Molecular Testing in Indeterminate Thyroid Nodules: Genomic Landscape, Diagnostic Performance, and Integrated Risk-Stratified Management

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This commentary by Tanaka and coworkers summarizes, on the basis of the molecular landscape of thyroid tumors derived from the studies appeared to date in the literature, the genetic and molecular aspects of indeterminate thyroid nodules in comparison with conventional diagnostic findings and in association with them, to improve the diagnosis and allow the most  appropriate management, in particular avoiding unnecessary surgical choice. 

COMMENTS.

I believe this commentary of some interest in clinical practice because the knowledge of  molecular aspects of indeterminate thyroid nodules may address the best risk-stratified  management of affected patients, especially  in those whose nodules show cytological FNAB-derived findings  falling within the TIR class 3(3a and 3b),  according to the Bethesda System for Reporting Thyroid Cytopatology, which still remains a challenge for the physician.  However, even if the commentary is focused on the molecular aspects of thyroid nodules , I think that in Discussion the Authors should not ignore  some pervious papers  deserving discussion ,as they may help the readers to complete their knowledge on this topic.  For example, I think useful to the discussion the review on the clinical and molecular aspects of the aggressive forms of thyroid carcinoma(Skipor S et al ,Int J Mol Sci,2025) and the study on the gene panel of pediatric thyroid cancer(Alzaharani A.S. et al, J Clin Endocrinol Metab, 2020) because they may contribute to address the physician to  the best diagnostic and therapeutic decision in some particular cases.

Author Response

Comment:

The reviewer suggested inclusion of additional literature regarding aggressive thyroid carcinoma and pediatric thyroid cancer molecular panels.

Response:

We thank the reviewer for these valuable suggestions. We have added the recommended references and incorporated them into the revised manuscript.

 

Specifically:

* The review regarding aggressive thyroid carcinomas (Schipor et al., Int J Mol Sci, 2025) was added to the discussion of advanced thyroid cancers and aggressive molecular alterations in Section 2.

* The pediatric thyroid cancer molecular panel study (Alzahrani et al., J Clin Endocrinol Metab, 2020) was incorporated into the discussion of targeted molecular panels in Section 3.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors made a comprehensive synthesis of the molecular landscape of thyroid tumors and the evolving role of molecular testing in the management of indeterminate thyroid nodules. Its central strength lies in its clear articulation of the biological continuum of thyroid neoplasia and the argument that molecular diagnostics must be interpreted within an integrated, context‑dependent framework. However, despite its breadth, the manuscript exhibits several conceptual and structural limitations that reduce its impact as a scholarly review.

A major strength is the detailed overview of genomic alterations relevant to thyroid tumorigenesis, including BRAF‑like and RAS‑like molecular subtypes, TERT promoter mutations, and gene fusions involving RET, NTRK, and ALK. The authors effectively highlight the clinical relevance of these alterations, particularly the synergistic risk associated with coexistent BRAF V600E and TERT promoter mutations. The discussion is well supported by contemporary literature and aligns with recent WHO and ATA frameworks. The manuscript also succeeds in emphasizing the probabilistic nature of molecular findings and the need to integrate them with cytomorphology, ultrasound risk stratification, and clinical context.

However, the review is largely descriptive and does not sufficiently critique the limitations of existing molecular platforms. While the authors acknowledge issues such as variable specificity, prevalence‑dependent predictive values, and the presence of mutations in both benign and malignant lesions, these points are not explored with the depth expected in a critical commentary. For example, the discussion of RAS mutations could benefit from a more nuanced examination of their low positive predictive value and the clinical consequences of over‑reliance on such markers. Similarly, the section on proteomics and multi‑omics integration is brief and lacks critical appraisal of methodological challenges, validation gaps, and real‑world feasibility. Moreover, these strategies could be useful in managing cases with giant thyroid tumors, reference this to doi: 10.3390/biomedicines12102204.

The manuscript’s repeated emphasis on “context‑dependent interpretation” is conceptually sound but becomes redundant without offering concrete clinical algorithms or decision pathways. Figures 1–3 illustrate conceptual frameworks, yet the text does not translate these into actionable guidance for clinicians. The review would be strengthened by including case‑based examples demonstrating how molecular results modify risk estimates in specific Bethesda III/IV scenarios.

Another limitation is the extensive focus on system‑level factors such as diagnostic thresholds, category utilization, and regional practice variation. While these are important considerations, the discussion becomes overly long and shifts the manuscript away from its stated purpose of reviewing molecular testing. The section on detection–intervention mismatch, although relevant to thyroid cancer epidemiology, feels tangential and insufficiently tied to molecular diagnostics.

Finally, the conclusion is underdeveloped. It reiterates generic expectations for a discussion section rather than synthesizing the manuscript’s key insights or proposing specific future research directions. This weakens the overall cohesion of the review.

 

Author Response

Comment:

The manuscript is largely descriptive and lacks sufficient critical appraisal of molecular testing limitations.

 

Response:

We appreciate this important comment and substantially strengthened the critical discussion throughout the manuscript.

 

The revised manuscript now includes additional discussion regarding:

 

* the limited specificity and variable positive predictive value of RAS mutations,

* overlap between benign and malignant follicular-patterned lesions,

* the probabilistic nature of molecular findings,

* the potential role of active surveillance in selected RAS-mutated nodules,

* selection bias in surgically resected cohorts,

* and variability in predictive values across healthcare systems.

 

These revisions were primarily added to Sections 2–4.

 

Comment:

The discussion of proteomics and multi-omics approaches lacked critical appraisal.

 

Response:

We agree and expanded this section by discussing important limitations of emerging multi-omics approaches, including:

 

* limited external validation,

* interplatform reproducibility,

* cost,

* lack of standardization,

* and variable availability across healthcare systems.

 

We also added discussion regarding potential applications in complex or biologically heterogeneous thyroid tumors.

 

Comment:

The manuscript repeatedly emphasizes “context-dependent interpretation” without actionable clinical examples.

 

Response:

To address this concern, we revised and reorganized Sections 6 and 7. We added quantitative and practice-based examples illustrating how differences in indeterminate category utilization influence baseline ROM and downstream molecular test performance.

 

For example, we added discussion indicating that in settings where FN utilization remains around 3%, malignancy rates among surgically resected nodules may exceed 60–70%, whereas broader FN utilization lowers baseline ROM and alters molecular test performance.

 

Comment:

The manuscript overemphasizes system-level issues and shifts away from molecular testing.

 

Response:

We appreciate this observation and attempted to improve balance by streamlining and reorganizing these sections. However, we respectfully believe that system-level interpretation is inseparable from real-world molecular test performance because predictive values and clinical utility are fundamentally influenced by disease prevalence, diagnostic thresholds, and healthcare structure.

 

Rather than removing this discussion, we revised the text to better connect system-level factors directly to molecular test interpretation and clinical decision-making.

 

Comment:

The conclusion was underdeveloped.

 

Response:

The Conclusions section was completely rewritten. The revised conclusion now provides a concise synthesis of the manuscript’s central concepts, including:

 

* integrated interpretation of molecular findings,

* biology-based risk stratification,

* context-dependent molecular testing,

* and future perspectives involving multimodal and AI-assisted risk assessment.

Reviewer 3 Report

Comments and Suggestions for Authors

This commentary reviews the evolving role of molecular testing in the management of cytologically indeterminate thyroid nodules, highlighting that genomic alterations should be interpreted within a broader framework including cytomorphology, ultrasound findings, and clinical risk assessment.

Issues to be addressed:

-The Conclusions section contains placeholder editorial instructions (“Authors should discuss the results…”) and not a finalized conclusion. This should be replaced with a synthesis of the review key messages and future perspectives.

- The manuscript lacks critical comparative analysis of the different molecular platforms. Although several molecular tests are summarized, the discussion remains descriptive. The manuscript would benefit from a more critical comparison of diagnostic accuracy, real-world performance, limitations across healthcare systems, and head-to-head differences between platforms.

-Given the emphasis on risk stratification, the review could be strengthened by discussing emerging AI-assisted cytology, digital pathology, or multimodal predictive models that integrate imaging, molecular, and histopathologic data.

-Paragraph 6 (“Diagnostic Thresholds, Disease Prevalence, and System Behavior”) is informative, but too concise. The concepts are valid and important, but the section would benefit from at least one concrete clinical example or quantitative illustration demonstrating how changes in malignancy prevalence impacts clinical decisions. As written, it sounds theoretical rather than clinically actionable.

-Paragraph 8 (“System-Level Interpretation of Molecular Testing”) is too short relative to the importance of the topic. It essentially repeats ideas already discussed in Sections 5–7 without adding new insight. The section could either be expanded or merged with Section 6. For example, Authors could discuss differences between Asian vs Western diagnostics, economic implications, multidisciplinary decision-making models, or how molecular testing alters real-world surgical rates and patient outcomes.

Author Response

Comment:

The Conclusions section contained placeholder editorial instructions.

 

Response:

We sincerely apologize for this oversight. The Conclusions section was completely rewritten and finalized.

 

Comment:

The manuscript lacked critical comparative analysis of molecular platforms.

 

Response:

We expanded the discussion of molecular platform limitations by addressing:

 

* variability in predictive values,

* prevalence-dependent performance,

* referral bias,

* and healthcare-system differences influencing clinical utility.

 

We also emphasized that molecular testing should not be interpreted as a universally interchangeable diagnostic solution.

 

Comment:

The reviewer suggested discussion of AI-assisted cytology, digital pathology, and multimodal predictive models.

 

Response:

We appreciate this excellent suggestion. We incorporated this perspective into the revised Conclusions section and future outlook discussion, where we now discuss emerging multimodal and artificial intelligence-assisted risk stratification approaches.

 

Comment:

Section 6 was too theoretical and required practical examples.

 

Response:

We revised and expanded Section 6 by adding quantitative and clinically contextualized examples demonstrating how differences in diagnostic thresholds and indeterminate category utilization influence molecular test performance and surgical selection.

 

Comment:

Section 8 was too short and repetitive.

 

Response:

Section 8 was substantially expanded and reorganized. The revised section now discusses:

 

* healthcare ecosystem differences,

* Asian versus Western thyroid nodule practice,

* multidisciplinary interpretation,

* disease prevalence,

* healthcare infrastructure,

* and context-dependent utilization of molecular testing.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

issues have been addressed