An approach for developing a blood-based screening panel for lung cancer based on clonal hematopoietic mutations

Ramu Anandakrishnan; Ryan Shahidi; Andrew Dai; Veneeth Antony; Ian J. Zyvoloski

doi:10.1371/journal.pone.0307232

Peer Review History

Original SubmissionMay 1, 2024
28 May 2024 Decision Letter - Francesco Bertolini, Editor PONE-D-24-17330Blood-based screening panel for lung cancer based on clonal hematopoietic mutationsPLOS ONE Dear Dr. Anandakrishnan, 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. In particular, I agree with Reviewer #2: "... Authors must ... be consistent with the results by using a title that clearly indicates that a method is presented in this manuscript and not an already defined panel."Abstract, introduction and discussion sections must be clear about the limitations of the proposed approach. Please submit your revised manuscript by Jul 12 2024 11:59PM. 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Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse." 3. Thank you for stating the following financial disclosure: "This work was funded by the VCOM 2023 REAP grant #1038624(RA)." Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. [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: No ******** 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: Interestingly, the authors show how mutations called on peripheral blood can raise a concrete signal useful to distinguish cancer vs healthy patients. The test devised by the authors could indeed complement ctDNA or other diagnostic tests. I wonder whether the presence of cancer in a patient could generate a peripheral blood mixture composed by a different percentage of cell types. Such different cells could harbour different mutations (or mutation rate) due to clonal hematopoiesis. Major points: 1) It would be useful to encapsulate the test in a software application (with suitable instructions) and provide the possibility to download/run such applications on new datasets. 2) It would be interesting to know if you still get a signal completely separating the studies used for training vs tests (i.e. removing the 25% test set). Reviewer #2: The authors want to determine whether clonal expansion of hematopoietic mutations in tumor-infiltrating immune cells in blood samples can predict lung cancer onset. Using sequencing data from publicly available lung cancer samples, the authors identify a set of 98 potentially pathogenic clonal hematopoietic mutations in tumor-infiltrating immune cells. They then use these mutations as predictors to develop a logistic regression machine learning model. Finally, the authors test the results on a separate sequencing dataset of >500 lung cancer samples and >500 noncancer samples from 18 different cohorts. The title suggests that the authors provide a panel of genes that can be used to predict the occurrence of lung cancer. The last sentence of the introduction (line 75) also suggests that a machine learning model is developed in this paper to predict the probability of cancer based on the detection of these mutations. However, the results obtained are in contrast in that the authors write that the model developed here may not represent an optimal lung cancer screening panel (line 340) and in that they describe the work as a starting point that needs further development (line 355). Authors must then be consistent with the results by using a title that clearly indicates that a method is presented in this manuscript and not an already defined panel. Consistency must be checked carefully throughout the manuscript. At the methodological level, it is not convincing that different alignment and variant calling approaches are used depending on the dataset used. This approach is risky as it could introduce batch effects that may not be corrected by the methods used by the authors. The authors should align reads and perform variant calling with one standardized approach for all datasets and re-run their method. In addition, the authors make a number of functional assumptions (line 356) about the identified mutations (Fig. 4) but they do not validate or show data to support the assumptions made. Additionally on line 349: 48/98 cannot be considered as “most,” since it is less than half. ****** 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.0307232.r001
Revision 1
11 Jun 2024 Author Response Overall, we concur with reviewers' comments and suggestions. Point-by-point response to reviewer comments Reviewer 1 comment: “The test devised by the authors could indeed complement ctDNA or other diagnostic tests. I wonder whether the presence of cancer in a patient could generate a peripheral blood mixture composed by a different percentage of cell types. Such different cells could harbour different mutations (or mutation rate) due to clonal hematopoiesis.” Response: This is an interesting question. Cancer is known to alter the peripheral blood immune cell composition, by triggering the expansion of specific immune cell subtypes. These immune cell subtypes may harbor different mutations, which would then be more abundant in the altered peripheral blood due to clonal hematopoiesis (CH). The CH mutations identified by our algorithm may indeed represent the result of CH expansion of specific mutations in specific immune cell subtypes, rather than the general presence of these mutations in hematopoietic stem and progenitor cells. Further investigation will be required to clarify the relationship. We have added a Discussion of this possibility in the manuscript. Reviewer 1 comment: “1) It would be useful to encapsulate the test in a software application (with suitable instructions) and provide the possibility to download/run such applications on new datasets.” Response: We have packaged the code for predicting the probability of cancer from an input VCF files, with instructions for execution. It is available for download from: https://sourceforge.net/projects/lung-cancer-screening-panel/ Reviewer 1 comment: “2) It would be interesting to know if you still get a signal completely separating the studies used for training vs tests (i.e. removing the 25% test set).” Response: The accuracy for the 8 independent test cohorts (89.88%) was comparable to the accuracy for the 10 cohorts where 25% of the data was set aside for testing (91.42%). These results are included in the Test Results subsection of the Results section. Reviewer 2 comment: “The title suggests that the authors provide a panel of genes that can be used to predict the occurrence of lung cancer. The last sentence of the introduction (line 75) also suggests that a machine learning model is developed in this paper to predict the probability of cancer based on the detection of these mutations. However, the results obtained are in contrast in that the authors write that the model developed here may not represent an optimal lung cancer screening panel (line 340) and in that they describe the work as a starting point that needs further development (line 355). Authors must then be consistent with the results by using a title that clearly indicates that a method is presented in this manuscript and not an already defined panel. Consistency must be checked carefully throughout the manuscript.” Response: We have updated the title to state that what we present here is “An approach for developing a blood-based screening panel for lung cancer based on clonal hematopoietic mutations”. We have also updated the manuscript throughout to indicate that we present an approach that needs to be further validated and optimized. Reviewer 2 comment: “At the methodological level, it is not convincing that different alignment and variant calling approaches are used depending on the dataset used. This approach is risky as it could introduce batch effects that may not be corrected by the methods used by the authors. The authors should align reads and perform variant calling with one standardized approach for all datasets and re-run their method.” Response: For the GDC datasets we used already aligned read files, whereas for the SRA read files we performed the alignment. However, we used the same alignment protocol for SRA dataset as was used for aligning the GDC datasets. Specifically, we used the Burroghs-Wheeler Aligner (BWA) with the GRCh38 reference genome for performing the alignment of SRA datasets, as was used to align the GDC datasets. The same variant calling protocol was then used for all aligned read files (Mutect2). In summary, the same approaches were used for sequence alignment and variant calling for all datasets. We have expanded the Methods section and updated other sections, to clarify. Reviewer 2 comment: “In addition, the authors make a number of functional assumptions (line 356) about the identified mutations (Fig. 4) but they do not validate or show data to support the assumptions made.” Response: On line 356 we stated that “The mutations selected as predictors for the LR model were based on their potentially pathogenic role.” The selection criteria are detailed in the Results section under the subsection “Potentially pathogenic CH mutations in TII cells”. The values for the selection criteria for each mutation are listed in Supplementary Table S2. The key word here is “potentially”. Although the selection criteria used suggest the possibility that these mutations are pathogenic, we do not claim that that is necessarily so. We only suggest that they warrant further investigation. We have expanded the text around line 356 to clarify. Reviewer 2 comment: “Additionally on line 349: 48/98 cannot be considered as “most,” since it is less than half.” Response: Most replaced with Many. Thank you. Attachments Attachment Submitted filename: Response to Reviewers plosone 20240528.pdf https://doi.org/10.1371/journal.pone.0307232.r002
2 Jul 2024 Decision Letter - Francesco Bertolini, Editor An approach for developing a blood-based screening panel for lung cancer based on clonal hematopoietic mutations PONE-D-24-17330R1 Dear Dr. Anandakrishnan, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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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: Yes 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: All my previous comments have been addressed. The authors responded to my comments and made necessary changes to the title and manuscript. ****** 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: Andrea Franceschini Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0307232.r003
Formally Accepted
8 Jul 2024 Acceptance Letter - Francesco Bertolini, Editor PONE-D-24-17330R1 PLOS ONE Dear Dr. Anandakrishnan, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Francesco Bertolini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307232.r004

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