A new clinical prognostic nomogram for liver cancer based on immune score

Qinyan Shen; Guinv Hu; JinZhong Wu; Liting Lv

doi:10.1371/journal.pone.0236622

Peer Review History

Original SubmissionMarch 4, 2020
23 Apr 2020 Decision Letter - Sai-Ching Jim Yeung, Editor PONE-D-20-05913 A new clinical prognostic nomogram for liver cancer based on immune score PLOS ONE Dear Mr.Shen Shen, 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. We would appreciate receiving your revised manuscript by Jun 06 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Sai-Ching Jim Yeung, MD, PhD Academic Editor PLOS ONE 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 2. Please provide the accession number of URL link to the specific TCGA dataset used in your study. Additional Editor Comments (if provided): The nomogram should be applicable to the entire range of possible score. The maximum of total points is 215, not 200. The font size of the labeling on Figure 3 and Figure 6 should be larger [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: No Reviewer #2: No ****** 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: This is a study to develop a clinical nomogram using immune score to predict prognosis based on 346 patient sample from TCGA. My following review is with a particular emphasis on the statistical methods and analyses. 1. The proportional hazard assumption for multivariate Cox regression is questionable with crossing survival curves. 2. A comparison to other clinical nomograms without using immune score for liver cancer is needed. 3. It could be risky to apply the proposed nomogram based on the limited sample size in this study without external validation. Reviewer #2: Overview: Incidence rates of HCC are growing worldwide with very poor survival. There are few available therapies for HCC, so clearly it is important to develop new ones. QinYan Shen and colleagues have conducted a study that aims to develop a novel prognostic immune nomogram for patients with HCC. They used a published immune score based on the gene expression of stromal and immune cells in tumor samples. They have made the interesting prognostic classifiers (three nomograms) for predict HCC patient survival. They only analyzed the TCGA dataset. Although the prognostic value of immune and/or stromal scores of liver cancer has not been sufficiently investigated, however, the authors did not well describe the methodology, significance and impact of the bioinformatics tool in this HCC study. Limitations of the study include the lack of large validation sample sets to confirm the results obtained from TCGA, and need for more detailed analysis of some specific immune and stromal cell genes, which they pointed out in this study. Specific comments: 1. Many statements throughout the manuscript lack appropriate references. In introduction section, authors should update most recent HCC clinical and basic research literatures. These should be added where appropriate. The ESTIMATE—a key method they used in this study to evaluate HCC samples but less described in details. Overall, the result section is also too simple and does not focus on what is the significance of the data on HCC study. 2. TCGA also provide normal liver samples either adjacent liver tissues to liver tumor in same patient or nonmalignant liver tissues they collected. It is important to compare the immunoscore not only in HCC but also in nonmalignant liver tissues that may reflect whole liver tumor microenvironment and immunity in liver. 3. It is important to provide and describe what the novelty of the present study is, not only somewhat incremental given that this immunoscore has been shown in other many cancer types. 4. What are the specific immune or stromal makers or immune genes in this HCC data set, such as inflammation cytokines, TGF-beta genes, and exclusive/exhaustive T cell markers? They only show a general correlation between the Score and patient survival in HCC samples. It would strengthen to show some specific immune marker correlations. These data will provide important information for clinical use. 5. Figure legend is too simple. The patients’ information they analyzed and the statistics should be included in. In the Fig.3, what is the detail method they used to get this p value 0.025? is it among 3 groups? Or only low group vs. high/medium groups? It looks like no significant between high and medium? ****** 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0236622.r001
Revision 1
16 May 2020 Author Response Dear Prof. Sai-Ching Jim Yeung Thanks a lot for having reviewed our manuscript. Now we have revised the manuscript according to the academic editor and reviewers’ comments. Most of the revisions are in the manuscript. Response to the academic editor Question1: 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 Answer1: We ensure that our manuscript meets PLOS ONE's style requirements. Question2: Please provide the accession number of URL link to the specific TCGA dataset used in your study. Answer2: We provide the accession number of URL link to the specific TCGA dataset used in our study. Question3: The nomogram should be applicable to the entire range of possible score. The maximum of total points is 215, not 200. The font size of the labeling on Figure 3 and Figure 6 should be larger Answer3: We have made changes according to the above requirements of the editor. Response to Reviewer #1: Question 1: The proportional hazard assumption for multivariate Cox regression is questionable with crossing survival curves. Answer1: Compared with the low immune score group, the p value of survival curve of medium or high immune scores group was less than 0.05, the difference was statistically significant. The cross of survival curve is mainly between the medium and high immune scores group, but there is no statistical significance between the medium and high immune scores groups. Kaplan–Meier curves depicting that in comparison with patients with low immune scores, those with medium and high immune scores had significantly improved survival time, so we think that the proportional hazard assumption for multivariate Cox regression is reliable. Question 2: A comparison to other clinical nomograms without using immune score for liver cancer is needed. Answer2: In clinic, we usually use tumor size, stage, lymph node metastasis to evaluate the prognosis of patients. Because of the lack of data about lymph node metastasis in TCGA data in this study, we did not build a clinical prediction model of tumor size, stage and lymph node metastasis for comparison. Our study aims to reveal the value of immune score in clinical prediction model and improve the basis for immunotherapy of liver cancer. Question 3: It could be risky to apply the proposed nomogram based on the limited sample size in this study without external validation. Answer3: Due to the small number of liver cancer samples in TCGA database, all data samples were in the experimental group and the validation group was not split. This is not only the deficiency of this study, but also the direction of the later study. Later, we plan to build a liver cancer data bank for further verification. Response to Reviewer #2: Question 1: Many statements throughout the manuscript lack appropriate references. In introduction section, authors should update most recent HCC clinical and basic research literatures. These should be added where appropriate. The ESTIMATE—a key method they used in this study to evaluate HCC samples but less described in details. Overall, the result section is also too simple and does not focus on what is the significance of the data on HCC study. Answer1: In introduction section, we add some clinical immune related studies of liver cancer and elaborate the ESTIMATE. In the results section, by analyzing the expression of different genes in the groups with high and low immune scores, we can screen out the target genes that may guide the clinical prognosis of liver cancer to further enrich our results. Question 2: TCGA also provide normal liver samples either adjacent liver tissues to liver tumor in same patient or nonmalignant liver tissues they collected. It is important to compare the immunoscore not only in HCC but also in nonmalignant liver tissues that may reflect whole liver tumor microenvironment and immunity in liver. Answer2: As the reviewer said, if there is an immune score of adjacent liver tissues to liver tumor in same patient, it will better reflect the whole tumor microenvironment. Although TCGA database has case data of normal liver tissue, there is no case data of adjacent liver tissues to liver tumor in same patient, and TCGA only has corresponding clinical data of cancer cases; secondly, The ESTIMATE database only has corresponding immune score of cancer patients, so the current data can only reveal immune score of microenvironment of liver cancer. Question 3: It is important to provide and describe what the novelty of the present study is, not only somewhat incremental given that this immunoscore has been shown in other many cancer types. Answer3: We further analyzed and screened the immune genes related to the prognosis of liver cancer, and provided theoretical basis for the research of liver cancer clinical targeted drugs. Question4: What are the specific immune or stromal makers or immune genes in this HCC data set, such as inflammation cytokines, TGF-beta genes, and exclusive/exhaustive T cell markers? They only show a general correlation between the Score and patient survival in HCC samples. It would strengthen to show some specific immune marker correlations. These data will provide important information for clinical use. Answer4: Through the analysis of differential gene expression in the high and low immune score groups, we screened out 10 target genes, and further analyzed that a and B are related to the prognosis of liver cancer, so as to provide basis for the research and development of clinical targeted drugs for liver cancer. Question5: Figure legend is too simple. The patients’ information they analyzed and the statistics should be included in. In the Fig.3, what is the detail method they used to get this p value 0.025? is it among 3 groups? Or only low group vs. high/medium groups? It looks like no significant between high and medium? Answer5: Figure 3 has been modified. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0236622.r002
12 Jun 2020 Decision Letter - Sai-Ching Jim Yeung, Editor PONE-D-20-05913R1 A new clinical prognostic nomogram for liver cancer based on immune score PLOS ONE Dear Dr. Shen, 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 Jul 27 2020 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Sai-Ching Jim Yeung, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (if provided): The responses to the Reviewers' comments were inadequate. Response to Reviewer#1 Question 1: Reviewer#1 is concerned about the proportional hazard assumption for the Cox regression model. Since Reviewer#1 raised the concern based on the appearance of the Kaplan-Meier curves, the authors should run diagnostic tests for Cox regression model: e.g., •Schoenfeld residuals to check the proportional hazards assumption •Martingale residual to assess nonlinearity. Response to Reviewer#1 Question 2: The inability to build a clinical prediction model for comparison should be discussed as a limitation of the study in the Discussion section. Response to Reviewer#1 Question 3: Again, this limitation of small number liver cancer samples is another limitation of the study that should be mentioned in the Discussion section. Response to Reviewer#2 Question 2: Although the ESTIMATE database only has the corresponding immune scores of the cancer samples, the scores for normal tissue samples can be generated. The TCGA gene expression data for the normal liver samples can be downloaded. The ESTIMATE R packages can be downloaded at https://bioinformatics.mdanderson.org/public-software/estimate/. The ESTIMATE scores can be generated for the normal liver samples and compared. [Note: HTML markup is below. Please do not edit.] 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 #2: All comments have been addressed Reviewer #3: (No Response) ******** 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 #2: Yes Reviewer #3: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: No ****** 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 #2: Yes Reviewer #3: 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 #2: Yes Reviewer #3: 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 #2: (No Response) Reviewer #3: I am sorry but I don't think the authors have addressed the methodological concerns provided by the previous reviewers. ****** 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 #2: Yes: Jian Chen Reviewer #3: Yes: Cielito Reyes-Gibby [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.0236622.r003
Revision 2
18 Jun 2020 Author Response Response to Reviewer #1: Answer1: Response to Reviewer#1 Question 1: Reviewer#1 is concerned about the proportional hazard assumption for the Cox regression model. Since Reviewer#1 raised the concern based on the appearance of the Kaplan-Meier curves, the authors should run diagnostic tests for Cox regression model: e.g., •Schoenfeld residuals to check the proportional hazards assumption •Martingale residual to assess nonlinearity. Question 1: Thanks to the reviewer's reminder, the reviewer's intention was not fully understood at the beginning, which resulted in the failure to answer the reviewer's questions well. Now, according to the reviewer's suggestion, we use R language to test the proportional hazard assumption for the Cox regression model. The results show that for age, stage, tumor grade and immune score, P＞0.05.(Figure in Response to Reviewers.docx) Question 2: Response to Reviewer#1 Question 2: The inability to build a clinical prediction model for comparison should be discussed as a limitation of the study in the Discussion section. Answer2: We put this part of the deficiency in the discussion part of the article. Question 3: Response to Reviewer#1 Question 3: Again, this limitation of small number liver cancer samples is another limitation of the study that should be mentioned in the Discussion section. Answer3: In the discussion part of the article, we will add some explanation on this point. Response to Reviewer #2: Question 1: Response to Reviewer#2 Question 2: Although the ESTIMATE database only has the corresponding immune scores of the cancer samples, the scores for normal tissue samples can be generated. The TCGA gene expression data for the normal liver samples can be downloaded. The ESTIMATE R packages can be downloaded at https://bioinformatics.mdanderson.org/public-software/estimate/. The ESTIMATE scores can be generated for the normal liver samples and compared. Answer1: The tumor microenvironment can be better reflected by the analysis of the immune score of the adjacent tissues of liver cancer. As the reviewer said, the immune score of the normal liver tissue sample data in TCGA can be obtained through the R package, but this part of the normal tissue sample is not the adjacent contrast tissue of the liver cancer tissue. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0236622.r004
13 Jul 2020 Decision Letter - Sai-Ching Jim Yeung, Editor A new clinical prognostic nomogram for liver cancer based on immune score PONE-D-20-05913R2 Dear Dr. Shen, 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, Sai-Ching Jim Yeung, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): The reviewers' comments have been addressed. Reviewers' comments: https://doi.org/10.1371/journal.pone.0236622.r005
Formally Accepted
17 Jul 2020 Acceptance Letter - Sai-Ching Jim Yeung, Editor PONE-D-20-05913R2 A new clinical prognostic nomogram for liver cancer based on immune score Dear Dr. Shen: 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. Sai-Ching Jim Yeung Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0236622.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .