ERβ Isoforms Have Differential Clinical Significance in Breast Cancer Subtypes and Subgroups

Round 1

Reviewer 1 Report

 The role ERβ in human breast cancer development and outcome is yet to be elucidated. The main challenge is the utilization of selective anti-ERβ antibodies. Choi et al assessed the expression of ERβ1, ERβ2, and ERβ5 isoforms(protein and mRNA) and correlated its expression with different subtypes of breast cancer in particular among the aggressive ER-Alpha negative and triple-negative subtypes. Consistent with previous studies, Choi et al found that ERβ mRNA are differentially expressed. Furthermore, they found that the ERβ isoforms and their mRNA and protein are associated with different clinical outcomes in subtypes and subgroups. 
 1)ERβ2 and ERβ5 mRNA expression are predictive of poor outcomes in ERα-negative BCa and TNBC. 
2) ERβ isoform expressing breast cancer cells are proliferating cells exhibiting high Ki-67 positivity. 

The paper presents an important topic and is well presented. However, I have some comments to improve the paper. 

1) Presentation of Table and figures needs editing to improve readability

For example:
Tables 2- 3: are you presenting row or column percentages? 
Table 3: what are the numbers in parentheses, cases?
Figure 3: It would be helpful to organise the mRNA and protein plots next to each other for each isoform 

2) The small number of cases and which is further reduced by sub-group analysis generating unstable effect estimates with wide confidence intervals.

 

 

Author Response

Responses to reviewer # 1.comments

 The role ERβ in human breast cancer development and outcome is yet to be elucidated. The main challenge is the utilization of selective anti-ERβ antibodies. Choi et al assessed the expression of ERβ1, ERβ2, and ERβ5 isoforms(protein and mRNA) and correlated its expression with different subtypes of breast cancer in particular among the aggressive ER-Alpha negative and triple-negative subtypes. Consistent with previous studies, Choi et al found that ERβ mRNA are differentially expressed. Furthermore, they found that the ERβ isoforms and their mRNA and protein are associated with different clinical outcomes in subtypes and subgroups. 
 1)ERβ2 and ERβ5 mRNA expression are predictive of poor outcomes in ERα-negative BCa and TNBC. 
2) ERβ isoform expressing breast cancer cells are proliferating cells exhibiting high Ki-67 positivity. 

The paper presents an important topic and is well presented. However, I have some comments to improve the paper. 

1) Presentation of Table and figures needs editing to improve readability

For example:
Tables 2- 3: are you presenting row or column percentages? Number of cases
Table 3: what are the numbers in parentheses, cases? Number of cases
Figure 3: It would be helpful to organise the mRNA and protein plots next to each other for each isoform 

Fig 3. Rearranged and the legend is revised

2) The small number of cases and which is further reduced by sub-group analysis generating unstable effect estimates with wide confidence intervals.

I do agree. However, it shows the significance of investigating ER beta in BCa subtypes and subgroups for its clinical relevance

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The study assessed ERb-negative tumors. Authors investigated both ERβ mRNA and ERβ1,2 and 5 isoform expression in breast cancers. ERβ isoform mRNA and proteins were a risk factor for poor outcome. The discussion suggests that standardization of ERβ testing protocol may be re- quired in clinical setting.

The topic is interesting, although the manuscript style is confusing and lacks logical aims. It seems that findings are also chaotic. Unfortunately, contradictory results were not discussed properly.

Abstract is too long and not clear. Sometimes it looks like an abstract for a review paper, sometimes- as an original research paper. It is a research paper. Therefore, the abstract should report the size of cohort, the number of tested patients etc.

Introduction does not reflect the substantial findings related to the role of ERbeta in ERalpha negative (and others) breast cancers.

 Methods: I could not find the statement about approval of the study by the human ethics committee. The study used patients’ samples. The approval of the human ethics committee is required.

Results: authors tested a good set of anti-ERbeta1 antibodies. It is a substantial contribution. Therefore, authors should focus on this part. However, only one type of anti-ERbeta 2 and 5were tested. Authors did not explain the choice of Abs properly.

The method of counting of ER beta positive cells requires clarification. It was not properly described what authors considered as ERbeta-positive cell. The indicated reference is about counting of ERalpha positive cells. ER can be found in the nucleus and in the cytoplasm. See the discussion of this problem: https://pubmed.ncbi.nlm.nih.gov/29358876/. Authors should discuss cytoplasmic vs nuclear expression of ERbeta.

Discussion section: many phrases /statements are not supported by relevant citations. For instance, the line 360 (“Limited studies are conducted on both ERβ mRNA and protein expression simultaneously of different ERβ isoforms”.) References should be shown. Authors should focus on the actual findings and avoid vacuous generalizations. Potential ERbeta targets associated with the treatment outcomes should be discussed.

Author Response

Responses to Reviewer #2 comments

The study assessed ERb-negative tumors. Authors investigated both ERβ mRNA and ERβ1,2 and 5 isoform expression in breast cancers. ERβ isoform mRNA and proteins were a risk factor for poor outcome. The discussion suggests that standardization of ERβ testing protocol may be required in clinical setting.

The topic is interesting, although the manuscript style is confusing and lacks logical aims. It seems that findings are also chaotic. Unfortunately, contradictory results were not discussed properly.

Following your comments, I made a major revision and restructured the MS in red ink highlighted sections. Consequently, all references are re-arranged and numbered.  

Abstract is too long and not clear. Sometimes it looks like an abstract for a review paper, sometimes- as an original research paper. It is a research paper. Therefore, the abstract should report the size of cohort, the number of tested patients etc.

Revised the abstract as for a research article format and also revised Simple Summary

Introduction does not reflect the substantial findings related to the role of ERbeta in ERalpha negative (and others) breast cancers.

Revised; The introduction is rearranged and revised add lines 93-105 for Bca types and the detailed description is under Discussion. As you know, this is not a review artilce.

Methods: I could not find the statement about approval of the study by the human ethics committee. The study used patients’ samples. The approval of the human ethics committee is required.

IRB approval was already placed under Compliance and Ethics ( page 17) but I also added it in Materials and Methods: 2.1. under Patients lines 117-118

Results: authors tested a good set of anti-ERbeta1 antibodies. It is a substantial contribution. Therefore, authors should focus on this part. However, only one type of anti-ERbeta 2 and 5were tested. Authors did not explain the choice of Abs properly.

Added in lines 194-199

The method of counting of ER beta positive cells requires clarification. It was not properly described what authors considered as ERbeta-positive cell. The indicated reference is about counting of ERalpha positive cells. ER can be found in the nucleus and in the cytoplasm. See the discussion of this problem: https://pubmed.ncbi.nlm.nih.gov/29358876/. Authors should discuss cytoplasmic vs nuclear expression of ERbeta.

Counting ER beta counting is same as that for ERa counting as described in lines 200-205. I added the information and description of cytoplasmic reaction in lines 205-213.

Discussion section: many phrases /statements are not supported by relevant citations. For instance, the line 360 (“Limited studies are conducted on both ERβ mRNA and protein expression simultaneously of different ERβ isoforms”.) References should be shown. Authors should focus on the actual findings and avoid vacuous generalizations. Potential ERbeta targets associated with the treatment outcomes should be discussed.

Revised by adding references and avoided vacuous generalizations.

Potential targets of ER beta is described in 4.3. lines 502-526

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

I want to start by congratulating the authors for the presented study. This kind of systematic study, evaluating different isoforms using the same methodology, allows a better understanding of the impact of the mRNA and protein expression in the clinical outcomes which is important for both patients and health professionals.

In my opinion, there are some issues that can still be changed to contribute to improve the presented article.

The discussion is presented as a brief review of previous studies but lacks a link with the results obtained in the work presented in this manuscript. Additionally, the favorable and adverse outcomes for each situation (expression of the different isoforms) could probably be easier to follow if presented in a table.

Considering the titles of the subsections of the discussion, what are the main differences between what is presented in subsections 4.1 and 4.2? Can the information from these sections be integrated into just one?

In the conclusion, the authors refer that the results are consistent with some of the previous studies (which ones?) and added further insight (what is new?). However, this is not easy to follow when the mentioned studies are cited in the discussion without mentioning the results presented in this work.

The authors present in the discussion several differences in the evaluation methods used in different studies and refer that “standardizing ERβ mRNA or protein testing protocol may be needed. Any specific proposal? What should be modified? In the authors’ opinion what are the best conditions for the different measurements needed?

Almost 3/4 of the cited references were published more than 10 years ago, and only about 7%hed in the last 5 years. Is there any particular reason why this topic has not been studied much recently? Or will there be more recent references that were not cited?

Additional typos that need to be corrected:

Please review the entire text as it has different font sizes (e.g., lines 60-61, 82-83, and many others)

Line 30: “lack” instead of “lacks”

Line 34: delete a space between “in” and “BCa”

Line 49: the authors should consider including “breast cancer” as a keyword (probably

Legend of table 2: delete a space between “Associations” and “between”

Line 298: “Overall, the association between on ERβmRNA and protein expression…” revise as it seems that the word “on” should be deleted

Lines 367-369: What do authors mean by “The studies reviewed herein are summarized.” followed by “The studies with different clinical outcomes reviewed herein are summarized in relation to ERβ isoforms, ERβ isoform mRNA and protein expression and testing protocols.”? Please revise these sentences.

Line 408: delete a space between “However,” and “the”

Lines 501-502: “The main findings of our studies are ERβ isoforms…”, should be “The main findings of our studies are that ERβ isoforms…”

Some references need to be reviewed because there are extra spaces between words; references 17, 22, 27, 28, 55 and 66 do not have the date in bold as the others; reference 36 written in a different color; references 56 and 86 in a different format.

Author Response

Reviewer #3 : responses to comments

I want to start by congratulating the authors for the presented study. This kind of systematic study, evaluating different isoforms using the same methodology, allows a better understanding of the impact of the mRNA and protein expression in the clinical outcomes which is important for both patients and health professionals.

Thank you for your understanding of the complexity of the topic. Indeed, it is a difficult topic and it is my sincere hope that ER-beta can be adopted in clinical setting in the near future.

In my opinion, there are some issues that can still be changed to contribute to improve the presented article.

The discussion is presented as a brief review of previous studies but lacks a link with the results obtained in the work presented in this manuscript. Additionally, the favorable and adverse outcomes for each situation (expression of the different isoforms) could probably be easier to follow if presented in a table.

Table 5: revised. Add Table 7

Considering the titles of the subsections of the discussion, what are the main differences between what is presented in subsections 4.1 and 4.2? Can the information from these sections be integrated into just one?

4.1 and 4.2 addressed two different clinical outcomes:

4.1 different ERβ isoforms and clinical outcomes

4.2 ERβ mRNA or ERβ protein expression, different testing target and clinical outcomes

In the conclusion, the authors refer that the results are consistent with some of the previous studies (which ones?) and added further insight (what is new?). However, this is not easy to follow when the mentioned studies are cited in the discussion without mentioning the results presented in this work.

Revised in line 507-509

The authors present in the discussion several differences in the evaluation methods used in different studies and refer that “standardizing ERβ mRNA or protein testing protocol may be needed. Any specific proposal? What should be modified? In the authors’ opinion what are the best conditions for the different measurements needed?

Revised in line 516-518

Almost 3/4 of the cited references were published more than 10 years ago, and only about 7%hed in the last 5 years. Is there any particular reason why this topic has not been studied much recently? Or will there be more recent references that were not cited?

The recent articles are more review or basic research on TNBC. I could not find any pertinent articles that are similar to our studies and addressing the inconsistent or conflicting results and testing both ERβ isoforms or mRNA or protein testing. However, I included the articles published up to 2021.

Additional typos that need to be corrected:

Please review the entire text as it has different font sizes (e.g., lines 60-61, 82-83, and many others)

done

Line 30: “lack” instead of “lacks” revised

Line 34: delete a space between “in” and “BCa” revised

Line 49: the authors should consider including “breast cancer” as a keyword ; done

Legend of table 2: delete a space between “Associations” and “between” done

Line 298: “Overall, the association between on ERβmRNA and protein expression…” revise as it seems that the word “on” should be deleted: revised

Lines 367-369: What do authors mean by “The studies reviewed herein are summarized.” followed by “The studies with different clinical outcomes reviewed herein are summarized in relation to ERβ isoforms, ERβ isoform mRNA and protein expression and testing protocols.”? Please revise these sentences. revised

Line 408: delete a space between “However,” and “the”; revised

Lines 501-502: “The main findings of our studies are ERβ isoforms…”, should be “The main findings of our studies are that ERβ isoforms…” revised

Some references need to be reviewed because there are extra spaces between words; references 17, 22, 27, 28, 55 and 66 do not have the date in bold as the others; reference 36 written in a different color; references 56 and 86 in a different format. revised

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript has been improved. However, not all my suggestions have been addressed properly. Many new phrases are not supported by relevant references. For instance line 583: " . The findings in our study are consistent with some of the previous studies which demonstrated adverse outcomes associated with high ERβ expression. " No references. Which studies indicated adverse outcome? it is missing.

Authors also used ideas from other papers without citation of the originals. For instance, line 87-88.

 English editing is still required, for instance line 512 " . ERβ expression are associated with good.." - this should be " ERβ expression is associated..."

Author Response

Responses to Reviewer #2 comments

The study assessed ERb-negative tumors. Authors investigated both ERβ mRNA and ERβ1,2 and 5 isoform expression in breast cancers. ERβ isoform mRNA and proteins were a risk factor for poor outcome. The discussion suggests that standardization of ERβ testing protocol may be required in clinical setting.

The topic is interesting, although the manuscript style is confusing and lacks logical aims. It seems that findings are also chaotic. Unfortunately, contradictory results were not discussed properly.

Following your comments, I made a major revision and restructured the MS in red ink highlighted sections. Consequently, all references are re-arranged and numbered.  

Abstract is too long and not clear. Sometimes it looks like an abstract for a review paper, sometimes- as an original research paper. It is a research paper. Therefore, the abstract should report the size of cohort, the number of tested patients etc.

Revised the abstract as for a research article format and also revised Simple Summary

Introduction does not reflect the substantial findings related to the role of ERbeta in ERalpha negative (and others) breast cancers.

Revised; The introduction is rearranged and revised add lines 93-105 for Bca types and the detailed description is under Discussion. As you know, this is not a review artilce.

Methods: I could not find the statement about approval of the study by the human ethics committee. The study used patients’ samples. The approval of the human ethics committee is required.

IRB approval was already placed under Compliance and Ethics ( page 17) but I also added it in Materials and Methods: 2.1. under Patients lines 117-118

Results: authors tested a good set of anti-ERbeta1 antibodies. It is a substantial contribution. Therefore, authors should focus on this part. However, only one type of anti-ERbeta 2 and 5were tested. Authors did not explain the choice of Abs properly.

Added in lines 194-199

The method of counting of ER beta positive cells requires clarification. It was not properly described what authors considered as ERbeta-positive cell. The indicated reference is about counting of ERalpha positive cells. ER can be found in the nucleus and in the cytoplasm. See the discussion of this problem: https://pubmed.ncbi.nlm.nih.gov/29358876/. Authors should discuss cytoplasmic vs nuclear expression of ERbeta.

Counting ER beta counting is same as that for ERa counting as described in lines 200-205. I added the information and description of cytoplasmic reaction in lines 205-213.

Discussion section: many phrases /statements are not supported by relevant citations. For instance, the line 360 (“Limited studies are conducted on both ERβ mRNA and protein expression simultaneously of different ERβ isoforms”.) References should be shown. Authors should focus on the actual findings and avoid vacuous generalizations. Potential ERbeta targets associated with the treatment outcomes should be discussed.

Revised by adding references and avoided vacuous generalizations.

Potnetial ER beta targets are added in 4.3. lines 502-526