Tumor-infiltrating lymphocytes and macrophages as a significant prognostic factor in biliary tract cancer

Ryota Tanaka; Shimpei Eguchi; Kenjiro Kimura; Go Ohira; Shogo Tanaka; Ryosuke Amano; Hiroaki Tanaka; Masakazu Yashiro; Masaichi Ohira; Shoji Kubo

doi:10.1371/journal.pone.0280348

Peer Review History

Original SubmissionAugust 11, 2022
18 Aug 2022 Decision Letter - Gianfranco D. Alpini, Editor PONE-D-22-22499Tumor-Infiltrating Lymphocytes and Macrophages as a Significant Prognostic Factor in Biliary Tract CancerPLOS ONE Dear Dr. Kenjiro Kimura, 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. The study has merit. Please submit your revised manuscript within 60 days. 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 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: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: Tumor-Infiltrating Lymphocytes and Macrophages as a Significant Prognostic Factor in Biliary Tract Cancer In this manuscript the authors study the impact of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) on the prognosis of biliary tract cancer (BTC), concepts that are not fully elucidated and understood. They study the effects of the various immune cells infiltration in tumor microenvironment (TME). For their in vivo experiments they used 130 patients with BTC who underwent surgical treatment and evaluated TILs and TAMs with immunohistochemical staining. In their results they show CD8-high, CD4-high, FOXP3-high, and CD68-low in TME as one factor, and they we calculated the immune score according to the number of factors. The high immune-score group showed significantly superior overall survival (OS) and recurrence-free survival (RFS) and the low immune-score group (median OS, 60.8 vs. 26.4 months, p = 0.001; median RFS not reached vs. 17.2 months, p 0.001). Furthermore, high immune-score was an independent good prognostic factor for OS and RFS (hazards ratio 2.05 and 2.41 and p = 0.01 and p = 0.001, respectively). Finally, they conclude that high immune-score group had significantly superior OS and RFS and was an independent good prognostic factor for OS and RFS. Despite the interesting points elucidated by the authors in this study, before proceeding with publication, the following questions must be addressed by the authors: 1. In the Figure 1, the authors shown representative immunohistochemistry images of high and low infiltration of CD8-high, CD4-high, and FOXP3-high TILs and CD68-low CD8+, CD4+, FOXP3+, and CD68+ cells, in human patients. First, the authors need to mention the kind of microscope by which these pictures are taken. Also, they please a graph shown a numerical evaluation of the staining it should be appreciable by the readers. Furthermore, need to add some arrows to show the infiltration of the above cells in the tissue. In the Figure1, the authors consider two groups of patients with low and high infiltration, it should be interesting if in the Figure 1 they add also images of the healthy control group. 2. In the Table1, the authors listed some of the clinicopathological characteristics of the 130 patients with BTC. Please, the authors must complete the list, give some more info such as age, eventually pharmacological treatment of the patients, stage of the illness ecc. 3. In the tissue microarray construction paragraph, the authors ensured that representative tumor cell-rich areas are HE stained with a light microscope and were sent to create TMA blocks. The authors must include some HE images of the tumor sections in their results. So the readers can appreciate the percentage of TILs on HE stained samples. 4. Is well known that immunohistochemical markers used to identify M1 and M2 TAMs are the keystones of TAM evaluation. In this manuscript the authors study the tumor-associated macrophages (TAMs) in BTC. It should be interesting if the authors extent their experiments (immunohistochemistry or PCR or blots) in other markers that characterized the TAMs, such as, CD11c, CD86, iNOS, pSTAT1 (M1 markers), CD163, CD204, CD206 (M2 markers) or at least some of them. Reviewer #2: This is a very interesting study looking at the correlation between TIL in tumors from Biliary Tract cancer. However, the authors only look at high and low levels of infiltrating immune cells, control/normal tissue analysis is needed. You may need to assess the co-staining of CD4+ and Foxp3 staining, since there may be overlap, to conclusively determine specific cell type, as well as CD3+ with CD8+/CD4+ cells (instead of them each individually). Images in Figure 2 are hard to visualize. It may be necessary to use Immunofluorescence to visualize co-staining. In the discussion, the authors state that role of CD8+ T cells as a cytotoxic role. This is not necessarily true. There are numerous subsets of CD8+ T cell and the authors did not access these. Although specific patient data may be restricted, the authors did not access potential cofounders that could alter results from this study in the discussion. ****** 6. 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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.0280348.r001
Revision 1
4 Dec 2022 Author Response Point by point response to Reviewer 1 Comments We are grateful for your comments which have helped to improve our manuscript. As indicated in the responses that follow, we have taken your comments into account in our manuscript. 1. In the Figure 1, the authors shown representative immunohistochemistry images of high and low infiltration of CD8-high, CD4-high, and FOXP3-high TILs and CD68-low CD8+, CD4+, FOXP3+, and CD68+ cells, in human patients. First, the authors need to mention the kind of microscope by which these pictures are taken. Also, they please a graph shown a numerical evaluation of the staining it should be appreciable by the readers. Furthermore, need to add some arrows to show the infiltration of the above cells in the tissue. In the Figure1, the authors consider two groups of patients with low and high infiltration, it should be interesting if in the Figure 1 they add also images of the healthy control group. Response: We added the information of microscope in page 9, line 92-93. We added the figure of the number of infiltrating immune cells as Supplementary Figure 2. We added arrows to the stained immune cells in Figure 1. In this current study, we evaluated the infiltration of immune cells into the tumor microenvironment in patients with biliary tract cancer. Also, we investigated how well the infiltration of immune cells can predict clinical prognosis. Because healthy patients do not have tumor, it is impossible to evaluate immune cell infiltrations into their tumor microenvironment. In addition, it is difficult to obtain biliary tract specimens from healthy non-cancer-bearing patients. 2. In the Table1, the authors listed some of the clinicopathological characteristics of the 130 patients with BTC. Please, the authors must complete the list, give some more info such as age, eventually pharmacological treatment of the patients, stage of the illness ecc. Response: We already listed age and stage in Table 1. We added the information of pharmacological treatment into Table 1. 3. In the tissue microarray construction paragraph, the authors ensured that representative tumor cell-rich areas are HE stained with a light microscope and were sent to create TMA blocks. The authors must include some HE images of the tumor sections in their results. So the readers can appreciate the percentage of TILs on HE stained samples. Response: We added the images of HE staining as Supplementary Figure 1. 4. Is well known that immunohistochemical markers used to identify M1 and M2 TAMs are the keystones of TAM evaluation. In this manuscript the authors study the tumor-associated macrophages (TAMs) in BTC. It should be interesting if the authors extent their experiments (immunohistochemistry or PCR or blots) in other markers that characterized the TAMs, such as, CD11c, CD86, iNOS, pSTAT1 (M1 markers), CD163, CD204, CD206 (M2 markers) or at least some of them. Response: I totally agree with your point. Performing double staining using various cell surface markers will allow us to understand immune cell infiltration in TME in more detail. However, our future goal is to examine how to apply this result to clinical practice, and we would like to replace it with a simple method. We evaluated CD3 positive cells as a pan-T cell marker. We found that CD3 is useful to understand the infiltration of immune cells in TME, although the accuracy decreased compared to each immune cell evaluation. In the future, we will investigate whether predicting prognosis is possible by evaluating immune cell infiltration with H E staining. We added the paragraph in page 31-32, line 305-325. Also, we added Supplementary Figure 4 and Supplementary Table 2. Point by point response to Reviewer 2 Comments We are grateful for your comments which have helped us greatly improve our manuscript. As indicated in the responses that follow, we have taken your comments into account in our manuscript. 1. However, the authors only look at high and low levels of infiltrating immune cells, control/normal tissue analysis is needed. Response: In this current study, we evaluated the infiltration of immune cells into the tumor microenvironment in patients with biliary tract cancer. Also, we investigated how well the infiltration of immune cells can predict clinical prognosis. Because healthy patients do not have tumor, it is impossible to evaluate immune cell infiltrations into their tumor microenvironment. In addition, it is difficult to obtain biliary tract specimens from healthy non-cancer-bearing patients. 2. You may need to assess the co-staining of CD4+ and Foxp3 staining, since there may be overlap, to conclusively determine specific cell type, as well as CD3+ with CD8+/CD4+ cells (instead of them each individually).　 Response: I totally agree with your point. Performing double staining using various cell surface markers will allow us to understand immune cell infiltration in TME in more detail. However, our future goal is to examine how to apply this result to clinical practice, and we would like to replace it with a simple method. We evaluated CD3 positive cells as a pan-T cell marker. We found that CD3 is useful to understand the infiltration of immune cells in TME, although the accuracy decreased compared to each immune cell evaluation. In the future, we will investigate whether predicting prognosis is possible by evaluating immune cell infiltration with H E staining. We added the paragraph in page 31-32, line 305-325. Also, we added Supplementary Figure 4 and Supplementary Table 2. 3. Images in Figure 2 are hard to visualize. It may be necessary to use Immunofluorescence to visualize co-staining. Response: We added arrows to the stained immune cells in Figure 1. Also, we changed the contrast and magnification to improve the clarity of visualization. 4. In the discussion, the authors state that role of CD8+ T cells as a cytotoxic role. This is not necessarily true. There are numerous subsets of CD8+ T cell and the authors did not access these. Response: We added the description of CD8 subsets in page 28, lines 264-267. Attachments Attachment Submitted filename: Response to Reviews.docx https://doi.org/10.1371/journal.pone.0280348.r002
27 Dec 2022 Decision Letter - Gianfranco D. Alpini, Editor Tumor-Infiltrating Lymphocytes and Macrophages as a Significant Prognostic Factor in Biliary Tract Cancer PONE-D-22-22499R1 Dear Dr. Kenjiro Kimura, 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, Gianfranco D. Alpini Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0280348.r003
Formally Accepted
4 Jan 2023 Acceptance Letter - Gianfranco D. Alpini, Editor PONE-D-22-22499R1 Tumor-Infiltrating Lymphocytes and Macrophages as a Significant Prognostic Factor in Biliary Tract Cancer Dear Dr. Kimura: 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. Gianfranco D. Alpini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0280348.r004

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