Immunohistochemical expression of substance P in breast cancer and its association with prognostic parameters and Ki-67 index

Maha S. Al-Keilani; Rana I. Elstaty; Mohammad A. Alqudah; Asem M. Alkhateeb

doi:10.1371/journal.pone.0252616

Peer Review History

Original SubmissionFebruary 2, 2021
28 Apr 2021 Decision Letter - Vahit Özmen, Editor PONE-D-21-03600 Immunohistochemical Expression of Substance P in Breast Cancer and its Association with Prognostic Parameters and Ki-67 Index. PLOS ONE Dear Dr. El Keillani, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 12 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. 4b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. We will update your Data Availability statement on your behalf to reflect the information you provide. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: - Please include the approval number of your instutional review board and the reason why you did not obtain form of consent from patients. - In line 49 "targeted" would be better instead of "biological". - In line 80 what do you mean by "depth of infiltration"? - How did you construct your tissue microarrays? What was the size of each core, how many cores did you take from each case? Which instrument did you use? - For ER and PR positivity what is your trashold? How did you evaluate HER2 by immunohistochemistry or FISH or both of them? Though you mentioned that you obtained thesse results pathology reports, please give some more detailed information. - In line 161 you wrote that you have whown SP expression levels in Table 1 but they were shown them in table 2 - In line 165 what do you mean by "negative expression of Ki67"? - In line 203 it would be better if you write "prognostic staging" instead of "full staging" with mentioning the date of last AJCC. - You did not give detailed histroy of DCIS of your cases. Then you suggest that there is high expression of SP in women with history of DCIS. We do not know the time period between diagnoisis of DCIS and invasive carcinoma, the site of DCIS, the type of treatment? It is quite speculative to draw such a conclusion. - Please re-write last sentences of discussion of your manuscript based on your results. As you have not evaluated the relation between SP expression and prognosis and there is nothing about the diagnosis of breast cancer as well. The last sentences is not relevant to your findings. Reviewer #2: Substance P in breast cancer is a relatively under-researched area. The article is interesting with this aspect. However, some correction is required for it to be better. Line 80. "Clinicopathological data including age, gender, tumor volume, degree of histological 80 differentiation (well / moderate / poor, WHO), depth of infiltration, staging, and status of lymph 81 nodes and distant metastasis, and hormone receptor (ER and PR) and Her2 / neu statuses were 82 retrospectively collected from patients' medical charts. " I could not understand what "depth of infiltration" is used for. Lİne 161. Table 1 should be changed to table 2. Figure 2 should be omitted. Table 2 desing is done incorrectly. The percentage of substance P level for each molecular subtype should be given in the table. For example Her2 / neu enriched; 13/42 (30.95%), 29/42 (69.05%). The table should be rearranged as shown in the example. This error continues with stage, grade and other parameters. Additionally, the correction regarding these numbers (percentage etc.) should also be made in the discussion. There is a difference in substance P expression level between which molecular subtypes are shown in Table 2. The p value for each molecular type should be given separately. Is there any relationship between Ki67 and substance P? I could not see it in the text and in the summary. However, in the study with the title it is written that investigated this relationship. "Immunohistochemical Expression of Substance P in Breast Cancer and its Association with Prognostic Parameters and Ki-67 Index." In the conclusion section, it should be written clearly in which molecular subtypes and at which stage the SP and ki67 levels are higher. The same is necessary for its relationship with ER, PR and lymph node metastasis. With this point of view, the summary should also be rearranged. ****** 6. 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Revision 1
16 May 2021 Author Response May 17th, 2021 PONE-D-21-03600 Dear Editor, We want to thank you and the reviewers for the generous comments on the manuscript titled “Immunohistochemical Expression of Substance P in Breast Cancer and its Association with Prognostic Parameters and Ki-67 Index”. We have edited the manuscript to address all the comments. Below please find our responses: A. Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: Done 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. Response: Done 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Response: The phrase that refers to the “data not shown” has been removed as it is not essential for this article. 4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. In your revised cover letter, please address the following prompts: 4a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. 4b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Response: The data has been uploaded as supplementary file (S1 File). This is notified in the statistical analysis subsection of materials and methods line 147 of the revised manuscript. B. Reviewers' comments: Reviewer #1: 1- Please include the approval number of your institutional review board and the reason why you did not obtain form of consent from patients. Response: The IRB approval reference number has been added as requested under a new subsection in the materials and methods section (lines 86-90). Due to the retrospective nature of the study and that all the data were fully anonymized, a consent form was waived by the IRB. 2- In line 49 "targeted" would be better instead of "biological". Response: “biological” has been replaced with “targeted” as recommended. 3- In line 80 what do you mean by "depth of infiltration"? Response: How deep tumor cells invade into the tissue, which is considered one of the prognostic factors in breast cancer. 4- How did you construct your tissue microarrays? What was the size of each core, how many cores did you take from each case? Which instrument did you use? Response: More details were added to the TMA subsection (Lines 94-105) 5- For ER and PR positivity what is your threshold? How did you evaluate HER2 by immunohistochemistry or FISH or both of them? Though you mentioned that you obtained these results pathology reports, please give some more detailed information. Response: The cut-off value for ER and PR positivity was set at 5%. Her2/neu status was evaluated according to the guidelines of IHC. FISH for Her2/neu status was not performed at the pathology department at KAUH. The guidelines consider 0 or +1 as negative, +3 as positive. All cases of +2 were sent to a more specialized center to do FISH and the results received were added to the pathology report as either positive or negative. The above information has been added to the materials and methods section lines 80-84. 6- In line 161 you wrote that you have shown SP expression levels in Table 1 but they were shown them in table 2 Response: The information are in Table 1 as cited, but for unknown reason the remaining of the table was omitted from the manuscript file. Thus we added the remaining of the table to the revised file. 7- In line 165 what do you mean by "negative expression of Ki67"? Response: Negative expression means no staining for Ki-67 (score 0) 8- In line 203 it would be better if you write "prognostic staging" instead of "full staging" with mentioning the date of last AJCC. Response: The term “full staging” was replaced by “prognostic staging” as recommended. The date of the last AJCC was added as recommended. 9- You did not give detailed history of DCIS of your cases. Then you suggest that there is high expression of SP in women with history of DCIS. We do not know the time period between diagnosis of DCIS and invasive carcinoma, the site of DCIS, the type of treatment? It is quite speculative to draw such a conclusion. Response: In almost all cases, DCIS is usually diagnosed at the same time in the same biopsy with invasive cancer. But in minority of cases, it is diagnosed alone. In such cases an immediate action to proceed with surgery is taken and duration does not exceed 2-3 weeks between both procedures 10- Please re-write last sentences of discussion of your manuscript based on your results. As you have not evaluated the relation between SP expression and prognosis and there is nothing about the diagnosis of breast cancer as well. The last sentences is not relevant to your findings. Response: The sentences have been rewritten as recommended. (lines 309-312) Reviewer #2: Substance P in breast cancer is a relatively under-researched area. The article is interesting with this aspect. However, some correction is required for it to be better. 1- Line 80. "Clinicopathological data including age, gender, tumor volume, degree of histological 80 differentiation (well / moderate / poor, WHO), depth of infiltration, staging, and status of lymph nodes and distant metastasis, and hormone receptor (ER and PR) and Her2 / neu statuses were retrospectively collected from patients' medical charts. " I could not understand what "depth of infiltration" is used for. Response: How deep tumor cells invade into the tissue, which is considered one of the prognostic factors in breast cancer. 2- Line 161. Table 1 should be changed to table 2. Response: The information is in Table 1 as cited, but for unknown reason the remaining of the table was omitted from the manuscript file. Thus we added the remaining of the table to the revised file. 3- Figure 2 should be omitted. Response: The figure has been omitted as recommended. 4- Table 2 design is done incorrectly. The percentage of substance P level for each molecular subtype should be given in the table. For example Her2 / neu enriched; 13/42 (30.95%), 29/42 (69.05%). The table should be rearranged as shown in the example. This error continues with stage, grade and other parameters. Additionally, the correction regarding these numbers (percentage etc.) should also be made in the discussion. Response: Table 2 has been adjusted as recommended. The paragraph describing the numbers has been omitted as all the data are available in the table. 5- There is a difference in substance P expression level between which molecular subtypes are shown in Table 2. The p value for each molecular type should be given separately. Response: Post hoc analysis by applying Bonferroni correction was performed. A figure to represent the results of the analysis are shown in an added figure (Fig. 3) 6- Is there any relationship between Ki67 and substance P? I could not see it in the text and in the summary. However, in the study with the title it is written that investigated this relationship. "Immunohistochemical Expression of Substance P in Breast Cancer and its Association with Prognostic Parameters and Ki-67 Index." Response: No significant relationship was found between SP and Ki-67 expression levels. This was mentioned in the results section (revised manuscript; line 203), and discussion section (revised manuscript; lines 264-265), and we added a sentence to the conclusion section (line 309). 7- In the conclusion section, it should be written clearly in which molecular subtypes and at which stage the SP and ki67 levels are higher. The same is necessary for its relationship with ER, PR and lymph node metastasis. With this point of view, the summary should also be rearranged. Response: Done. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0252616.r002
19 May 2021 Decision Letter - Vahit Özmen, Editor Immunohistochemical expression of substance P in breast cancer and its association with prognostic parameters and Ki-67 index. PONE-D-21-03600R1 Dear Dr. Al-Keilani, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Vahit Özmen, MD, FACS Professor of Surgery Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0252616.r003
Formally Accepted
24 May 2021 Acceptance Letter - Vahit Özmen, Editor PONE-D-21-03600R1 Immunohistochemical expression of substance P in breast cancer and its association with prognostic parameters and Ki-67 index. Dear Dr. Al-Keilani: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Vahit Özmen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252616.r004

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