Association between KRAS and PIK3CA Mutations and Progesterone Resistance in Endometriotic Epithelial Cell Line

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The manuscript by Kanno and co-authors focuses on a study conducted on the cell line HMOsisEC10, which was previously established in their laboratory. The study has several limitations, all of which are listed in the manuscript. In my opinion, the most significant limitation is the lack of a control endometriotic cell line. However, the authors stated that the unmodified HMOsisEC10 is a relevant control for their study. Another important limitation is the absence of supportive data from clinical material. Again, the authors wrote that they are currently working on establishing further patient-derived endometriotic cell lines to provide more evidence. Therefore, the conclusions drawn in the manuscript should be considered preliminary, and further research is needed to strengthen them.

Additionally, while reading this manuscript, two additional questions arose.

1.      Statistical significance in Figure 2C, Figure 3C and Figure 4C needs to be rechecked. Can you please present these data in a diagram with individual values?

2.      The use of Dunnett's test should also be justified.

Author Response

April 9th, 2024

 

Prof. Dr. Madhav Bhatia

Editor-in-Chief

Current Issues in Molecular Biology

 

Dear Editor:

 

We wish to re-submit the manuscript titled “Association between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line.” The manuscript ID is cimb-2949032, the resubmission of cimb-2900397.

 

We thank you and the reviewers for their thoughtful suggestions and insights, by which, we believe, the manuscript has apparently improved, and hopefully being acceptable for publication in Current Issues in Molecular Biology.

 

The manuscript has been rechecked, and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been indicated in red text as attached below and reflected in the revised version of the manuscript shown in red text.

 

Responses to reviewer 1 comments

 

1. The manuscript by Kanno and co-authors focuses on a study conducted on the cell line HMOsisEC10, which was previously established in their laboratory. The study has several limitations, all of which are listed in the manuscript. In my opinion, the most significant limitation is the lack of a control endometriotic cell line. However, the authors stated that the unmodified HMOsisEC10 is a relevant control for their study. Another important limitation is the absence of supportive data from clinical material. Again, the authors wrote that they are currently working on establishing further patient-derived endometriotic cell lines to provide more evidence. Therefore, the conclusions drawn in the manuscript should be considered preliminary, and further research is needed to strengthen them.

 

As the reviewer mentioned, we consistently think that unmodified HMOsisEC10 cells sufficiently responded to progestin treatment in our study and they can therefore be a relevant control for our study. The reviewer suggested to incorporate more cell lines established from additional patients to improve scientific accuracy. We greatly appreciate to the review for helpful advice, but it will take further 6-12 months for the completion of such experiments, that is unrealistic and unsuitable for this revision. We greatly appreciate to the review for his (her) advice, of which critical point was mentioned in the discussion section.

 

2. Statistical significance in Figure 2C, Figure 3C and Figure 4C needs to be rechecked. Can you please present these data in a diagram with individual values?

 

Thank you for your question. We made Figure s1 to present each value of the figures you mentioned. We presented means and SDs on the top of each graph.

 

Figure s1.

 

 

3. The use of Dunnett's test should also be justified.

Thank you for this suggestion. Our experiment is three group comparison. If we conduct Student’s t test, it is multiple comparison and may lead to false positive of significance. Therefore, we must have conduct multiple comparison procedure. Since our experiment is focusing on progestin resistance/sensitivity and we needed to compare the DNG group and MPA group to the control group, we chose Dunnett test for our multiple comparison procedure.

 

Responses to reviewer 2 comments

 

1. Please prepare list of abbreviations used in manuscript.

Thank you for your suggestion. Because the format of abbreviation list is not defined in the author instruction, we just show the list in this cover letter.  If they are necessary in the text, we are welcome that they are incorporated in the text.

 

Table. Abbreviation List

 

2. Please mention the role of the KRAS mutation and PIK3CA mutation in other gynecological / pregnancy related diseases.

Thank you for this suggestion. To our knowledge, these mutations have not been reported in pregnancy related diseased, while they have recently been reported in normal endometrial epithelial and endometriotic epithelial cells, in relation to endometrial cancer as well as ovarian endometrioid and clear cell carcinoma.  However, it remains to be fully elucidated how these mutations mechanistically contribute to multistep carcinogenesis or even the development of endometriosis.  This information was added in the introduction section on pages 2, lines 60–68.

 

3.Please add the paragraphs about potential clinical usage of your result in the endometriosis diagnosis, differential diagnosis and/or treatment.

Thank you for this suggestion. We added the description of clinical usage of our result focusing on treatment in the discussion section on pages 13, lines 321–326.

 

Again, thank you for giving us the opportunity to improve our manuscript with your valuable comments and queries.

 

Sincerely,

Kosuke Kanno, MD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

Kentaro Nakayama, MD, PhD

Nagoya City University East Medical Center

Chikusaku Wakamizu 1-2-23, Nagoya, Aichi, Japan 4648547

Phone: 81-52-721-7171

Fax: 81-52-721-1308 (fax)

E-mail: [email protected]

 

Satoru Kyo, MD, PhD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

I appreciate the opportunity to review the manuscript entitled “Association between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line” submitted in journal CIMB.  

The authors reported that KRAS mutation and PIK3CA mutation in endometriotic cells are not be associated with progesterone resistance in terms of aggressiveness of endometriosis buy  KRAS mutations are  associated with progesterone resistance possibly in the context of pain

 

Reviewer Comments:

1.      Please prepare list of abbreviations used in manuscript.

2.      Please mention the role of the KRAS mutation and PIK3CA mu-
tation in other gynecological / pregnancy related diseases.

3.      Pleas add the paragraphs about potential clinical usage of the your result in the endometriosis diagnosis, differential diagnosis and/or treatment.

 

Taking into account the pathology reviewed in above mentioned manuscript, my opinion is that this submission meet the criteria to be published in journal CIMB after major revisions and inclusion of the data I suggested.

Author Response

April 9th, 2024

 

Prof. Dr. Madhav Bhatia

Editor-in-Chief

Current Issues in Molecular Biology

 

Dear Editor:

 

We wish to re-submit the manuscript titled “Association between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line.” The manuscript ID is cimb-2949032, the resubmission of cimb-2900397.

 

We thank you and the reviewers for their thoughtful suggestions and insights, by which, we believe, the manuscript has apparently improved, and hopefully being acceptable for publication in Current Issues in Molecular Biology.

 

The manuscript has been rechecked, and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been indicated in red text as attached below and reflected in the revised version of the manuscript shown in red text.

 

Responses to reviewer 1 comments

 

1. The manuscript by Kanno and co-authors focuses on a study conducted on the cell line HMOsisEC10, which was previously established in their laboratory. The study has several limitations, all of which are listed in the manuscript. In my opinion, the most significant limitation is the lack of a control endometriotic cell line. However, the authors stated that the unmodified HMOsisEC10 is a relevant control for their study. Another important limitation is the absence of supportive data from clinical material. Again, the authors wrote that they are currently working on establishing further patient-derived endometriotic cell lines to provide more evidence. Therefore, the conclusions drawn in the manuscript should be considered preliminary, and further research is needed to strengthen them.

 

As the reviewer mentioned, we consistently think that unmodified HMOsisEC10 cells sufficiently responded to progestin treatment in our study and they can therefore be a relevant control for our study. The reviewer suggested to incorporate more cell lines established from additional patients to improve scientific accuracy. We greatly appreciate to the review for helpful advice, but it will take further 6-12 months for the completion of such experiments, that is unrealistic and unsuitable for this revision. We greatly appreciate to the review for his (her) advice, of which critical point was mentioned in the discussion section.

 

2. Statistical significance in Figure 2C, Figure 3C and Figure 4C needs to be rechecked. Can you please present these data in a diagram with individual values?

 

Thank you for your question. We made Figure s1 to present each value of the figures you mentioned. We presented means and SDs on the top of each graph.

 

Figure s1.

 

 

3. The use of Dunnett's test should also be justified.

Thank you for this suggestion. Our experiment is three group comparison. If we conduct Student’s t test, it is multiple comparison and may lead to false positive of significance. Therefore, we must have conduct multiple comparison procedure. Since our experiment is focusing on progestin resistance/sensitivity and we needed to compare the DNG group and MPA group to the control group, we chose Dunnett test for our multiple comparison procedure.

 

Responses to reviewer 2 comments

 

1. Please prepare list of abbreviations used in manuscript.

Thank you for your suggestion. Because the format of abbreviation list is not defined in the author instruction, we just show the list in this cover letter.  If they are necessary in the text, we are welcome that they are incorporated in the text.

 

Table. Abbreviation List

 

2. Please mention the role of the KRAS mutation and PIK3CA mutation in other gynecological / pregnancy related diseases.

Thank you for this suggestion. To our knowledge, these mutations have not been reported in pregnancy related diseased, while they have recently been reported in normal endometrial epithelial and endometriotic epithelial cells, in relation to endometrial cancer as well as ovarian endometrioid and clear cell carcinoma.  However, it remains to be fully elucidated how these mutations mechanistically contribute to multistep carcinogenesis or even the development of endometriosis.  This information was added in the introduction section on pages 2, lines 60–68.

 

3.Please add the paragraphs about potential clinical usage of your result in the endometriosis diagnosis, differential diagnosis and/or treatment.

Thank you for this suggestion. We added the description of clinical usage of our result focusing on treatment in the discussion section on pages 13, lines 321–326.

 

Again, thank you for giving us the opportunity to improve our manuscript with your valuable comments and queries.

 

Sincerely,

Kosuke Kanno, MD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

Kentaro Nakayama, MD, PhD

Nagoya City University East Medical Center

Chikusaku Wakamizu 1-2-23, Nagoya, Aichi, Japan 4648547

Phone: 81-52-721-7171

Fax: 81-52-721-1308 (fax)

E-mail: [email protected]

 

Satoru Kyo, MD, PhD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Please, see in attachement

Comments for author File: Comments.pdf

Author Response

April 16th, 2024

 

Prof. Madhav Bhatia

Editor-in-Chief

Current Issues in Molecular Biology

 

Dear Editor:

 

We wish to re-submit the manuscript titled “Association between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line.” The manuscript ID is cimb-2949032, the resubmission of cimb-2900397.

 

We greatly thank you and the reviewers for their thoughtful suggestions and insights, by which, we believe, the manuscript has apparently been improved, and hopefully being acceptable for publication in Current Issues in Molecular Biology.

 

The manuscript has been rechecked, and the changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been indicated in red text as attached below and reflected in the revised version of the manuscript shown in red text.

 

Responses to reviewer 1 comments

 

1. I appreciate your efforts to improve the manuscript. It may be accepted that HMOsisEC10 cell line is used as a control and it may be accepted that currently your conclusions are not supported by clinical data but it seems that you are not fully meet my concerns on data processing and presentations. It is highly recommended to present your graphs as scatter plots. An example is given below:

 

Thank you for showing an example of scatter plot. We changed Figures 2 to 7 according to your comment.

During the process of the plotting, we found our careless mistake on Figure 2-C. When we made this figure in the 1^st draft, a few columns of the excel data in DNG group were wrongly selected so that a few columns of control groups were partially incorporated into DNG group.  Eventually, the mean values have now been slightly altered in the revised version, but the statistical significance has not been affected by this mistake. We apologize for our careless mistake.

 

Responses to reviewer 2 comments

 

1. The authors improved the manuscript in concordance to the reviewer comments. Accordingly, the paper is suitable to be published in the CIMB.

We appreciate to the reviewer for the positive comment.

 

Again, thank you for giving us the opportunity to improve our manuscript with your valuable comments and queries.

 

Sincerely,

Kosuke Kanno, MD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

Kentaro Nakayama, MD, PhD

Nagoya City University East Medical Center

Chikusaku Wakamizu 1-2-23, Nagoya, Aichi, Japan 4648547

Phone: 81-52-721-7171

Fax: 81-52-721-1308 (fax)

E-mail: [email protected]

 

Satoru Kyo, MD, PhD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors improved the manuscript in concordance to the reviewer comments. Accordingly, the paper is suitable to be published in the CIMB. 

Author Response

April 16th, 2024

 

Prof. Madhav Bhatia

Editor-in-Chief

Current Issues in Molecular Biology

 

Dear Editor:

 

We wish to re-submit the manuscript titled “Association between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line.” The manuscript ID is cimb-2949032, the resubmission of cimb-2900397.

 

We greatly thank you and the reviewers for their thoughtful suggestions and insights, by which, we believe, the manuscript has apparently been improved, and hopefully being acceptable for publication in Current Issues in Molecular Biology.

 

The manuscript has been rechecked, and the changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been indicated in red text as attached below and reflected in the revised version of the manuscript shown in red text.

 

Responses to reviewer 1 comments

 

1. I appreciate your efforts to improve the manuscript. It may be accepted that HMOsisEC10 cell line is used as a control and it may be accepted that currently your conclusions are not supported by clinical data but it seems that you are not fully meet my concerns on data processing and presentations. It is highly recommended to present your graphs as scatter plots. An example is given below:

 

Thank you for showing an example of scatter plot. We changed Figures 2 to 7 according to your comment.

During the process of the plotting, we found our careless mistake on Figure 2-C. When we made this figure in the 1^st draft, a few columns of the excel data in DNG group were wrongly selected so that a few columns of control groups were partially incorporated into DNG group.  Eventually, the mean values have now been slightly altered in the revised version, but the statistical significance has not been affected by this mistake. We apologize for our careless mistake.

 

Responses to reviewer 2 comments

 

1. The authors improved the manuscript in concordance to the reviewer comments. Accordingly, the paper is suitable to be published in the CIMB.

We appreciate to the reviewer for the positive comment.

 

Again, thank you for giving us the opportunity to improve our manuscript with your valuable comments and queries.

 

Sincerely,

Kosuke Kanno, MD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

Kentaro Nakayama, MD, PhD

Nagoya City University East Medical Center

Chikusaku Wakamizu 1-2-23, Nagoya, Aichi, Japan 4648547

Phone: 81-52-721-7171

Fax: 81-52-721-1308 (fax)

E-mail: [email protected]

 

Satoru Kyo, MD, PhD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

 

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Now the authors addressed all the issues of the manuscript. It can be published in the present form.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors tried to establish link between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line that was previously developed in their lab. Unfortunately, some serious flaws must be removed before making decision on publishing of this manusript.

MAJOR

1.     The design of the presented study lacks clarity.Endometriosis is not an inflammatory disease, but a hormone-dependent pathology. Proliferation of endometriotic tissue, in the form of heterotopias outside the uterine cavity (external genital endometriosis), and inside the uterus (adenomyosis), can provoke a pain syndrome in various ways. This is not only due to the hormonal cascade that triggers an inflammatory reaction, but also through other mechanisms. For example, histological examination of certain heterotopias reveals nerve endings.

2.     The relationship between progesterone and pain in the disease has not been discussed enough in the introduction.

3.     There are several endometriotic cell lines that are commercially available, including epithelial type 12-Z, 49-Z,
108-Z and 11-Z. In my opinion experiments with the newly established cell line should be done in parallel with control cell line to make conclusions more clear.

4.     There are inaccuracies in the description of nosology in the discussion.

5.     The discussion and conclusions are written in an evasive manner.

6.     The limitations listed in the discussion cast doubt on the relevance of the study.

MINOR

1.     The design of the drawings does not follow a unified style.

2.     Figure captions do not include information about statistical analysis and the number of samples.

Author Response

March 13th, 2024

 

Prof. Dr. Madhav Bhatia

Editor-in-Chief

Current Issues in Molecular Biology

 

Dear Editor:

We wish to re-submit the manuscript titled “Association between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line.” The manuscript ID is cimb-2900307.

 

We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We look forward to working with you and the reviewers to move this manuscript closer to publication in Current Issues in Molecular Biology.

 

The manuscript has been rechecked, and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared, indicated in red text, and attached below. Additionally, the revisions are indicated with in red text.

 

 

Responses to reviewer 1 comments

 

MAJOR

1. The design of the presented study lacks clarity. Endometriosis is not an inflammatory disease, but a hormone-dependent pathology. Proliferation of endometriotic tissue, in the form of heterotopias outside the uterine cavity (external genital endometriosis), and inside the uterus (adenomyosis), can provoke a pain syndrome in various ways. This is not only due to the hormonal cascade that triggers an inflammatory reaction, but also through other mechanisms. For example, histological examination of certain heterotopias reveals nerve endings.

Thank you for this suggestion. As you mentioned, we did not describe the association between endometriosis, hormonal cascade, and pain. Therefore, we have added the description of endometriosis-related pain in the discussion section on pages 10–11, lines 234–254.

 

2. The relationship between progesterone and pain in the disease has not been discussed enough in the introduction.

Thank you for this suggestion. As you pointed out, we apologize for omitting the description of the relationship between progesterone and pain in the introduction section. We have added it in the introduction section, on pages 1–2, lines 46–57.

 

3. There are several endometriotic cell lines that are commercially available, including epithelial type 12-Z, 49-Z, 108-Z and 11-Z. In my opinion experiments with the newly established cell line should be done in parallel with control cell line to make conclusions more clear.

Thank you for this suggestion. We apologize for our omission in using other established and commercially available endometriotic cell lines. As you indicated, we should have used a control cell line. Regrettably, due to constraints within the allotted revision timeframe, we find ourselves unable to undertake further experiments. We are currently advancing an ongoing project aimed at addressing the limitations identified in this report. To enhance the scope of our investigation, we intend to incorporate commercially available endometriotic cell lines into the ongoing project.

 

4. There are inaccuracies in the description of nosology in the discussion.

Thank you for this suggestion. We have added the description of nosology in the discussion section on page 10, lines 234–243.

 

5. The discussion and conclusions are written in an evasive manner.

Thank you for this suggestion. We have revised our discussion and conclusion sections to provide a more detailed and concrete explanation.

On page 12, lines 323–336

On page 12, lines 338–342

6. The limitations listed in the discussion cast doubt on the relevance of the study.

Thank you for this suggestion. As you mentioned, our reports have several limitations. We regret we could not overcome those limitations this time. This is an in vitro experiment, and we position it as the first step of ongoing projects.

 

MINOR

1. The design of the drawings does not follow a unified style.

Thank you for this suggestion. We have deleted Figure 8.

 

2. Figure captions do not include information about statistical analysis and the number of samples.

Thank you for this suggestion. We apologize for this omission. We have added information about statistical analysis (Dunnett’s test), number of samples, and standard deviation to the figures (Figures 2–6).

 

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

 

Sincerely,

Kosuke Kanno, MD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

Kentaro Nakayama, MD, PhD

Nagoya City University East Medical Center

Chikusaku Wakamizu 1-2-23, Nagoya, Aichi, Japan 4648547

Phone: 81-52-721-7171

Fax: 81-52-721-1308 (fax)

E-mail: [email protected]

 

Reviewer 2 Report

Comments and Suggestions for Authors

This text presents a study on the association between mutations in the KRAS and PIK3CA genes and progesterone resistance in an epithelial cell line of endometriosis. We conducted an analysis of the study, highlighting relevant points and others that in our opinion should be improved.

 

Key Points:

 

Focus on an important issue: The study addresses a relevant problem in the field of endometriosis, which is resistance to hormonal therapy, specifically to progesterone. This is a crucial aspect as endometriosis can have a significant impact on women's quality of life due to chronic pain and other complications.

 

Detailed analysis of molecular mechanisms: The study provides a detailed understanding of the possible molecular mechanisms involved in progesterone resistance, focusing on mutations in the KRAS and PIK3CA genes. This contributes to knowledge about the pathogenesis of endometriosis and could have important implications for the development of new therapeutic strategies.

 

Exploration of factors associated with pain: In addition to examining progesterone resistance, the study also investigates the association between genetic mutations and pain associated with endometriosis. This expands the scope of the study by considering important clinical aspects beyond hormonal treatment response.

 

Areas for Improvement:

 

Expand sample diversity: The study relies on a single cell line, limiting the generalizability of the results. It would be beneficial to include a variety of cell lines or patient samples to validate the findings and increase the robustness of the results.

 

Clinical validation: Although the study provides valuable information about molecular mechanisms, it lacks clinical validation in real patients with endometriosis. It would be crucial to complement in vitro findings with clinical studies evaluating the relevance of these mutations in medical practice.

 

Improve discussion of limitations: While some limitations of the study are mentioned, such as the use of a single cell line, a more detailed discussion of these limitations and how they could affect the interpretation of results would be helpful.

 

Optimize drug concentrations: The study uses significantly higher concentrations of DNG and MPA than those found in plasma, which may not adequately reflect physiological conditions. It would be important to adjust concentrations to be more representative of clinical conditions.

 

Explore other mechanisms: Although the study focuses on genetic mutations, it would be valuable to explore other mechanisms that could contribute to progesterone resistance in endometriosis, such as epigenetic modifications or interaction with the endometriotic lesion microenvironment.

 

In summary, the study offers a significant contribution to understanding endometriosis and progesterone resistance, but it needs to address some limitations and consider additional aspects to strengthen its validity and clinical relevance.

Author Response

March 13th, 2024

 

Prof. Dr. Madhav Bhatia

Editor-in-Chief

Current Issues in Molecular Biology

 

Dear Editor:

We wish to re-submit the manuscript titled “Association between KRAS and PIK3CA mutations and progesterone resistance in endometriotic epithelial cell line.” The manuscript ID is cimb-2900307.

 

We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We look forward to working with you and the reviewers to move this manuscript closer to publication in Current Issues in Molecular Biology.

 

The manuscript has been rechecked, and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared, indicated in red text, and attached below. Additionally, the revisions are indicated with tracked changes in the manuscript.

 

 

Responses to reviewer 2 comments

 

1. Expand sample diversity: The study relies on a single cell line, limiting the generalizability of the results. It would be beneficial to include a variety of cell lines or patient samples to validate the findings and increase the robustness of the results.

Thank you for this suggestion. We regret that we did not use other endometriotic cell lines. As you indicated, we should have used a control cell line; however, conducting additional experiments within the confines of the revision limit is not feasible. Currently, we are conducting an ongoing project that overcomes this report’s limitations. We would add commercially available endometriotic cell lines to the ongoing project.

 

2. Clinical validation: Although the study provides valuable information about molecular mechanisms, it lacks clinical validation in real patients with endometriosis. It would be crucial to complement in vitro findings with clinical studies evaluating the relevance of these mutations in medical practice.

Thank you for this suggestion. We have added a report regarding the clinical features of KRAS-mutated endometriosis in the discussion section on page12, lines 300-302. In our ongoing project, we aim to compare the effect of mutations on progesterone resistance between patients and in vitro models.

 

3. Improve discussion of limitations: While some limitations of the study are mentioned, such as the use of a single cell line, a more detailed discussion of these limitations and how they could affect the interpretation of results would be helpful.

Thank you for this suggestion. We have added descriptions in the limitation paragraph about using single cell lines and the concentration on page 12, lines 323–336.

 

4. Optimize drug concentrations: The study uses significantly higher concentrations of DNG and MPA than those found in plasma, which may not adequately reflect physiological conditions. It would be important to adjust concentrations to be more representative of clinical conditions.

Thank you for this suggestion. As you indicated, the concentrations of DNG and MPA are significantly higher than those in plasma. Regrettably, we are unable to conduct additional experiments within the constraints of the revision limit. However, we intend to optimize this aspect in the ongoing project.

 

5. Explore other mechanisms: Although the study focuses on genetic mutations, it would be valuable to explore other mechanisms that could contribute to progesterone resistance in endometriosis, such as epigenetic modifications or interaction with the endometriotic lesion microenvironment.

Thank you for this suggestion. As described in our discussion, epigenetic modifications, such as PR promoter hypermethylation or microRNA dysregulation, are reported to be associated with progesterone resistance via decreased PR expression. However, we could not assess it in our present experiments. Therefore, we transduced PRB to our HMOsisEC10 cell lines, and the effect of decreased PR expression via epigenetic mechanism was eliminated in our experiment. We also did not assess the epigenetic effect of PR signaling downstream. In our ongoing project, we are examining the correlation between KRAS or PIK3CA and progesterone resistance through the utilization of endometriotic epithelial organoids. Additionally, we plan to delve into the epigenetic aspect of this investigation using organoids.

 

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

 

Sincerely,

Kosuke Kanno, MD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

Kentaro Nakayama, MD, PhD

Nagoya City University East Medical Center

Chikusaku Wakamizu 1-2-23, Nagoya, Aichi, Japan 4648547

Phone: 81-52-721-7171

Fax: 81-52-721-1308 (fax)

E-mail: [email protected]

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I appreciate your effort to improve the manuscript. Although there have been some changes to the introduction and discussion sections, nothing has been done to improve the experimental part. Unfortunately, this means that your conclusions are not based on the results. I would recommend that you continue this study with proper controls in order to obtain better data.

Author Response

March 19th, 2024

 

Prof. Dr. Madhav Bhatia

Editor-in-Chief

Current Issues in Molecular Biology

 

Dear Editor:

We sincerely tried to respond reviewer’s major concern that control cell lines should be used in parallel.  However, we found several difficulties to use them. The reasons are mentioned below.

 

The reviewer insisted that experiments with the newly established cell line should be done in parallel with control cell line to make conclusions more clear.  However, in the manuscript, we have already showed that our cell line with PR overexpression responded to P4 very well, suggesting that they maintained hormonal responses as a good positive control. 

To make the best effort to respond the reviewer’s request, we have searched endometriotic epithelial cells available, some are commercially available.  However, to our knowledge, most of them were created by the introduction of SV40 LT or other oncogenes to immortalize.  As you know, this causes serious problems because SV40LT strongly induces chromosomal instability in normal cells, resulting in highly frequent chromosomal abnormalities, especially in high proportion doublings.  Eventually, such cells will not maintain biological characteristics of normal endometriotic epithelial cells.  This is why we selected non-SV40 method to create immortalized endometriotic epithelial cells.  Most importantly, there is no guarantee that these cells can response to P4 expectedly, potentially due to the above reasons.  Thus, we are very negative to use such cells as positive control cells.  Alternatively, it might be ideal to create additional cell lines immortalized by our methods, originated from other patients, but unfortunately it will take long term periods, probably in a year or more

One of the foci of this study is the establishment of model cell line with highly defined genetic elements, including KRAS-MT and PIK3CA-MT, that we believe the readers of your journal will be interested in. No such cell lines have not been reported so far.   These genetic elements were introduced by rentiviral transfection, with polyclonal growth features, minimizing the selection bias, therefore may cover disadvantage of the usage of one cell line.

In the revised manuscript, we mention the limitation of the study that only one cell line was used for examination, but we currently attempt to establish further cell lines from additional patients, which is highlighted in the revised version.  Other minor alterations due to our careless mistakes are also highlighted.

Based on several reasons listed above, we greatly appreciate if you could finally reconsider your decision and provide us an opportunity to publish our rare model in your journal by the above modification.

 

Sincerely,

Kosuke Kanno, MD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]

 

Satoru Kyo, MD, PhD

Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine

Enyacho 89-1, Izumo, Shimane, Japan 693-8501

Phone: 81-853-20-2268

Email: [email protected]