Role of HER2 in Response to Neoadjuvant Endocrine Therapy in Luminal Breast Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Using Ki67 change, tumor size change, PEPI score, and survival outcomes, this single-center retrospective cohort (2017–2023) assesses whether HER2-low status (IHC 1+ or 2+/ISH–) affects response to neoadjuvant endocrine therapy (NET) in luminal, HER2-negative breast cancer. Although the dataset is clinically relevant and the question is timely, interpretability and reproducibility are currently limited by design, reporting, and statistical issues. 

-Provide a STROBE-style flow diagram: screened (included → excluded with explanations)

-HER2 testing methodology requires reproducible details. Indicate which ASCO/CAP HER2 guideline was used, the platforms and antibodies used, the fixation parameters, and the IHC scorers. Since "HER2-low 2+" necessitates ISH–, clearly state that all IHC 2+ cases underwent ISH and were negative.

-P-values play a significant role in the main conclusion, "no differences between HER2 groups." Could you supply 95% CIs for effect sizes? To account for confounding, use multivariable models for every endpoint. 

-Ki67 is frequently lab-variable and skewed. In addition to means, consider reporting medians and IQRs for Ki67 change, as well as transformation, using robust methods.

-Describe how negative changes were handled and whether "difference" refers to an absolute percentage-point change (baseline–interim).

-Could you describe whether PEPI was computed for every patient and how missing nodal/tumor variables were taken into account?

Author Response

We are very grateful for the time and effort the reviewer dedicated to evaluating our manuscript, “Role of HER2 in response to neoadjuvant endocrine therapy in luminal breast cancer” (curroncol-4047229). The feedback we received was extremely thoughtful, constructive, and helpful in improving the clarity and scientific rigor of our work.

We have carefully considered each suggestion. Wherever possible, and in order to address the reviewer’s thoughtful comments thoroughly, we performed additional analyses and reviewed several patient records to ensure that our responses were accurate and comprehensive. In a few cases where direct changes were not feasible for this submission, we have provided clarifications in our responses and will certainly take these valuable recommendations into account for future work. We also welcome the opportunity to clarify the limitations* in study design, reporting, and statistical methodology identified during the review process. We have addressed each point in detail below. We are grateful for the opportunity to revise the manuscript and believe these efforts have strengthened the work.

*In the manuscript, we acknowledge the study’s limitations as follows: its retrospective design, small sample size, short follow-up duration, and incomplete availability of complete staging data (ypT and ypN) for all 173 patients.

Comment 1. Provide a STROBE-style flow diagram: screened (included → excluded with explanations)

We have added a STROBE-style flow diagram (new Figure 2) detailing patient selection, including the number of patients screened, included, and excluded, along with explicit reasons for exclusion at each stage (e.g., patients who did not undergo surgery, patients who received less than one month of NET, patients who underwent cryoablation procedure instead of surgery, patients without necessary postoperative pathological data available, patients who received NCT). This diagram is referenced in the Methods section - Study design.

Comment 2. HER2 testing methodology requires reproducible details. Indicate which ASCO/CAP HER2 guideline was used, the platforms and antibodies used, the fixation parameters, and the IHC scorers. Since "HER2-low 2+" necessitates ISH–, clearly state that all IHC 2+ cases underwent ISH and were negative.

We have modified the Methods section to include detailed information on HER2 assessment. This study followed the 2018 - Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update (J Clin Oncol) [Reference 9], as per hospital protocol.

The classification and therapeutic framework applied in this study are summarized and simplified in Figure 1 for clarity.

• Tumors with IHC scores of 0 (no staining) were considered HER2-zero tumors. Tumors with IHC scores of 0 (faint incomplete membrane staining in <10% of cells) were considered HER2-Ultra-low tumors, although treated and classified in the same group as HER2-zero tumors.
• Tumors with IHC scores of 1+ (faint incomplete membrane staining in >10% of cells) were considered HER2-low tumors.
• Tumors with IHC scores of 2+ (weak-moderate complete membrane staining in >10% of cells or intense membrane staining in <10% of cells) all underwent ISH testing*. If no amplification was found in ISH, the tumor was considered HER2-low.

Clarification: both “HER2-low” and “HER2-zero” tumors currently remain categorized as HER2-negative.

• Tumors with IHC scores of 2+ (weak-moderate complete membrane staining in >10% of cells or intense membrane staining in <10% of cells) with HER2 amplification in ISH, the tumor was considered HER2-positive.
• Tumors with IHC score of 3+ (intense complete membrane staining in <10% of cells) were considered HER2-positive tumors.

Clarification: HER2-positive tumors (whether IHC score 2+ with positive ISH or IHC score 3+) were not included in this study.

*HER2 gene amplification was evaluated with single-probe ISH or dual-probe ISH assay following ASCO/CAP 2018 Guideline algorithms.

Comment 3. P-values play a significant role in the main conclusion, "no differences between HER2 groups." Could you supply 95% CIs for effect sizes? To account for confounding, use multivariable models for every endpoint. 

We have reviewed the results of the statistical analysis for every endpoint. As the only statistically significant variable was the greater difference in Ki67 levels between diagnosis and interim biopsy (p < 0.05), there are no sufficient data to construct a complete table with hazard ratios and confidence intervals for all variables. The statistical software used only provides HR and CI values for parameters that reach statistical significance. While we can provide the output table generated by the program, it includes only the p-values for non-significant variables and the HR, CI and p-value for the variation in Ki67 levels. We await the reviewer’s guidance on whether inclusion of this limited table would be preferred.

	HR	CI†	p
Ki67 variation between diagnosis and interim CNB	0.85	0.76 – 0.95	0.004*
Ki67 variation between diagnosis and surgical specimen	NA	NA	0.611
PEPI-score	NA	NA	0.168
* p values <0.05 are considered statistically significant. † CI at 95%. Abbreviations: CNB, core needle biopsy; NA, non-available; PEPI, Preoperative Endocrine Prognostic Index.

Comment 4. Ki67 is frequently lab-variable and skewed. In addition to means, consider reporting medians and IQRs for Ki67 change, as well as transformation, using robust methods.

We thank the reviewer for this comment. We agree that Ki67 values are lab-variable and skewed. In Table 1, Ki67 levels are presented for the overall cohort and stratified by HER2 status (HER2-0, HER2-low 1+, and HER2-low 2+), using medians and interquartile ranges (IQR).

In addition, as described in the previous response (Comment 3), we have developed an additional table reporting hazard ratios (HRs) with their corresponding 95% confidence intervals, which can be incorporated into the manuscript if the reviewers consider it appropriate.

Comment 5. Describe how negative changes were handled and whether "difference" refers to an absolute percentage-point change (baseline–interim).

We appreciate the opportunity to clarify the data presented in Table 5. The “mean difference” refers to the variation in Ki67 levels between the diagnostic biopsy and the interim CNB, calculated for each patient. Specifically, the table represents the average decrease in Ki67 expression following NET. As explained in the manuscript text (“Ki67 level variation between diagnosis and interim CNB…”), this value was derived by subtracting the interim Ki67 level from the diagnostic Ki67 level for each patient. [For example, Patient 1 had Ki67 levels of 40% at diagnosis and 10% at interim biopsy, resulting in a difference of 30%; Patient 2 had Ki67 levels of 25% and 15%, respectively, yielding a difference of 10%. These individual differences were then used to calculate the mean decrease, along with the corresponding confidence interval.] We have now explained in the table legend what the mean difference represents.

Negative changes were handled automatically by the statistical software provided by the hospital.

Comment 6. Could you describe whether PEPI was computed for every patient and how missing nodal/tumor variables were taken into account?

We have profusely reviewed all patient medical records. In a small number of cases, the final surgical pathology reports did not specify tumor size or the exact number of affected lymph nodes. As a result, complete ypT and ypN data were not available for all 173 patients. We verified that PEPI score values were calculated for all patients with complete data on post-NET tumor size, nodal status, ER status, and Ki67. For patients with missing nodal or tumor size data, PEPI was considered not computable and these cases were excluded from PEPI-based analyses only. This limitation has now been explicitly acknowledged in the Discussion section, within the paragraph addressing the methodological constraints of the study.

We hope that the revisions and clarifications we have made in response to the thoughtful feedback will improve the quality of our manuscript. We again thank the reviewer for their insightful and constructive suggestions, which have greatly strengthened our work.

We look forward to your further consideration of our revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for this thoughtful analysis of outcomes for Her2 low and Her2 negative patient populations. I find that interestingly there is no difference between the groups in response to therapy. The Ki67 analysis is helpful because it validates what others have shown about cancer proliferation.

If you are asked to revise, it would be good to explicitly state whether changes in current practice would lead to a different standard of care for any patients with greater than 1+ Her 2 staining. Maybe your work suggests that no change might be necessary considering the additional risk of side effects and the added costs.

Author Response

We sincerely thank the reviewer for the thoughtful, generous, and encouraging comments, and for the careful attention given to our work. We are truly grateful for the reviewer’s insightful perspective on the clinical relevance of our findings and for highlighting the value of the Ki67 analysis in the context of tumor proliferation.

We have carefully considered the suggestion and we are grateful for the opportunity to add this explanation to our manuscript.

Comment 1. It would be good to explicitly state whether changes in current practice would lead to a different standard of care for any patients with greater than 1+ Her 2 staining. Maybe your work suggests that no change might be necessary considering the additional risk of side effects and the added costs.

We agree that an important implication of this work is its potential impact on current clinical practice. In response to the reviewer’s suggestion, we have added a paragraph to the Discussion section explicitly addressing whether HER2 expression should influence standard-of-care management in patients receiving neoadjuvant endocrine therapy.

Given the absence of significant differences in response to NET between HER2-low and HER2-zero tumors across multiple endpoints, we hypothesize that low-level HER2 expression does not meaningfully affect responsiveness to NET in ER-positive/HER2-negative breast cancer. Accordingly, our findings do not support a change in current clinical practice for patients with greater than 1+ HER2 staining in the NET setting, particularly in light of the additional costs and potential toxicities associated with HER2-targeted therapies.

We further note that these results raise the question of whether HER2 expression may similarly have limited impact on endocrine responsiveness in ER-positive/HER2-positive tumors. Although this remains speculative at present, this hypothesis warrants further investigation. If confirmed, it could have important therapeutic implications. We emphasize, however, that such an approach would require validation in prospective clinical trials before any modification of standard-of-care recommendations.

We hope that the clarification we have made in response to the thoughtful feedback will improve the quality of our manuscript. We again thank the reviewer for their insightful and constructive suggestions, which have greatly strengthened our work.

We look forward to your further consideration of our revised manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This is a well written paper in an area of growing clinical relevance - the importance of her 2 low in therapeutic descision making 

the study is modest in size, single centre and with relatively short follow up for a tumor where delayed relapse is common- limitations which are acknowledged by the authors in the discussion. the paper is comprehensive with detailed statistical analysis section and up to date references which are well integrated into the text 

 

Author Response

We sincerely thank the reviewer for the generous and thoughtful evaluation of our manuscript and for highlighting the growing clinical relevance of HER2-low status in therapeutic decision-making. We are very grateful for the reviewer’s positive assessment of the clarity of the manuscript, the comprehensiveness of the analyses, and the integration of up-to-date references.

We also appreciate the reviewer’s acknowledgment of the study’s limitations, including its modest sample size, single-center design, and relatively short follow-up duration in the context of luminal breast cancer, in which delayed relapse is common. As noted, these limitations were explicitly recognized and discussed in the manuscript. We agree that these factors underscore the need for larger, multicenter studies with longer follow-up to further validate and extend our findings.

We thank the reviewer again for the constructive and encouraging feedback, which we believe supports the relevance of this work and highlights important avenues for future research.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Summary

This retrospective single-centered study evaluated whether low levels of HER2 expression influence response to NET in luminal BC. The authors analyzed 175 tumors and found that HER2-low status didn’t affect treatment response or survival outcomes, while factors such as ER level, histologic features, Ki67 at interim biopsy, and treatment duration were associated with response. The recognition of HER2-low BC is relatively recent and has become clinically relevant. Most published work on HER2-low to date has focused on NACT rather than NET. Currently, there’s limited evidence on whether small differences in HER2 protein levels influence endocrine therapy sensitivity. Although this is a single-centered study with a relatively small sample size, it addresses a gap in the literature and therefore adds incremental novelty.

Below are my suggestions for improvement –

1. Figure 1 is missing: Line 60 and Line 112 mentioned Figure 1, but this figure is not included in the manuscript or supplementary material. Please clarify.
2. Introduction: suggest adding information 1) clarifying HER2-low gained relevance is not just for pathology reclassification, but also due to new drug activity; 2) about why HER2-low BC matters now; 3) stating HER2-low BC has been studied in chemotherapy settings, but its role in endocrine response is unclear to strengthen the novelty.
3. Table 1: The authors state that “This study analyzed data from 175 tumors belonging to a total of 173 patients.” However, in Table 1, the N number for “Clinical primary tumor (cT)” is 172 and for “Clinical node status (cN)” is 169. Please clarify these discrepancies.
4. Figure 2 is missing: Line 233-241 mentioned Figure 2, but this figure is not included in the manuscript or supplementary material. Please clarify.
5. Discussion: suggest adding the discussion 1) how clinicians should interpret HER2-low status when deciding NET vs. other treatments based on their findings; 2) why HER2-low might not influence NET response despite signaling links.

Minor:

1. Line 88 and 366: “single-centre” should be corrected to “single-centered”.
2. Line 88: “Data was collected” should be corrected to “Data were collected”.
3. Line 131 : “Kaplan Meyer” should be corrected to “Kaplan–Meier”
4. Consistency: Use either NET or neoadjuvant endocrine therapy consistently. Use breast cancer (BC) or full term consistently after first definition.
5. Line 303: “Trastuzumab-Deruxtecan (T-D)” should be corrected to “Trastuzumab-Deruxtecan (T-DXd)”, which is the widely accepted shorthand.

Author Response

We sincerely thank the reviewer for their detailed and thoughtful evaluation of our manuscript and for highlighting both the relevance and novelty of our study in the context of HER2-low breast cancer. We are especially grateful for the recognition of the clinical importance of assessing HER2-low status in therapeutic decision-making, and for acknowledging the thoroughness of our statistical analyses and the integration of up-to-date references. Your constructive comments have been extremely valuable in helping us improve the clarity, completeness, and precision of our manuscript.

We have addressed each of your specific suggestions below:

Comment 1. Figure 1 is missing: Line 60 and Line 112 mentioned Figure 1, but this figure is not included in the manuscript or supplementary material. Please clarify.

We apologize for the oversight. This was a mistake made during the submission process, as the figures had been included in separate files and uploaded at a later date. All figures and supplementary materials have now been uploaded and are available for review.

Comment 2. Introduction: suggest adding information 1) clarifying HER2-low gained relevance is not just for pathology reclassification, but also due to new drug activity; 2) about why HER2-low BC matters now; 3) stating HER2-low BC has been studied in chemotherapy settings, but its role in endocrine response is unclear to strengthen the novelty.

We appreciate the reviewer’s suggestion. We have revised the Introduction to incorporate all suggestions with brief sentences, highlighting the clinical relevance of HER2-low status and the novelty of assessing HER2-low in the context of endocrine therapy. However, we have explored these topics more comprehensively in the Discussion section, combining scientific evidence -when available-  and backing it up or testing it with our results.

Comment 3. Table 1: The authors state that “This study analyzed data from 175 tumors belonging to a total of 173 patients.” However, in Table 1, the N number for “Clinical primary tumor (cT)” is 172 and for “Clinical node status (cN)” is 169. Please clarify these discrepancies.

We thank the reviewer for raising this important issue. The two patients with multiple tumors (175 tumors belonging to 173 patients) presented with synchronous primaries diagnosed at the same time, rather than metachronous lesions. We have reviewed all patient records thoroughly. In a very small number of cases, initial or final surgical pathology reports did not specify tumor size or exact nodal involvement (maximum one data point unavailable per patient). Consequently, complete cT, cN, ypT, and ypN data were not available for all 173 patients. This limitation is now explicitly acknowledged in the Discussion, within the paragraph addressing methodological constraints. Additionally, a “data not available” (DNA) indicator has been added to Tables 1 and 2 wherever information was missing. We appreciate the reviewer’s attention to potential confounding and have clarified this in the manuscript.

Comment 4. Figure 2 is missing: Line 233-241 mentioned Figure 2, but this figure is not included in the manuscript or supplementary material. Please clarify.

We again apologize for the oversight. As with Figure 1, this was a submission error. Figure 2 has now been included and is available for review.

Comment 5. Discussion: suggest adding the discussion 1) how clinicians should interpret HER2-low status when deciding NET vs. other treatments based on their findings; 2) why HER2-low might not influence NET response despite signaling links.

We appreciate the reviewer’s suggestion. We have revised the Discussion to incorporate all suggestions on how to interpret HER2-low status when deciding treatment and potential causes for limited impact of HER2 expression in NET response.

Minor corrections

We apologize for the minor errors identified by the reviewer. All have been addressed in the revised manuscript.

We hope that the revisions and clarifications we have made in response to the thoughtful feedback will improve the quality of our manuscript. We sincerely thank the reviewer again for the careful reading, insightful comments, and constructive suggestions, which have greatly contributed to improving the quality and clarity of our manuscript.

We look forward to your further consideration of our revised manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No additional adjustments are needed, and all issues have been satisfactorily addressed.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the clarification of this point. I'm satisfied with the additional explanation and background that you provide.