Overexpression of PD-1 and CD39 in tumor-infiltrating lymphocytes compared with peripheral blood lymphocytes in triple-negative breast cancer

Asmaa M. Zahran; Amal Rayan; Zeinab Albadry M. Zahran; Wael M. Y. Mohamed; Dalia O. Mohamed; Mona H. Abdel-Rahim; Omnia El-Badawy

doi:10.1371/journal.pone.0262650

Peer Review History

Original SubmissionApril 29, 2021
16 Jul 2021 Decision Letter - Sushanta K Banerjee, Editor PONE-D-21-14265 Overexpression of PD-1 and CD39 in tumor-infiltrating lymphocytes compared with peripheral blood lymphocytes in triple-negative breast cancer PLOS ONE Dear Dr. Rayan, 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 Aug 28 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 upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript (PONE-D-21-14265) by Asmaa M. Zahran et al, had investigated the expression of PD-1 and CD39 on CD4+ and CD8+ cells infiltrating tumor tissue compared to their counterparts in peripheral blood. They also explored their association with tumor characteristics, disease progression, and prognosis in females with TNBC. My specific comments are as follows- 1. The authors must mention how they have normalized the flow data for the patient and healthy subset. 2. The single cell suspension protocol from surgically resurrected tumors should be elaborated and properly cited. 3. The authors have shown that the level of peripheral CD4+ T lymphocytes was lower in patients than in controls. An observation by Bushra Sikandar et al contradicts this observation. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648386/ . is there any reason that this patient subset behaved differently ? 4. In the discussion section, the authors quote “Although the level of peripheral CD4+ T lymphocytes was lower in patients than in controls, the percent of PD1 and CD39 expression among these cells was higher in patients. The frequency of CD4+ T lymphocytes in breast cancer tissue was higher than in the periphery and higher than that in the healthy tissue.” This statement is self contradictory. 5. Overall the images do not look well organized and could be improved by including multiple graphs in one figure. 6. The manuscript needs formatting and multiple typological errors were detected that could be taken care of. 7. The experimental design is robust and the data supports their hypothesis that the CD39 and PD1 inhibitory pathways are synergistically utilized by TNBC cells for evading host immune response causing poor survival, and hence combined immunotherapy blocking these pathways might be a promising treatment strategy in this type of cancers. ****** 6. 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Revision 1
24 Aug 2021 Author Response Dear reviewers Thank you for your timely thorough revisions of our manuscript, we answered your questions and made the corresponding changes in the manuscript, all changes were yellow highlighted. The authors must mention how they have normalized the flow data for the patient and healthy subset. Normalization of flow data was done by using natural logarithms of their values, however we repeated their statistical analysis using the corresponding non-parametric tests as declared in the section of statistics. The single cell suspension protocol from surgically resurrected tumors should be elaborated and properly cited. We added reference for the detailed steps of preparation in the methods The authors have shown that the level of peripheral CD4+ T lymphocytes was lower in patients than in controls. An observation by Bushra Sikandar et al contradicts this observation. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648386/ . is there any reason that this patient subset behaved differently ? Our findings are not contradictory to the above mentioned that of Bushra Sikandar et al. Although level of peripheral CD4+ T lymphocytes in our study was lower in patients than in controls, the frequency of CD4+ T lymphocytes in breast cancer tissue was higher than in the periphery and higher than that in the healthy tissue. Bushra Sikandar et al. studied the percentages of lymphocyte subsets including CD4 lymphocytes only in tissue by immunohistochemistry. They compared the percentages of CD4+ T lymphocytes between breast cancer tissue between patients with TNBC and non-TNBC patients and their results showed significantly increased tumour infiltration of lymphocytes (T and B-lymphocytes) in TNBC compared to the patients with non-TNBC. In the discussion section, the authors quote “Although the level of peripheral CD4+ T lymphocytes was lower in patients than in controls, the percent of PD1 and CD39 expression among these cells was higher in patients. The frequency of CD4+ T lymphocytes in breast cancer tissue was higher than in the periphery and higher than that in the healthy tissue.” This statement is self-contradictory. In our study we compared the level of CD4 and CD8 lymphocytes and their expression of PD1 and CD39 between patients with TNBC and healthy control, both in peripheral blood and breast tissue. level of peripheral CD4+ T lymphocytes in our study was lower in patients than in controls. On the contrary, the frequency of CD4+ T lymphocytes in breast cancer tissue was higher than that in the healthy tissue and was even higher than that in the periphery. We explained this in the discussion “Our findings collectively indicate robust trials of the immune system to mount sufficient tumor-specific immune response, possibly by inducing CD4+ migration and accumulation in breast cancer tissue”. Overall the images do not look well organized and could be improved by including multiple graphs in one figure. Three figures were joined in one figure (figures 3A, B, and 4) The manuscript needs formatting and multiple typological errors were detected that could be taken care of. Done as much possible The experimental design is robust and the data supports their hypothesis that the CD39 and PD1 inhibitory pathways are synergistically utilized by TNBC cells for evading host immune response causing poor survival, and hence combined immunotherapy blocking these pathways might be a promising treatment strategy in this type of cancers. The predominant systemic therapy for most metastatic TNBC is chemotherapy, but responses are often short-lived, and patients have a median overall survival of 12 to 18 months. Therefore, improved therapies are urgently needed. Immunotherapy has prolonged survival in many solid tumors and represents a promising treatment strategy for TNBC. The most successful immunotherapeutic agents consist of immune checkpoint inhibitors, such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and PD-1 inhibirors, to improve the cytotoxicity and proliferative capacity of tumor-infiltrating lymphocytes. Immune checkpoint inhibitors, including monoclonal antibodies against PD-1 (ie, pembrolizumab, nivolumab), PD-L1 (ie, atezolizumab, durvalumab, avelumab), and CTLA-4 (ie, ipilimumab), have generated durable responses across many tumor types. Attachments Attachment Submitted filename: response to reviewers PD1CD39.docx https://doi.org/10.1371/journal.pone.0262650.r002
4 Jan 2022 Decision Letter - Sushanta K Banerjee, Editor Overexpression of PD-1 and CD39 in tumor-infiltrating lymphocytes compared with peripheral blood lymphocytes in triple-negative breast cancer PONE-D-21-14265R1 Dear Dr. Rayan, 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, Sushanta K Banerjee, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0262650.r003
Formally Accepted
10 Jan 2022 Acceptance Letter - Sushanta K Banerjee, Editor PONE-D-21-14265R1 Overexpression of PD-1 and CD39 in tumor-infiltrating lymphocytes compared with peripheral blood lymphocytes in triple-negative breast cancer Dear Dr. Rayan: 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 Professor Sushanta K Banerjee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0262650.r004

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