Utility of High-Frequency Ultrasound in Preoperative Evaluation of the Thickness of Cutaneous Melanoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.The introduction states that “almost all CMs necessitate a preoperative biopsy,” yet current dermatologic practice increasingly relies on non-invasive diagnostic tools—including dermoscopy, reflectance confocal microscopy, high-frequency ultrasound and line field confocal optical coherence tomography, which are explicitly acknowledged in recent guidelines such as the European consensus-based melanoma guidelines and the EADV/ESMO recommendations. Could the authors clarify or update this statement to reflect the growing role of non-invasive diagnostic modalities

2.In the introduction, the authors suggest that biopsy may promote metastasis. Could they clarify which studies provide the strongest evidence for this claim and whether they consider it speculative or established?

3.The introduction refers to HFUS but does not define the frequency thresholds used in dermatology for this designation. Could the authors clarify how they define “high frequency” ?

4.The authors state that few studies exist on HFUS for measuring Breslow thickness. Would they consider briefly summarizing the main findings of these limited studies to better justify the rationale for their investigation?

5.The methods refer to “partial biopsy” in general terms. Could the authors clarify which biopsy techniques (shave, punch, incisional) were actually used?

6.The methods do not explain how the specific site for partial biopsy was selected on each lesion. Could the authors clarify whether biopsy placement was guided by dermoscopy, clinical suspicion of maximal thickness, or operator judgment alone? Given the subsequent finding that partial biopsies frequently underestimated Breslow thickness, is it possible that suboptimal site selection may have contributed to this underestimation? 

7.Was the pathologist blinded to HFUS measurements and biopsy thickness? If not, could this have introduced interpretive bias?

8.Thirteen out of seventeen lesions were ulcerated. Could the authors comment on whether ulceration may have influenced the accuracy of HFUS or partial biopsy in this cohort? Was any stratified analysis attempted comparing ulcerated vs non-ulcerated lesions?

9.HFUS overestimated thickness in two cases. Could the authors clarify whether these cases shared common features (e.g., inflammation, ulceration, fibrosis) that might explain the mismatch?

10.The discussion states that underestimation of Breslow thickness by partial biopsy may “lead to inaccurate clinical staging, resulting in inadequate follow-up and treatment.” However, clinical T staging and subsequent follow-up decisions are typically based on the postoperative histopathologic Breslow thickness, not on preoperative estimations. Could the authors clarify how a preoperative underestimation would influence staging or follow-up in real practice, given that definitive management relies on the final excision specimen? Would the authors consider revising this paragraph to reflect that biopsy underestimation mainly affects initial surgical planning, rather than long-term staging or follow-up pathways?

11.The discussion states that HFUS can offer valuable insights into TNM staging by evaluating satellite nodules, lymph node metastases, and distant metastasis. However, the methods and results of this study focus exclusively on HFUS measurement of Breslow thickness of the primary tumor. Could the authors clarify the relevance of this statement to their study, and reconsider whether it should be removed or rephrased?

 

 

Author Response

Response to Reviewer #1:

Comments 1.The introduction states that “almost all CMs necessitate a preoperative biopsy,” yet current dermatologic practice increasingly relies on non-invasive diagnostic tools—including dermoscopy, reflectance confocal microscopy, high-frequency ultrasound and line field confocal optical coherence tomography, which are explicitly acknowledged in recent guidelines such as the European consensus-based melanoma guidelines and the EADV/ESMO recommendations. Could the authors clarify or update this statement to reflect the growing role of non-invasive diagnostic modalities

Response 1:

We thank the reviewer for this important clarification. We agree that non-invasive diagnostic modalities—such as dermoscopy, reflectance confocal microscopy (RCM), high-frequency ultrasound (HFUS), and line-field confocal optical coherence tomography—have become increasingly important in current dermatologic practice and are explicitly addressed in recent European and EADV/ESMO guidelines. Our original wording was indeed too absolute.

Accordingly, we have revised the introduction to reflect this trend. The updated text now acknowledges the expanding role of non-invasive imaging in melanoma evaluation, while also clarifying that histopathological assessment from biopsy remains the current gold standard for definitive diagnosis and Breslow thickness determination. The revised sentence reads as follows:

“While contemporary dermatologic practice increasingly incorporates non-invasive imaging tools (e.g., dermoscopy, RCM, HFUS, line-field confocal OCT) as recommended by current international guidelines, histopathological examination from biopsy remains the reference standard for definitive diagnosis and Breslow thickness assessment in most clinical settings.”

We believe this revision accurately reflects current clinical practice and aligns with contemporary guideline recommendations.

 

 

Comments 2.In the introduction, the authors suggest that biopsy may promote metastasis. Could they clarify which studies provide the strongest evidence for this claim and whether they consider it speculative or established?

Response 2:

We appreciate the reviewer’s question and agree that the relationship between biopsy-induced tissue disruption and melanoma progression requires careful contextualization. In our original text, the statement regarding biopsy potentially promoting metastasis was intended as a hypothesis grounded in evidence rather than a definitive conclusion.

The most relevant mechanistic evidence derives from experimental models such as “Surgical damage to the lymphatic system promotes tumor growth via impaired adaptive immune response,” in which disruption of lymphatic drainage in mice reduced CD4⁺ and CD8⁺ T-cell infiltration, decreased intratumoral cytotoxic activity, and ultimately facilitated tumor progression (Nakamura Y et al., J Dermatol Sci. 2018). Additionally, retrospective clinical analyses such as “Incisional Biopsy Technique Is Associated With Decreased Overall Survival for Cutaneous Melanoma” (n=42,272 cases) have suggested an association between incisional biopsy and reduced 5-year overall survival (Liszewski W et al., Dermatol Surg. 2022).

However, we fully acknowledge that these findings do not establish biopsy-related metastasis as a proven causal effect in humans. Therefore, we have revised the introduction to clearly frame this concept as a hypothesis rather than a settled conclusion. The updated text reads:

“Some experimental and retrospective data suggest that surgical disruption of the tumor–lymphatic environment may theoretically influence melanoma progression; however, this remains investigational and is not definitively established in clinical practice.”

If the you consider this topic insufficiently supported or potentially misleading in this context, we remain entirely willing to further limit or remove this discussion.

 

 

Comments 3.The introduction refers to HFUS but does not define the frequency thresholds used in dermatology for this designation. Could the authors clarify how they define “high frequency” ?

Respons 3:

We thank the reviewer for this valuable comment. In dermatology, HFUS commonly refers to ultrasound frequencies ≥15 MHz, with >20–30 MHz sometimes described as very high frequency according to established guidelines such as those from the DERMUS Group (Wortsman et al., J Ultrasound Med, 2016). In our study, lesion localization was performed using a 10–22 MHz probe, while Breslow thickness measurements were obtained using a 22–38 MHz transducer, which falls firmly within accepted HFUS ranges. We have added this clarification to the Introduction to avoid ambiguity.

 

 

Comments 4.The authors state that few studies exist on HFUS for measuring Breslow thickness. Would they consider briefly summarizing the main findings of these limited studies to better justify the rationale for their investigation?

Response 4:

We thank the reviewer for this helpful suggestion. We have now added a brief contextual sentence in the Introduction noting that prior studies have demonstrated good correlation between HFUS-measured thickness and histologic Breslow depth, particularly for thicker lesions, and that HFUS may assist in surgical planning. We then clarify that these studies did not directly compare HFUS with partial biopsy, which is the specific focus of our investigation.

“Previous studies have reported a good correlation between HFUS-measured melanoma thickness and histologic Breslow depth, particularly in thicker lesions, but have not directly compared HFUS with partial biopsy.”

This highlights the gap our study addresses:

“Our work specifically compares HFUS with partial biopsy to determine whether HFUS can provide a more reliable preoperative thickness estimate.”

 

 

Comments 5.The methods refer to “partial biopsy” in general terms. Could the authors clarify which biopsy techniques (shave, punch, incisional) were actually used?

Response 5:

We thank the reviewer for this question. In our cohort, the majority of partial biopsies were performed using incisional biopsy, with a smaller number conducted using punch biopsy. Shave biopsy was not used in any of the cases. We have now clarified this explicitly in the Methods section.

“Partial biopsies were performed predominantly using incisional biopsy, with a minority using punch biopsy.”

 

 

Comments 6.The methods do not explain how the specific site for partial biopsy was selected on each lesion. Could the authors clarify whether biopsy placement was guided by dermoscopy, clinical suspicion of maximal thickness, or operator judgment alone? Given the subsequent finding that partial biopsies frequently underestimated Breslow thickness, is it possible that suboptimal site selection may have contributed to this underestimation? 

Response 6:

We thank the reviewer for this insightful question. In our clinical setting, although dermoscopy was routinely performed for all patients, dermoscopy does not provide information on lesion thickness. Therefore, the selection of the biopsy site relied primarily on the operator’s clinical judgment, focusing on the area that appeared most suspicious or most elevated on visual and palpatory examination. We have added this clarification to the Methods section.

The revised text reads:

“Although dermoscopy was routinely performed, it does not provide thickness assessment; therefore, the biopsy site was selected based mainly on the operator’s clinical judgment of the most suspicious or clinically thickest-appearing area.”

Regarding the reviewer’s question about potential bias, we agree that operator-dependent site selection may contribute to the underestimation of Breslow thickness observed in partial biopsies. We have now added this point to the Discussion as an additional limitation:

“Because biopsy site selection relied on operator judgment, suboptimal targeting of the true maximal-thickness area may have contributed to the underestimation of Breslow thickness observed in partial biopsies.”

We believe these additions improve the clarity and completeness of the methodology and interpretation.

 

 

Comments 7.Was the pathologist blinded to HFUS measurements and biopsy thickness? If not, could this have introduced interpretive bias?

Response 7:

We thank the reviewer for raising this important point. In our study, the dermatopathologist was blinded to both the HFUS measurements and the partial biopsy thickness when evaluating the postoperative specimens. Thus, no information from the imaging assessment or biopsy results was available during histological measurement, minimizing the risk of interpretive bias.

We have added the following clarification to the Methods section:

“The dermatopathologist assessing postoperative specimens was blinded to both HFUS measurements and partial biopsy thickness to avoid interpretive bias.”

We believe this addition adequately addresses the reviewer’s concern.

 

 

Comments 8.Thirteen out of seventeen lesions were ulcerated. Could the authors comment on whether ulceration may have influenced the accuracy of HFUS or partial biopsy in this cohort? Was any stratified analysis attempted comparing ulcerated vs non-ulcerated lesions?

Response 8:

We appreciate the reviewer’s thoughtful question. In our cohort, 13 of 17 melanomas were ulcerated. Importantly, ulceration did not negatively impact HFUS accuracy because—similar to histopathologic Breslow measurement—we measured depth from the base of the ulceration to the deepest point of tumor infiltration, rather than from the disrupted surface. The ultrasound gel provides uniform acoustic coupling over the ulcerated area, enabling clear visualization of the dermis and lesion base.

Regarding partial biopsy, ulceration did not systematically affect the measured thickness, as biopsies were taken from the clinically or dermoscopically most suspicious region, independent of ulceration status.

Given the small sample size, a stratified statistical comparison between ulcerated vs. non-ulcerated lesions was not feasible; however, descriptively, we did not observe any difference in HFUS performance between these groups. We have added a clarifying note to the Discussion to explain this.

 

 

Comments 9.HFUS overestimated thickness in two cases. Could the authors clarify whether these cases shared common features (e.g., inflammation, ulceration, fibrosis) that might explain the mismatch?

Response 9:

Thank you for this thoughtful question. In our cohort, HFUS measurements were on average ~0.3 mm higher than histopathology. In two cases, the measurement value by HFUS even exceeded 4 millimeters, resulting in an overestimation of the T stage. We propose two plausible explanations:

• Tissue shrinkage during histologic processing — It is well established that dehydration and fixation can lead to measurable contraction of melanoma tissue, resulting in a histologic thickness slightly lower than the in-vivo value. We have already discussed this mechanism in the manuscript and it is supported by prior literature.
• Local inflammatory activity — Upon reviewing the histology of these cases, we noted that both exhibited relatively prominent lymphocytic infiltration beneath the lesion. HFUS cannot distinguish inflammatory infiltrate from tumor cells, and this may contribute to a mild overestimation of thickness.

Given the small sample size of these specific cases, these interpretations remain speculative. We agree that additional studies with a larger cohort will be needed to determine whether inflammatory activity or other features (e.g., fibrosis) systematically contribute to HFUS overestimation.

 

 

Comments 10.The discussion states that underestimation of Breslow thickness by partial biopsy may “lead to inaccurate clinical staging, resulting in inadequate follow-up and treatment.” However, clinical T staging and subsequent follow-up decisions are typically based on the postoperative histopathologic Breslow thickness, not on preoperative estimations. Could the authors clarify how a preoperative underestimation would influence staging or follow-up in real practice, given that definitive management relies on the final excision specimen? Would the authors consider revising this paragraph to reflect that biopsy underestimation mainly affects initial surgical planning, rather than long-term staging or follow-up pathways?

Response 10:

We thank the reviewer for this important clarification. We fully agree that the final clinical T staging and long-term follow-up recommendations are ultimately based on the postoperative histopathologic Breslow thickness, rather than preoperative estimates. Therefore, the paragraph in our Discussion has been revised to avoid implying that preoperative biopsy alone determines definitive staging.

“While definitive T staging and follow-up decisions are based on the postoperative histopathologic Breslow thickness, preoperative underestimation by partial biopsy can still affect initial surgical management. An inaccurately low thickness estimate may lead to suboptimal excision margins or omission of a sentinel lymph node biopsy, potentially necessitating secondary procedures. Thus, the clinical impact of biopsy underestimation lies primarily in early surgical planning rather than long-term staging. HFUS, by providing a more accurate preoperative thickness measurement, may help mitigate this limitation.”

 

 

Comments 11.The discussion states that HFUS can offer valuable insights into TNM staging by evaluating satellite nodules, lymph node metastases, and distant metastasis. However, the methods and results of this study focus exclusively on HFUS measurement of Breslow thickness of the primary tumor. Could the authors clarify the relevance of this statement to their study, and reconsider whether it should be removed or rephrased?

Response 11:

Thank you for this insightful comment. We agree that the sentence regarding HFUS assessment of satellite lesions or metastasis is not directly supported by the methods or results of the present study, which focused exclusively on HFUS evaluation of primary tumor thickness. Although HFUS has potential applications in regional staging, this extends beyond the scope of our dataset.

To maintain the coherence and focus of the manuscript, we have rephrased this statement to limit the discussion to findings directly supported by our data. The revised sentence now reads:

“In this study, HFUS demonstrated strong accuracy in assessing Breslow thickness of the primary melanoma, supporting its value as a noninvasive tool for preoperative characterization of the primary lesion.”

We believe this revision addresses the reviewer’s concern by removing unsupported implications regarding TNM staging while preserving the relevance of our findings.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a very interesting study, and I would be happy to review it again when it's done on a bigger group. I think the observation that HFUS measurements were very similar to the histopathological ones, whereas they weren't as precise in terms of partial biopsies, was very interesting. However, that needs to be verified on the bigger group.

I agree with the authors regarding the limitations of the study, specifically that in HFUS, we cannot differentiate between lymphocytes and tumor cells. I strongly think that HFUS will be an essential part of the melanoma treatment process, but that requires more experience.

The images are great quality and very informative, as well as the statistics part.

Author Response

Response to Reviewer #2:

Comment: This is a very interesting study, and I would be happy to review it again when it's done on a bigger group. I think the observation that HFUS measurements were very similar to the histopathological ones, whereas they weren't as precise in terms of partial biopsies, was very interesting. However, that needs to be verified on the bigger group.

I agree with the authors regarding the limitations of the study, specifically that in HFUS, we cannot differentiate between lymphocytes and tumor cells. I strongly think that HFUS will be an essential part of the melanoma treatment process, but that requires more experience.

The images are great quality and very informative, as well as the statistics part.

Response:

We sincerely thank the reviewer for the positive and encouraging feedback. We truly appreciate the recognition of the clinical relevance of our findings and the quality of the imaging and statistical analyses. We fully agree that validation in a larger cohort is essential, and we are currently planning follow-up studies with expanded patient numbers to further confirm and refine these observations.

We also concur with the reviewer’s remark that HFUS cannot reliably distinguish lymphocytes from tumor cells at this stage, and that further experience and technical refinement are needed for optimal implementation. We are grateful for the reviewer’s constructive perspective regarding the potential future role of HFUS in melanoma management, which aligns with our own clinical vision. We look forward to improving this work and hope it will contribute to advancing noninvasive melanoma assessment.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript can be published in the current form.