-->PONE-D-26-18193-->-->68Ga-FAPI-04 PET/CT has high diagnostic efficacy in Tg-positive and Tg-negative differentiated thyroid cancer-->-->PLOS One

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This retrospective study aimed to evaluate the diagnostic performance of 68Ga-FAPI-04 PET/CT in 147 patients with differentiated thyroid cancer (DTC) after surgery and 131I therapy. The authors compare this emerging imaging modality specifically with the clinical standard of serum thyroglobulin (Tg) testing in different thyrotropin (TSH) states. Strengthens: Clinical Utility in Tg-Negative Cases: The most significant contribution is the identification of metastatic lesions in eight Tg-negative patients, primarily in lymph nodes. This addresses a critical "blind spot" in standard DTC follow-up where low Tg levels might provide a false sense of security. Performance Across TSH States: The data demonstrates that 68Ga-FAPI-04 maintains high accuracy in both TSH-suppressed (95.96%) and TSH-stimulated (100%) states. This suggests patients may not need to undergo the discomfort of levothyroxine withdrawal to achieve high-quality imaging. Quantitative Threshold Optimization: The authors performed a sophisticated analysis of Target-to-Background Ratios (TBR). Identifying the mediastinum as the optimal background reference and establishing a threshold of 1.428 provides clinicians with actionable quantitative criteria for interpreting these scans. Comparison to Standard of Care: By directly comparing PET/CT metrics against Tg sensitivity and specificity, the study frames the new modality within the context of current clinical workflows. Limitations: Retrospective Design and Potential Bias: As a retrospective study, there is inherent selection bias. Furthermore, only 29 of the 147 participants had their PET/CT findings confirmed via histopathology; the remainder relied on clinical follow-up, which is less definitive. The "Inflammation" Hurdle: Consistent with 68Ga-FAPI's known limitations, the study reported false positives in lymph nodes due to inflammatory changes. While the authors addressed this through TBR optimization, the risk of "over-calling" reactive nodes remains a concern for surgical planning. Lack of Direct Comparison with 18F-FDG: Although the authors cite literature comparing FAPI to FDG, this specific cohort was not tested head-to-head. Given that FDG is the current alternative for Tg-positive/Iodine-negative (TENIS) cases, a direct comparison would have strengthened the argument for switching to FAPI. The authors should make clear in the introduction that ⁶⁸Ga-FAPI may be useful for TENIS or for any state of DTC. Please note that the quality of images in the manuscript is poor, so the reviewer could not see lesions clearly.

Reviewer #2: This retrospective study examines the diagnostic efficacy of 68Ga-FAPI-04 PET/CT in patients with differentiated thyroid carcinoma (DTC) following 131I therapy, with a particular focus on its clinical value in localizing lesions among thyroglobulin (Tg)-negative patients. The inclusion of 147 patients (99 TSH-suppressed, 48 TSH-stimulated) provides a robust sample size for assessing an emerging radiotracer. The quantitative analysis, specifically determining a target-to-background ratio (TBR) threshold of 1.428 utilizing the mediastinum as a reference, is a notable contribution that could aid in standardizing image interpretation. However, several methodological and statistical limitations regarding the reference standard and threshold validation must be addressed before the manuscript can be considered for publication.

Major Comments

1. The study reports that only 29 of the 147 patients (19.7%) underwent histopathological biopsy as the definitive reference standard. For the remaining patients, serial serum Tg monitoring over a minimum of one year was utilized alongside imaging to confirm disease status. Because the primary objective of this study is to compare the diagnostic accuracy of 68Ga-FAPI-04 PET/CT directly against Tg testing, utilizing Tg as the ground truth to subsequently calculate the sensitivity and specificity of Tg creates a significant incorporation bias.

Recommendation: The authors should perform a subgroup analysis restricting the diagnostic performance comparison (FAPI vs. Tg) exclusively to the 29 patients with biopsy-proven results. Furthermore, the median follow-up time for the clinically monitored cohort should be explicitly stated, and this bias must be discussed in the Limitations section.

2. The study derives an optimal TBR threshold of 1.428 (AUC = 0.982) for predicting lymph node metastases using the entire patient cohort. Deriving a diagnostic cutoff and reporting its performance metrics on the exact same dataset carries a high risk of statistical overfitting.

Recommendation: To strengthen the reliability of this threshold, the authors should perform an internal validation (e.g., K-fold cross-validation or bootstrapping with 1,000 resamples) and report the validated accuracy. If internal validation is not feasible, the lack of an independent validation cohort must be explicitly stated as a major limitation in the Discussion.

3. The positive 68Ga-FAPI-04 scan rate in this cohort is relatively high at 38.8% (57/147). This suggests that the enrolled population may inherently possess a higher pre-test probability for recurrence or metastasis than a standard post-therapy follow-up population.

Recommendation: Please clarify the specific clinical indications for ordering the 68Ga-FAPI-04 imaging in the Methods section (e.g., were patients scanned due to suspected recurrence on ultrasound, or as a routine pre-therapy assessment?). The authors should also discuss how this potential selection bias might influence the reported Positive Predictive Value (PPV) and Negative Predictive Value (NPV).

4. The manuscript frequently references the limitations of 18F-FDG PET/CT and cites literature suggesting FAPI's superiority in DTC. However, the current study does not provide direct comparative data.

Recommendation: If a subset of the enrolled patients underwent 18F-FDG PET/CT within a similar timeframe, incorporating a head-to-head comparative analysis would significantly elevate the manuscript's impact. If such data is entirely unavailable, the authors should soften claims regarding comparative superiority and list the absence of 18F-FDG data as a limitation.

5. There are some spelling mistakes and inconsistent formats in the original manuscript.

(1). Table 1: Under the "Pathologic subtype" category, the term "Follicula" is misspelled and should be corrected to "Follicular".

(2). The manuscript uses inconsistent formatting and abbreviations for thyroglobulin states, interchanging Tgoff, Tgofr, and Tgoff. Please rigorously standardize these terms (e.g., consistently using Tgoff and Tgon) throughout the text and figures.

(3). Line 166: Correct "fndings" to "findings". Line 167: Correct "classifed" to "classified".

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Reviewer #1: Yes:  Mai Hong Son

Reviewer #2: No

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68Ga-FAPI-04 PET/CT has high diagnostic efficacy in Tg-positive and Tg-negative differentiated thyroid cancer

PONE-D-26-18193R1

Dear Dr. wang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Francesco Dondi

Academic Editor

PLOS One

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