Integrated analysis of long non-coding RNAs and mRNA profiles reveals potential sex-dependent biomarkers of bevacizumab/erlotinib response in advanced lung cancer

Chao Tu; Yingqi Pi; Shan Xing; Yang Ling

doi:10.1371/journal.pone.0240633

Peer Review History

Original SubmissionJune 7, 2020
10 Aug 2020 Decision Letter - Rama Krishna Kancha, Editor PONE-D-20-16411 Integrated analysis of long non-coding RNAs and mRNA profiles reveals potential sex-dependent biomarkers of bevacizumab/erlotinib response in advanced lung cancer PLOS ONE Dear Dr. Tu, 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 Sep 24 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. 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Additional Editor Comments (if provided): The manuscript entitled "Integrated analysis of long non-coding RNAs and mRNA profiles reveals potential sex-dependent biomarkers of bevacizumab/erlotinib response in advanced lung cancer" by Tu et al. demonstrates sex-specific differential expression of biomarkers in relation to drug response in lung cancer. The reviewers are of the opinion that the work is of significant interest. However, the reviewers raised important questions that need to be answered to bring more clarity and highlight the importance of the study. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript sounds 1. non coding genes transcriptional control is elusive and the work provides expression differences by sex dependent markers and 2. lung cancer dependent alterations with noncoding and mRNA profiles by a clinical drug perturbation is elaborated. Therefore, the manuscript is interest to the literature and I recommend the ms. Reviewer #2: Gist/Summary: The authors come up with identification of several differentially expressed (DE) lncRNAs and mRNAs from publicly available gene expression omnibus (GEO) datasets associated with NSCLC with bevacizumab and erlotinib (BE) treatment. Then they make enrichment/pathway analyses based on gender and identify unique DEs Strengths: Novel in what they claim that the authors mention on the the first of its kind of work on BE treatment ( which invariably is not so!) Figures Weaknesses: The work is based on GEO datasets and not original Hazy texts in between Major comments: Wei et al. 2016 and Saito eta l. 2019, 2020 indeed mention this and identify DE genes ( if NOT lncRNAs) in BE treatment. The authors must crosscheck their works and cite them. Lines 95-99: The authors must glorify lncRNAs and on why they could be used as biomarkers Some of the texts go hand-i-hand with clinicaltrials.gov. The authors must check it Lines 130 in Methods must be renamed. The methodology is out of vigour. As the work is public interpretation of GEO., I wonder why the authors mention about the ethics statement etc., which gives a falsehood to the story. There must be a methodological flowchart Have the authors carried out the bidirectional best blast hits whence inferring lncRNAs from BLAST? The authors must mention whether or not the exon array enrichment they infer uniquely mapped to genes and the lncRNAs were indeed inferred from those genes. Line 209 on interactions. The combined score of 0.4 doesn't make sense The NONHSAT095695.2 appears to be largely expressed in liver/adrenal glands NOT lungs. How do the authors justify this? The whole purpose of making a story for DEs for male and females is not warranted and justified in haste. For example, why males have SLE related DEs is not discussed. The same applies to the coalesce of pathways between males and females. The multivenn diagram with union of intersection of venns showing 1 and 2 respectively for DE lncRNAs and De mRNAs were not discussed in great detail. For example, where are they localized to and their expression patterns The conclusions and discussions must herald have point son what if multi-omic studies are integrated to it or say, RNA-Seq expression data is used instead of exon array Minor comments The abstract must have an expansion of EGFR Lines 385 and line 296: Please rewrite or use comma All figures must be high resolution ****** 6. 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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.0240633.r001
Revision 1
21 Sep 2020 Author Response Dear Editor, We have now revised our manuscript PONE-D-20-16411 taking into account the useful comments of the academic editor and the two reviewers. We thank you and the reviewers for your careful reading and the suggestions to improve our initial manuscript. We took some time to respond to each point raised by the academic editor and reviewers, the detailed responses to the reviewers’ comments are explained below. And we also uploaded our figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool (https://pacev2.apexcovantage.com/) to ensure that all of our figures except Supporting Information files meet PLOS requirements. And we checked all themanuscript to make sure that it meets PLOS ONE’s styl requirements, including those for file naming according to the PLOS ONE style templates. We are confident that these additional modifications strenthened and ameliorated this manuscript. In addition, the author information was modified according to the contribution of all the authors after the revision. We hope that the reviewers and yourself will find our manuscript suitable for publication in PLOS ONE. Best regards, Chao TU The detailed responses to the reviewers’ comments are explained below: Wei et al. 2016 and Saito et al. 2019, 2020 indeed mention this and identify DE genes ( if NOT lncRNAs) in BE treatment. The authors must crosscheck their works and cite them. We thank the reviewer for providing the references regarding BE treatment. We carefully searched these references, and found that Wei et al. 2018 (PMID:30032815) and Saito et al.2019 (PMID:30975627) did mention about the treatment of erlotinib plus bevacizumab(BE) in non-small cell lung cancer (NSCLC). Both papers investigated the efficacy and safety of erlotinib plus bevacizumab treatment in patients with non-small-cell lung cancer. Instead, the reference of Saito et al.2020 studied the effectiveness and safety of nivolumab treatment, not BE treatment, in NSCLC patients in real-world setting in Japan. However, none of these papers did the study on the profiling of differentially expressed genes or non-coding RNAs after BE treatment in NSCLC patients. Nevertheless, we discussed these papers in the manuscript and cited them as references according to the reviewer’s suggestion. Lines 95-99: The authors must glorify lncRNAs and on why they could be used as biomarkers. We agree with the reviewer that we should introduce more about lncRNAs and why they could be used as biomarkers. LncRNA is a class of non-coding RNAs of more than 200 nucleotides with biological functions. They are widely involved in biological development and pathological process through chromatin remodelling, cis-regulation at enhancers and post transcriptional regulation. LncRNAs have been found to be specifically expressed in the oncogenesis and metastasis of many tumor tissues (June 2014, Nature Genetics, kiyer). Therefore, these lnRNAs are also considered to be associated with carcinogenesis or tumor inhibition, and are potential biomarkers that can predict the tissue carcinogenesis. And we added the above introduction concerning why lncRNAs could be used as biomarkers in the manuscript. Some of the texts go hand-i-hand with clinicaltrials.gov. The authors must check it. We are sorry that we are not so sure about what the reviewer mentions in this point. We have written the text independent of clinicaltrials.gov. And we did the duplicate checking for our manuscript, and no similar content to clinicaltrials.gov was found. Lines 130 in Methods must be renamed. The methodology is out of vigour. As the work is public interpretation of GEO., I wonder why the authors mention about the ethics statement etc., which gives a falsehood to the story. We are sorry for the redundancy of Line 130 in Methods, and it has been renamed to “Data sets”. We agree with the reviewer that it’s not necessary to mention about the ethics statement in Methods as our work is based on the public GEO dataset, therefore, we removed those concerning ethics statement and written informed consent in this part (Please refer to the end of “Data sets” in Materials and Methods). There must be a methodological flowchart We have added the flowchart in “Supplementary Figure 1.tif” for the sample selection and data analysis used in the manuscript. Have the authors carried out the bidirectional best blast hits whence inferring lncRNAs from BLAST? Yes, we have carried out blast with the bidirectional best hit information method, which is more precise and possesses higher matching degree compared with the single-directional best hit information method. The authors must mention whether or not the exon array enrichment they infer uniquely mapped to genes and the lncRNAs were indeed inferred from those genes. We have removed those probes that were not uniquely mapped to genes, therefore, the exon array enrichment we used was uniquely mapped to genes and the lncRNAs were indeed inferred from those genes. We have added this information in Materials and Methods (Please refer to the part of “mRNA and lncRNA profiling” in Materials and Methods). Line 209 on interactions. The combined score of 0.4 doesn't make sense We thank the reviewer for this comment. However, we are not so sure about what the reviewer means here. When performing protein-protein interaction (PPI) network analysis, we used the interaction score of 0.4 according to the recommendation of the official developping team for STRING database in this paper from Nucleic Acids Research, 2017: The STRING database in 2017: quality-controlled protein–protein association networks, made broadly accessible. And this confidence cutoff of 0.4 is used widely by many other groups, below are some example references that used the interaction score of 0.4 when PPI network: ①Deciphering miRNA transcription factor feed-forward loops to identify drug repurposing candidates for cystic fibrosis. Genome Medicine 2014, 6:94 ②Identifification of key pathways and genes in colorectal cancer using bioinformatics analysis. Med Oncol (2016) 33:111. DOI 10.1007/s12032-016-0829-6 ③Screening and identification of key biomarkers in hepatocellular carcinoma: Evidence from bioinformatic analysis. The NONHSAT095695.2 appears to be largely expressed in liver/adrenal glands NOT lungs. How do the authors justify this? We thank the reviewer for this comment. In our present study, we only detected the differential expression of NONHSAT095695.2 through bioinformatic analysis by gene annotation, this needs to be validated by experiments. In our future work, we will detect the expression level of NONHSAT095695.2 in lungs of both male and female patients before and after BE treatment by RT-QPCR. The whole purpose of making a story for DEs for male and females is not warranted and justified in haste. For example, why males have SLE related DEs is not discussed. The same applies to the coalesce of pathways between males and females. We thank the reviewer for this point. Gender differences play an important role in the drug response in cancer, especially in lung cancer, for example, in the following papers they all focus their work on sex-dependent drug response in lung cancer: ①Conforti F, Pala L, Bagnardi V, et al. Sex-Based Heterogeneity in Response to Lung Cancer Immunotherapy: A Systematic Review and Meta-Analysis. J Natl Cancer Inst. 2019;111(8):772-781. doi:10.1093/jnci/djz094. ②Wang S, Zhang J, He Z, Wu K, Liu XS. The predictive power of tumor mutational burden in lung cancer immunotherapy response is influenced by patients' sex. Int J Cancer. 2019;145(10):2840-2849. doi:10.1002/ijc.32327. ③Tsiouda T, Sardeli C, Porpodis K, et al. Sex Differences and Adverse Effects between Chemotherapy and Immunotherapy for Non-Small Cell Lung Cancer. J Cancer. 2020;11(11):3407-3415. Published 2020 Mar 5. doi:10.7150/jca.40196. We added the discussion about the coalesce of pathways between males and females in the manuscript. However, we didn't mention SLE-related DEs because no research in our lab is involved in the area of SLE. Sorry for this and thank you for your comprehension. The multivenn diagram with union of intersection of venns showing 1 and 2 respectively for DE lncRNAs and De mRNAs were not discussed in great detail. For example, where are they localized to and their expression patterns Thank you very much for pointing out this issue. We have checked the venn diagram in Fig 5A and Fig 5B, respectively, and found that the union intersection of the significantly enriched two GO terms overlapping the female-upregulated, male-upregulated, female-downregulated and male-downregulated DE mRNA datasets in Fig 5A are : “positive regulation of transcription from RNA polymerase II promoter ”and “negative regulation of transcription from RNA polymerase II promoter”, while the union intersection of the significantly enriched one shared pathway among those four groups in Fig 5B is : “Metabolic pathways”. The detailed mRNAs of the localization in the union intersection of shared GO terms and pathway mentioned above and their expression patterns are provided in Supplementary Table 7 and Supplementary Table 8, respectively. And we have added the discussion about the union of intersection of venns showing 1 and 2 respectively for shared GO functions and pathways in the manuscript according to the reviewer’s suggestion. The conclusions and discussions must herald have point son what if multi-omic studies are integrated to it or say, RNA-Seq expression data is used instead of exon array We thank the reviewer for this comment. It is sure that more potential differentially expressed long non-coding RNAs and coding genes can be discovered if RNA-seq data is used instead of exon array data. In future, we will integrate more multi-omic studies to our work in order to discover more possible lncRNA-mRNA correlation. We have added these points in the Discussion and Conclusion part. Please refer to the manuscript for the modifications. Minor comments The abstract must have an expansion of EGFR We have added the full name of EGFR : “Epidermal growth factor receptor” in the abstract. Please refer to the abstract in the manuscript for the modification. Lines 385 and line 296: Please rewrite or use comma We have rewritten line 296 and line 385, please refer to the manuscript for the modifications. All figures must be high resolution. We have adjusted the figures with low resolution according to the PLOS ONE guidelines: https://journals.plos.org/plosone/s/figures. Now the resolution of all the submitted figures is at least 600dpi. We hope that the newly submitted figures will meet the requirements of PLOS ONE. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0240633.r002
30 Sep 2020 Decision Letter - Rama Krishna Kancha, Editor I ntegrated analysisof long non-coding RNAs and mRNA profiles reveal s potential sex-dependent biomarkers of bevacizumab/erlotinib response in advanced lung cancer PONE-D-20-16411R1 Dear Dr. Tu, 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, Rama Krishna Kancha Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0240633.r003
Formally Accepted
2 Oct 2020 Acceptance Letter - Rama Krishna Kancha, Editor PONE-D-20-16411R1 Integrated analysis of long non-coding RNAs and mRNA profiles reveals potential sex-dependent biomarkers of bevacizumab/erlotinib response in advanced lung cancer Dear Dr. Tu: 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. Rama Krishna Kancha Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0240633.r004

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