Spatial clustering of CD68+ tumor associated macrophages with tumor cells is associated with worse overall survival in metastatic clear cell renal cell carcinoma

Nicholas H. Chakiryan; Gregory J. Kimmel; Youngchul Kim; Ali Hajiran; Ahmet M. Aydin; Logan Zemp; Esther Katende; Jonathan Nguyen; Neale Lopez-Blanco; Jad Chahoud; Philippe E. Spiess; Michelle Fournier; Jasreman Dhillon; Liang Wang; Carlos Moran-Segura; Asmaa El-Kenawi; James Mulé; Philipp M. Altrock; Brandon J. Manley

doi:10.1371/journal.pone.0245415

Peer Review History

Original SubmissionDecember 18, 2020
10 Feb 2021 Decision Letter - Pankaj K Singh, Editor PONE-D-20-39779 Spatial Clustering of CD68+ Tumor Associated Macrophages with Tumor Cells is Associated with Worse Overall Survival in Metastatic Clear Cell Renal Cell Carcinoma PLOS ONE Dear Dr. Chakiryan, 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 5th Aug 2021. 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 include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. 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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: The authors conducted a study aiming to assess cellular density and cellular clustering for myeloid cell markers in 129 regions of interest from 55 samples from 35 patients with metastatic ccRCC. CD68+ density was not associated with OS, while high tumor/CD68+ cell clustering was associated with significantly worse OS. I have some concerns on the study design and statistical analysis. 1. The authors need to provide the sample size calculation and power analysis to justify the design of the study. 2. The definition of overall survival is starting from the sample collection time which may varies across patients. The starting time is suggested to use either diagnosis time or treatment starting time. 3. Have authors conducted assessment to test the Cox proportional hazards assumption? 4. The major limitation of the study is that, there is no validation cohort of the study, it needs to be discussed in the manuscript. Reviewer #2: In this manuscript by Chakriyan et. al., the authors have performed spatial clustering analysis of macrophage markers in metastatic clear cell renal cell carcinoma samples using multiplex immunohistochemistry. The authors have analyzed spatial clusters of macrophage markers with tumor and stromal sections of the tumor tissue to predict the effect on overall survival. It is an interesting approach to analyze and predict overall survival in patients. As mentioned by authors the main limitation of the study is small sample size. The authors show that CD68 spatial distribution has a role to play in predicting OS. The CD163 and CD206 are M2 macrophage markers and are also associated with worse OS in multiple cancer studies. The experimental data would have been more informative and relevant if public database-based OS were compared with the multiplex immunohistochemistry-based spatial clustering of markers. Overall, the article presents an interesting approach to predict overall OS based on spatial distribution of markers. The authors should address the concerns listed: 1. The authors should discuss the relevance of CD163 and CD206 marker clustering in the stromal regions. Since tumor cells secrete a variety of factors that polarize macrophages to M2 type, it is intriguing to note why M2 markers are localized in stromal compartment. 2. Have the authors checked the spatial distribution of CD80 and CD86? Is the staining pattern distribution of these markers similar to CD68? 3. In the Figure 2, authors have stained the tissues for PD-L1 also. The authors should describe the PD-L1 expression in results. 4. Did the authors observe any difference in spatial distribution of CD68 in tissues when segregated based on gender and does it have any effect on OS also? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0245415.r001
Revision 1
26 Feb 2021 Author Response NOTE: The response to reviewers is also included as an attached file, which includes an appendix with supporting information to accompany the responses. RESPONSE TO REVIEWERS We would like to thank the reviewers for their careful reading of the manuscript and thoughtful feedback. Reviewer #1: The authors conducted a study aiming to assess cellular density and cellular clustering for myeloid cell markers in 129 regions of interest from 55 samples from 35 patients with metastatic ccRCC. CD68+ density was not associated with OS, while high tumor/CD68+ cell clustering was associated with significantly worse OS. I have some concerns on the study design and statistical analysis. 1. The authors need to provide the sample size calculation and power analysis to justify the design of the study. This study was conceived as a pilot feasibility study to explore the effect of TAM/tumor spatial clustering on overall survival, a relationship that has not been previously assessed. As such, our study design was not based on an explicit statistical hypothesis but we were interested in discovering large effect sizes. A power/sample size analysis using a power of 0.80, and alpha of 0.05, with an 80% event rate indicates that the 35-patient cohort in our analysis would be adequate to detect a minimum detectable hazard ratio (MDHR) of 1.7 for a standardized continuous variable in a univariate Cox proportional hazards regression analysis. This has been included in the methods, results, and discussion sections of the manuscript (lines 143 to 144, and 181 to 184). We consider this MDHR to be appropriate, as we were interested in discovering large effect sizes. We certainly acknowledge that with a small cohort we are at risk of making Type-II errors in outcomes which were not reported as significant in the manuscript. This limitation has been added to the discussion section of the manuscript (lines 223 to 227). 2. The definition of overall survival is starting from the sample collection time which may varies across patients. The starting time is suggested to use either diagnosis time or treatment starting time. We explicitly chose OS from the time of sample collection when the study was designed. The specific variables of interest in this study were immune cell clustering and density, which are identified in a snapshot that occurs exactly at the time of sample collection. As such, this reflects survival from the moment in time that these variables were measured, which we feel is the appropriate timeframe. Additionally, all samples included in this study were collected in the brief pre-treatment window from patients with metastatic ccRCC, and thus the time from diagnosis to sample collection is very short. The median time from diagnosis to sample collection was 26 days (IQR 9 – 42). 3. Have authors conducted assessment to test the Cox proportional hazards assumption? We have included an assessment of the Cox proportional hazards assumption of the primary Cox regression model (Figure 3C). None of the individual covariates or the Schoenfeld’s global chi-square test violated the proportional hazards assumption. This is included at the bottom of this file as an Appendix. 4. The major limitation of the study is that, there is no validation cohort of the study, it needs to be discussed in the manuscript. We agree completely. This analysis certainly warrants prospective validation in a larger cohort. We have added this into the limitations section (lines 227 to 228). Reviewer #2: In this manuscript by Chakriyan et. al., the authors have performed spatial clustering analysis of macrophage markers in metastatic clear cell renal cell carcinoma samples using multiplex immunohistochemistry. The authors have analyzed spatial clusters of macrophage markers with tumor and stromal sections of the tumor tissue to predict the effect on overall survival. It is an interesting approach to analyze and predict overall survival in patients. As mentioned by authors the main limitation of the study is small sample size. The authors show that CD68 spatial distribution has a role to play in predicting OS. The CD163 and CD206 are M2 macrophage markers and are also associated with worse OS in multiple cancer studies. The experimental data would have been more informative and relevant if public database-based OS were compared with the multiplex immunohistochemistry-based spatial clustering of markers. Overall, the article presents an interesting approach to predict overall OS based on spatial distribution of markers. The authors should address the concerns listed: 1. The authors should discuss the relevance of CD163 and CD206 marker clustering in the stromal regions. Since tumor cells secrete a variety of factors that polarize macrophages to M2 type, it is intriguing to note why M2 markers are localized in stromal compartment. We agree that this is a highly interesting finding. We do not have an answer as to why CD163+ and CD206+ TAMs preferentially cluster into the stromal compartment. However, it is worth noting that this relationship was remarked upon in a prior study, though it was not the purpose of that study and was not quantified (Reference #24 in the manuscript). Further work regarding macrophage chemotaxis into the tumor microenvironment is warranted. For our part, we will first pursue validation of these findings in a larger IF cohort, to confirm this association. 2. Have the authors checked the spatial distribution of CD80 and CD86? Is the staining pattern distribution of these markers similar to CD68? This is an interesting question, but unfortunately the myeloid panel used in this study did not include the CD80 or CD86 markers. 3. In the Figure 2, authors have stained the tissues for PD-L1 also. The authors should describe the PD-L1 expression in results. Thank you for noticing this discrepancy. We have included the PDL1 distribution results in the results section (lines 168 to 170). 4. Did the authors observe any difference in spatial distribution of CD68 in tissues when segregated based on gender and does it have any effect on OS also? These are interesting questions. We have included this data below as an Appendix to this file. There was no difference in CD68/Tumor clustering or OS between genders in our cohort. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0245415.r002
6 Apr 2021 Decision Letter - Pankaj K Singh, Editor Spatial Clustering of CD68+ Tumor Associated Macrophages with Tumor Cells is Associated with Worse Overall Survival in Metastatic Clear Cell Renal Cell Carcinoma PONE-D-20-39779R1 Dear Dr. Chakiryan, 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, Pankaj K Singh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. 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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. 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 ****** 5. 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: (No Response) Reviewer #2: Yes ****** 6. 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: (No Response) Reviewer #2: The authors have addressed all the comments satisfactorily by either performing the experiments or providing justifications. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Wei Xu Reviewer #2: No https://doi.org/10.1371/journal.pone.0245415.r003
Formally Accepted
8 Apr 2021 Acceptance Letter - Pankaj K Singh, Editor PONE-D-20-39779R1 Spatial Clustering of CD68+ Tumor Associated Macrophages with Tumor Cells is Associated with Worse Overall Survival in Metastatic Clear Cell Renal Cell Carcinoma Dear Dr. Chakiryan: 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. Pankaj K Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0245415.r004

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