Clinical and molecular characteristics associated with high PD-L1 expression in EGFR-mutated lung adenocarcinoma

Jeremy Slomka; Hugo Berthou; Audrey Mansuet-Lupo; Hélène Blons; Elizabeth Fabre; Ivan Lerner; Bastien Rance; Marco Alifano; Jeanne Chapron; Gary Birsen; Laure Gibault; Jennifer Arrondeau; Karen Leroy; Marie Wislez

doi:10.1371/journal.pone.0307161

Peer Review History

Original SubmissionJanuary 21, 2024
3 Apr 2024 Decision Letter - Fumihiro Yamaguchi, Editor PONE-D-24-01682Clinical and Molecular Characteristics Associated with High PD-L1 expression in EGFR-Mutated Lung AdenocarcinomaPLOS ONE Dear Dr. Wislez, 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 reviewers have recommended publication, but also suggest significant revisions to your manuscript. Therefore, I invite you to respond to the reviewers' comments and revise your manuscript. Please submit your revised manuscript by May 18 2024 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. 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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. In the online submission form, you indicated that All the data from this study have been imported into a REDCAP database, which cannot be shared publicly as it is protected by our institution. However, at your request, this data can be extracted and sent to you. All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either a. In a public repository, b. Within the manuscript itself, or c. Uploaded as supplementary information. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Additional Editor Comments: This is a well-designed study, but it needs significant revision. [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 Reviewer #3: Yes Reviewer #4: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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: Fascinating article. Would really appreciate a breakdown as to which TKIs patients were treated with though. I'm not comfortable supporting some of the conclusions (ex: rates of CNS relapse, OS) without knowing how many patients were treated with osimertinib vs something like gefitinib which has significantly worse CNS penetration and OS. A discussion as to the biology of EGFR and the mechanism for why PD-L1 can be upregulated would be interesting and useful here (for example EGFR signaling may induce PD-L1 expression in bronchial cells). Reviewer #2: In this manuscript, the authors analyzed the prognostic significance of PD-L1 expression in a retrospective cohort of patients with metastatic EGFR-mutated NSCLC. Although the authors provided important data and suggestions for clinical practice, these data have already been published in other papers. Comments: 1) The authors show that high PD-L1 expression (≥50% TPS) is significantly associated with decreased OS and not significantly associated with shorter PFS in multivariate analysis. Please provide the information of second-line therapy such as chemotherapy, immunotherapy, or the other EGFR-TKIs in each PD-L1 < 50% and PD-L1 ≥ 50% groups. 2) The authors show a higher frequency of de novo resistance in the PD-L1 ≥ 50% group compared with PD-L1 < 50% group. Please provide the information of first-line therapy such as 1st, 2nd or 3rd generation EGFR-TKI in each PD-L1 < 50% and PD-L1 ≥ 50% groups. 3) If possible, please provide the toxicity profiles of the first-line EGFR-TKI treatment in each PD-L1 < 50% and PD-L1 ≥ 50% groups. Reviewer #3: This is a retrospective observational study that aims to explore the relationship between the level of PDL1 expression and the paterns of EGFR mutation, as well as outcomes of the treatment. Although the small sample size makes it difficult to draw the compellent conclusions, the current study had been designed strictly, conducted well, and analyzed properly. Some findings as high PDL1 expression is associated with complex EGFR mutations may give the resonable cause that PDL1 over expression leads to the poorer outcome in patients with EGFR mutation. The shortcomings of the study had been discussed deeply and adequately. The above points make this article readable and could give some enlightenment to the readers. I have just minor comments for the authors. 1. Materials and methods-Assessment of PD-L1 level and molecular characterization: “EGFR mutation and co-mutation status (specifically TP53, CTNNB1, SMAD4, or 129 PIK3CA) weas identified during routine care using next-generation sequencing.”. I don’t understand the word “WEAS”, should it be “were” ? 2. S1 table can not be displayed. Since the number of complex mutation is not big, I suggest describing them in the RESULTS part. 3. TABLE1-SMOKING STATUS: “ABSENT” means never smoke? Or quit smoking? 4. Conclusions in the tail of the text is too complex, it is better putting it at the end of DISCUSSION part. I suggest use the conclusions similar with that in the ABSTRACT. Reviewer #4: Slomka et al. investigated the association between high PD-L1 expression and overall survival (OS) in a retrospective EGFR-mutant lung adenocarcinoma cohort. They observed that high PD-L1 was associated with complex EGFR mutations, poor clinical characteristics (liver and brain metastasis, poor performance), primary resistance, and shorter OS. The manuscript is well written, and the analyses are well conducted; however, it does not add new information compared to previous reports investigating the same question. Major comments The authors need to make the novelty of their work apparent. Various previous works have investigated the association between PD-L1 expression and survival in EGFR-mutant NSCLC and, in general, demonstrated that PD-L1 is associated with worse PFS or OS (Tang et al. Oncotarget. 2025; Yang et al. Eur J cancer. 2020; Peng et al. World J Surgery Oncol. 2021; Shiozawa et al. Anticancer Res. 2022; Ji et al. Cancer Bill Therapy. 2016; Bai et al. Cancer Biol Med. 2018; Liu et al. Lung Cancer. 2021) or primary resistance (Lan et al. Medicine (Baltimore). 2021). The authors state they used Fisher's exact test to test the association between PD-L1 expression and other categorical variables; nevertheless, they show various p-values in Table 1 when there should be only one p-value for each variable. How do they explain that? Besides, some variables have too many categories. I recommend collapsing some of them to reduce dimensionality. There are some hints that the worse prognosis associated with high PD-L1 expression can reflect its association with tumors that are genetically more complex, probably due to smoking exposure. It would be interesting to test if there is an association (Wilcoxon's test) or correlation (Spearman's correlation test) between smoking load (pack-years) and PD-L1 expression and type of EGFR mutation. Besides, the authors included smoking in univariate and multivariate only as a categorical variable. I recommend testing its impact on OS and PFS as a continuous variable (pack-years). The impact of PD-L1 on OS might reflect different treatment patterns. The authors did not describe the treatment given to the patients (1st Gen TKI and 2nd Gen TKI x 3rd Gen TKI x QT) and its association with OS. Were the tumor samples submitted to comprehensive genomic analysis? If that is the case, it would be interesting to describe the frequency of co-mutations, investigate their impact and of TMB on OS, and their association with the EGFR mutation type and PD-L1 expression. Minor comments The authors declare they used different antibody clones to test PD-L1 expression, but they do not show what clones were employed or how many samples were tested with each one. ****** 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: Yes: Whitney Elizabeth Lewis Reviewer #2: No Reviewer #3: No Reviewer #4: 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. 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Revision 1
16 May 2024 Author Response Dear Dr Yamagushi, Thank you for considering our manuscript titled "Clinical and Molecular Features Associated with High PD-L1 Expression in EGFR-Mutated Metastatic Lung Adenocarcinoma" (PONE-D-24-01682). We have diligently revised the text and tables in response to the reviewers' comments (detailed responses provided below). We believe these revisions have significantly enhanced the quality of our manuscript. We have ensured that our manuscript and figure files comply with PLOS ONE's style guidelines. Additionally, we have updated the ORCID iD of the corresponding author and included anonymized study data as a supplementary file, aligning with the journal's request to facilitate access for fellow researchers. Gary Birsen had participated to this study, as was mentioned in the author contribution section, and was unfortunately removed from the list of co-authors in the first submission. The list of authors has been corrected in the revised version. We are confident that this revised manuscript meets the standards for publication in PLOS ONE. Thank you for considering our work. Yours sincerely, Professor Marie Wislez, on behalf the coauthors Corresponding author: Professor Marie Wislez Pneumology, Cochin Hospital Paris, Université Paris Cité, Faculty of Medicine, Paris, France; telephone: 33 158413091 e-mail: marie.wislez@aphp.fr Subject: Reponse to Reviewers - "Clinical and molecular features associated with high PD-L1 expression in EGFR-mutated metastatic lung adenocarcinoma" Reviewer #1: Fascinating article. Would really appreciate a breakdown as to which TKIs patients were treated with though. I'm not comfortable supporting some of the conclusions (ex: rates of CNS relapse, OS) without knowing how many patients were treated with osimertinib vs something like gefitinib which has significantly worse CNS penetration and OS. Thank you for your thoughtful and constructive feedback. We acknowledge the importance of detailing the treatments received in the first line of therapy to enhance the clarity and comprehension of our manuscript. As per your suggestion, we have incorporated a comprehensive breakdown of the different treatments administered to patients during the first line into Table 2 (formerly Table 1). Additionally, in the paragraph "Progression after the First Line of Treatment" (page 14, lines 270-275), we have provided a description of the various treatments utilized in the second line. Upon analysis, we did not identify significant differences in the type of first-line treatment received between the two groups. Nonetheless, we recognize the need for caution in interpreting these findings due to the limitations posed by our relatively small sample sizes. A discussion as to the biology of EGFR and the mechanism for why PD-L1 can be upregulated would be interesting and useful here (for example EGFR signaling may induce PD-L1 expression in bronchial cells). As noted by the reviewer, we discussed in the discussion section (page 16, lines 316-328) the complexity of PD-L1 regulation, which is influenced by both endogenous EGFR signaling and exogenous signals such as interferon that are associated with the activation of anti-tumor immunity. To support this point, we have incorporated a specific reference in the field (Sun C, Mezzadra R, Schumacher TN. Regulation and Function of the PD-L1 Checkpoint. Immunity. 2018 Mar 20;48(3):434-452. doi: 10.1016/j.immuni.2018.03.014. PMID: 29562194; PMCID: PMC7116507). The observed variation in PD-L1 expression among EGFR-mutated tumors may indeed be attributed to differences in anti-tumor immunity and immune cell infiltration. However, it is important to note that we were unable to assess this aspect in our retrospective study. _______________________________________________________________________________________________ Reviewer #2: In this manuscript, the authors analyzed the prognostic significance of PD-L1 expression in a retrospective cohort of patients with metastatic EGFR-mutated NSCLC. Although the authors provided important data and suggestions for clinical practice, these data have already been published in other papers. Comments: 1) The authors show that high PD-L1 expression (≥50% TPS) is significantly associated with decreased OS and not significantly associated with shorter PFS in multivariate analysis. Please provide the information of second-line therapy such as chemotherapy, immunotherapy, or the other EGFR-TKIs in each PD-L1 < 50% and PD-L1 ≥ 50% groups. Thank you for this very interesting remark; second-line treatments could be a relevant avenue to explain the association of PD-L1 with OS, and absence of difference in PFS. Therefore, we have added in the paragraph 'Progression after the First Line of Treatment' on page 14, lines 270-275 the description of the different treatments used in the second line. There was not obvious difference that could explain our results. 2) The authors show a higher frequency of de novo resistance in the PD-L1 ≥ 50% group compared with PD-L1 < 50% group. Please provide the information of first-line therapy such as 1st, 2nd or 3rd generation EGFR-TKI in each PD-L1 < 50% and PD-L1 ≥ 50% groups. The treatments administered in the first line of therapy are indeed crucial for a comprehensive understanding of our manuscript. To address this, we have incorporated a detailed breakdown of the different treatments administered to patients during the first line in Table 2 (previously Table 1). Notably, our analysis did not reveal any significant difference in the type of first-line treatment received between the two groups. However, we recognize the importance of interpreting these findings cautiously given the limitations posed by our relatively small sample sizes. 3) If possible, please provide the toxicity profiles of the first-line EGFR-TKI treatment in each PD-L1 < 50% and PD-L1 ≥ 50% groups. Regarding the toxicities of the treatments, it would have been very pertinent to describe the side effects. However, due to the retrospective nature of this study involving routine care data, it was not possible to obtain this information reliably; therefore, this data was not collected. _______________________________________________________________________________________________ Reviewer #3: This is a retrospective observational study that aims to explore the relationship between the level of PDL1 expression and the paterns of EGFR mutation, as well as outcomes of the treatment. Although the small sample size makes it difficult to draw the compellent conclusions, the current study had been designed strictly, conducted well, and analyzed properly. Some findings as high PDL1 expression is associated with complex EGFR mutations may give the resonable cause that PDL1 over expression leads to the poorer outcome in patients with EGFR mutation. The shortcomings of the study had been discussed deeply and adequately. The above points make this article readable and could give some enlightenment to the readers. I have just minor comments for the authors. 1. Materials and methods-Assessment of PD-L1 level and molecular characterization: “EGFR mutation and co-mutation status (specifically TP53, CTNNB1, SMAD4, or 129 PIK3CA) weas identified during routine care using next-generation sequencing.”. I don’t understand the word “WEAS”, should it be “were”? Thank you for these very relevant remarks. Regarding the sentence about the changes, the term "WEAS" is indeed a typographical error and should have been "were." This modification has been made on page 6 line 129. 2. S1 table can not be displayed. Since the number of complex mutation is not big, I suggest describing them in the RESULTS part. As requested by the reviewer, the complex mutations have been displayed in a specific table. Revised manuscript: Table 1, page 3, line 140. The former Table 1 becomes Table 2 and Table 2 becomes Table 3, respectively. 3. TABLE1-SMOKING STATUS: “ABSENT” means never smoke? Or quit smoking? For clarity and as suggested, we have replaced smoking status in Table 2 (former Table 1) with "never smoked" or "current/former smoker." 4. Conclusions in the tail of the text is too complex, it is better putting it at the end of DISCUSSION part. I suggest use the conclusions similar with that in the ABSTRACT. As requested by the reviewer, we have simplified the conclusions as follows: “High PD-L1 expression was associated with uncommon and complex EGFR mutations, as well as a higher frequency of de novo resistance and independently predicted poor survival. Further investigations with larger prospective cohorts are warranted to validate and expand upon our findings, as well as to unravel the complex interplay between EGFR mutations, immune signaling, and PD-L1 expression in EGFR-mutated NSCLC”. _______________________________________________________________________________________________ Reviewer #4: Slomka et al. investigated the association between high PD-L1 expression and overall survival (OS) in a retrospective EGFR-mutant lung adenocarcinoma cohort. They observed that high PD-L1 was associated with complex EGFR mutations, poor clinical characteristics (liver and brain metastasis, poor performance), primary resistance, and shorter OS. The manuscript is well written, and the analyses are well conducted; however, it does not add new information compared to previous reports investigating the same question. Major comments The authors need to make the novelty of their work apparent. Various previous works have investigated the association between PD-L1 expression and survival in EGFR-mutant NSCLC and, in general, demonstrated that PD-L1 is associated with worse PFS or OS (Tang et al. Oncotarget. 2025; Yang et al. Eur J cancer. 2020; Peng et al. World J Surgery Oncol. 2021; Shiozawa et al. Anticancer Res. 2022; Ji et al. Cancer Bill Therapy. 2016; Bai et al. Cancer Biol Med. 2018; Liu et al. Lung Cancer. 2021) or primary resistance (Lan et al. Medicine (Baltimore). 2021). Thank you for your valuable feedback. The sources you provided are indeed highly relevant to our study. We have incorporated these references into the article as references 10-15, 17 and 18, specifically on page 4, line 83 and page 15, line 308. The authors state they used Fisher's exact test to test the association between PD-L1 expression and other categorical variables; nevertheless, they show various p-values in Table 1 when there should be only one p-value for each variable. How do they explain that? Besides, some variables have too many categories. I recommend collapsing some of them to reduce dimensionality. We conducted Fisher's tests on the categorical variables, presenting a p-value for each. For the continuous variables, we included p-values from the Mann-Whitney test. To enhance clarity, we have revised the Materials and Methods section, specifically on page 7, line 149. Notably, we reorganized the categories, such as performance status below or above 2, and combined certain metastatic sites in Table 2 (formerly Table 1). Statistical tests were performed for each type of EGFR mutation, which was critical for addressing the study's key question. There are some hints that the worse prognosis associated with high PD-L1 expression can reflect its association with tumors that are genetically more complex, probably due to smoking exposure. It would be interesting to test if there is an association (Wilcoxon's test) or correlation (Spearman's correlation test) between smoking load (pack-years) and PD-L1 expression and type of EGFR mutation. This observation is indeed pertinent. We investigated the relationship between pack-years and EGFR common mutations (p=0.2) as well as EGFR complex mutations (p=0.5) among the 88 patients for whom pack-year data was available using a Wilcoxon test. Our analysis did not reveal any significant differences in these associations. With respect to PD-L1 expression, no significant difference was observed based on smoking status, and a similar trend was observed for the average number of pack-years (p=0.1). It's worth noting that these subgroups consisted of a limited number of patients, which may have contributed to the lack of observed differences. Besides, the authors included smoking in univariate and multivariate only as a categorical variable. I recommend testing its impact on OS and PFS as a continuous variable (pack-years). We encountered 25 missing data points regarding the amount of tobacco smoked by patients in the study. As suggested, we performed both univariate and multivariate analyses among the 88 patients for whom we had complete data. We observed a correlation between progression-free survival (PFS) and overall survival (OS) and the number of pack-years. These findings have been incorporated into the section titled "Univariate and Multivariate Evaluation of Survival" on page 13, lines 246-255. Additionally, the table summarizing the univariate and multivariate analyses for this group of 88 patients has been included in the supplementary data (S1 Table). The impact of PD-L1 on OS might reflect different treatment patterns. The authors did not describe the treatment given to the patients (1st Gen TKI and 2nd Gen TKI x 3rd Gen TKI x QT) and its association with OS. The treatments administered in the first line of therapy are crucial for a comprehensive understanding of the manuscript. To address this, we have included a detailed summary of the different treatments administered to patients during the first line in Table 2 (previously Table 1). Despite our analysis not revealing any significant differences in the type of first-line treatment received between the two groups, it is important to interpret these results cautiously due to the relatively small sample sizes. Were the tumor samples submitted to comprehensive genomic analysis? If that is the case, it would be interesting to describe the frequency of co-mutations, investigate their impact and of TMB on OS, and their association with the EGFR mutation type and PD-L1 expression. As described in the methods section (page 6, line 128), tumor samples were analyzed using a next-generation sequencing (NGS) panel targeting hotspot regions of 22 genes. Recurrent co-mutations identified in this analysis included mutations in TP53, CTNNB1, and SMAD4, with one case exhibiting a BRAF exon 11 co-mutation. However, it's important to note that this panel does not allow the evaluation of TMB. Minor comments The authors declare they used different antibody clones to test PD-L1 expression, but they do not show what clones were employed or how many samples were tested with each one. Regarding the antibodies used for PD-L1 evaluation, we have modified the text to make it more explicit. Specifically, the E1L3N antibody was used in 38 cases, the 22C3 antibody in 31 cases, and the QR1 antibody in 34 cases. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0307161.r002
9 Jun 2024 Decision Letter - Fumihiro Yamaguchi, Editor PONE-D-24-01682R1Clinical and Molecular Characteristics Associated with High PD-L1 expression in EGFR-Mutated Lung AdenocarcinomaPLOS ONE Dear Dr. Wislez, 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. ============================== ACADEMIC EDITOR: The reviewers have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the reviewers' comments and revise your manuscript. ============================== Please submit your revised manuscript by Jul 24 2024 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Fumihiro Yamaguchi Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: (No Response) Reviewer #3: All comments have been addressed Reviewer #4: 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 #2: (No Response) Reviewer #3: Yes Reviewer #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) Reviewer #3: Yes Reviewer #4: 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 #2: (No Response) Reviewer #3: Yes Reviewer #4: 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: (No Response) Reviewer #3: Yes Reviewer #4: 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: The authors have responded to my review comments by adding some analytical results, demonstrating that they do not affect the original conclusions of the authors. However, toxicities by EGFR-TKI according to PD-L1 expressions had not been presented in the revised manuscript because they do not have representative data. This should be noted in the discussion as a limitation. Reviewer #3: my comments had been addressed well by the authors. Reviewer #4: The points raised by all the reviewers were adequately addressed by the authors. I have no further comments. ****** 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: No Reviewer #3: Yes: Jiang Zhu Reviewer #4: 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.0307161.r003
Revision 2
17 Jun 2024 Author Response Reviewer #2: The authors have responded to my review comments by adding some analytical results, demonstrating that they do not affect the original conclusions of the authors. However, toxicities by EGFR-TKI according to PD-L1 expressions had not been presented in the revised manuscript because they do not have representative data. This should be noted in the discussion as a limitation. Thank you for your feedback. Indeed, this is a limitation that we did not address in the text. We have therefore added the following sentence: "Given the retrospective nature of this study, the analysis of the side effects of the different treatments received, according to PD-L1 expression, could not be cataloged and analyzed in this article, which represents a potential bias to consider when interpreting these results" on page 15, line 315-318. ________________________________________________________________________________ Reviewer #3: my comments had been addressed well by the authors. We thank you for your review and your various comments. ________________________________________________________________________________ Reviewer #4: The points raised by all the reviewers were adequately addressed by the authors. I have no further comments. We thank you for your review and your various comments. Attachments Attachment Submitted filename: Reponse to reviewer.docx https://doi.org/10.1371/journal.pone.0307161.r004
2 Jul 2024 Decision Letter - Fumihiro Yamaguchi, Editor Clinical and Molecular Characteristics Associated with High PD-L1 expression in EGFR-Mutated Lung Adenocarcinoma PONE-D-24-01682R2 Dear Dr. Wislez, 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. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Fumihiro Yamaguchi 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 #2: All comments have been addressed Reviewer #3: All comments have been addressed Reviewer #4: 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 #2: Yes Reviewer #3: Yes Reviewer #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: 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 #2: Yes Reviewer #3: Yes Reviewer #4: 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 Reviewer #4: 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: Thank you for revising the manuscript according to my comments. All concerns have been addressed well. Reviewer #3: (No Response) Reviewer #4: My comments were properly addressed in the previous submission. I have no addtional comments or concerns. ****** 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: Satoshi Watanabe Reviewer #3: Yes: Jiang Zhu Reviewer #4: No ******** https://doi.org/10.1371/journal.pone.0307161.r005
Formally Accepted
9 Sep 2024 Acceptance Letter - Fumihiro Yamaguchi, Editor PONE-D-24-01682R2 PLOS ONE Dear Dr. Wislez, 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. Fumihiro Yamaguchi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307161.r006

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