and Translational Limitations)

Comment 2: Insufficient methodological rigor and transparency

“The manuscript lacks transparency regarding study selection, evidence evaluation, and handling of conflicting data.”

Response 2
We agree that transparency is essential, even for a narrative review. Accordingly, the Materials and Methods section has been substantially revised to improve methodological clarity and reproducibility.

The revised version now includes:

explicit reporting of the number of records screened (n= 335),
detailed inclusion and exclusion criteria,
a stepwise description of title/abstract screening and full-text evaluation,
prioritization criteria emphasizing clinical and translational relevance,
and acknowledgment that the review is narrative rather than systematic, with rationale provided.

Additionally, throughout the manuscript, we now explicitly stratify evidence as:

guideline-endorsed / FDA-approved,
clinically validated but optional,
investigational / hypothesis-generating.

Location of changes:

Section 2 (Materials and Methods), entire section revised
Tables 1-3, 6-7 (Evidence status clarified)

Comment 3: Inadequate discussion of CTC technical and quantitative limitations

“CTCs are presented as promising without sufficient discussion of biological constraints, tumor burden requirements, and quantitative reliability.”

Response 3
We agree that these limitations are critical and wish to emphasize that they are now explicitly and extensively discussed in the revised manuscript.

A dedicated subsection (Section 5.1) now clearly states that:

CTCs are not suitable for early detection or screening due to extreme rarity and dependence on tumor burden,
quantitative reliability at low CTC counts is limited,
inter-platform and inter-operator variability remains a major barrier,
and CTC assays are clinically useful only in advanced disease settings.

These points are further summarized in Table 5, which explicitly links biological constraints to clinical implications.

Location of changes:

Section 5.1 (CTCs Are Not Suitable for Screening)
Table 5 (Technical limitations summary)

Comment 4: RNA biomarker normalization, reproducibility, and analytical trade-offs

“Key issues related to PCA3 normalization, miRNA hemolysis sensitivity, and analytical sensitivity versus tumor heterogeneity are not addressed.”

Response 4
We appreciate this technically precise critique and would like to clarify that these issues are now explicitly addressed in the revised manuscript, including:

PCA3 normalization to PSA mRNA rather than creatinine, with discussion of urine dilution and sampling variability (Section 5.4),
miRNA susceptibility to hemolysis, platelet contamination, and lack of consensus normalization strategies, including the limitations of miR-16 (Section 5.3),
the trade-off between increasing analytical sensitivity and amplifying biological noise in heterogeneous, multifocal tumors (Section 5.5).

These discussions directly address the Reviewer’s concerns regarding quantitative reliability and translational realism.

Location of changes:

Sections 5.3-5.5
Table 5 (RNA-related limitations)

Comment 5: Pre-analytical, logistical, and SOP-related challenges

“The manuscript overlooks sample handling, preservation, transport, batch effects, and SOP standardization.”

Response 5
We agree that these issues are central to real-world implementation. A new dedicated subsection (Section 5.7) now discusses:

pre-analytical variability in blood and urine collection,
impact of processing delays and storage conditions,
lack of harmonized SOPs across centers,
and how these factors can outweigh biological signal.

We also emphasize that these challenges are a major reason for the cautious stance of clinical guidelines, which is explicitly discussed in Section 5.8.

Location of changes:

Sections 5.7 and 5.8
Table 5 (Pre-analytical variability)

Comment 6: Multi-marker panels, cost, and reproducibility

“The discussion of multi-marker panels lacks a balanced assessment of cost, complexity, and reproducibility.”

Response 6
We agree and have revised the manuscript to present a balanced, critical assessment of multi-marker panels, explicitly stating that:

while they may better capture heterogeneity,
they are associated with higher cost, bioinformatic complexity, and reduced scalability,
and their long-term reproducibility and reimbursement remain uncertain.

This discussion is now integrated into both Sections 5.6 and 6.

Location of changes:

Section 5.6 (Cost and Complexity of Multimarker Panels)
Section 6 (Clinical Use)

Comment 7: Population-level screening and PSA as a cautionary framework

“The manuscript does not sufficiently engage with population screening failures and risk enrichment strategies.”

Response 7
We appreciate this important contextual point. The revised manuscript now explicitly frames PSA-based screening as a historical cautionary example, emphasizing:

overdiagnosis,
limited mortality benefit in certain populations,
and the risks of deploying biomarkers without appropriate pre-test risk enrichment.

We further discuss the role of clinical risk models, family history, age, and imaging as necessary complements to molecular biomarkers.

Location of changes:

Section 6 (Clinical Implementation)
Conclusions

While we acknowledge that the review does not introduce new biomarkers per se, we respectfully emphasize that Cancers is a journal that publishes high-quality integrative and translational reviews, and the revised manuscript now provides:

a clear conceptual framework centered on tumor heterogeneity,
transparent methodology appropriate for a narrative review,
and an unusually detailed, explicit critique of technical, quantitative, and translational barriers, which directly addresses the Reviewer’s core concerns.

We therefore believe that, following substantial restructuring and expansion, the manuscript now meets the scientific and clinical standards of Cancers and offers realistic, practice-oriented guidance to both clinicians and researchers.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all comments from the previous reviewers, reflecting careful attention to detail and a strong commitment to manuscript improvement. The work now meets the expected standards of scientific quality and rigor for publication.

Author Response

We sincerely thank the reviewer for the positive and encouraging evaluation of our manuscript. We greatly appreciate the recognition of our efforts to carefully address all comments and improve the quality of the work. We are pleased that the revised version now meets the expected standards of scientific quality and rigor for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is scientifically sound, wellstructured, and all major concerns have been adequately addressed.

I have only two minor, language-related suggestions to improve clarity and readability further:

Sentence length and readability: In a few sections (particularly in the discussion of technical and translational limitations), some sentences are relatively long and contain multiple ideas. While the meaning is clear, splitting a small number of these sentences or simplifying their structure could improve readability for a broad clinical and research audience.
Terminology consistency: The terms “clinical implementation”, “clinical use”, and “routine clinical use” are sometimes used interchangeably. Although the intended meaning is generally clear from context, using more consistent terminology (or briefly clarifying distinctions where relevant) would improve precision.

These are minor stylistic points and do not affect the scientific content or conclusions of the manuscript.

Author Response

We thank the reviewer for their positive assessment of the manuscript. In response to the minor language-related suggestions, we have revised selected long sentences - particularly in the sections discussing technical and translational limitations - to improve readability. In addition, terminology referring to “clinical implementation,” “clinical use,” and “routine clinical use” has been harmonized throughout the manuscript, with consistent usage aligned to guideline status and clinical context. These changes do not affect the scientific content or conclusions of the work.