Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM)

Julia González-Rincón; José A. Garcia-Vela; Sagrario Gómez; Belén Fernández-Cuevas; Sara Nova-Gurumeta; Nuria Pérez-Sanz; Miguel Alcoceba; Marcos González; Eduardo Anguita; Javier López-Jiménez; Eva González-Barca; Lucrecia Yáñez; Ernesto Pérez-Persona; Javier de la Serna; Miguel Fernández-Zarzoso; Guillermo Deben; Francisco J. Peñalver; María C. Fernández; Jaime Pérez de Oteyza; M. Ángeles Andreu; M. Ángeles Ruíz-Guinaldo; Raquel Paz-Arias; M. Dolores García-Malo; Valle Recasens; Rosa Collado; Raúl Córdoba; Belén Navarro-Matilla; Margarita Sánchez-Beato; José A. García-Marco

doi:10.1371/journal.pone.0257353

Peer Review History

Original SubmissionApril 23, 2021
8 Jun 2021 Decision Letter - Francesco Bertolini, Editor PONE-D-21-13505 Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM) PLOS ONE Dear Dr. Sanchez-Beato, 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 by the 3 Reviewers, experts in the CLL field. Please submit your revised manuscript by Jul 09 2021 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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Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes Reviewer #3: 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 ****** 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 ****** 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 authors sequenced the coding exons and UTR regions of over 20 candidate genes of chronic lymphocytic leukemia (CLL) in 71 CLL patients. They evaluated association between the mutations identified and cytogenetic, phenotypic features and survival rates of the patients. I have some questions about the data analysis: Age is a strong player of survival and a dichotomized age variable as a covariate may not be enough to account for age effects. What if age is used as the time scale in Cox regression? Many of these patients have cytogenetic abnormalities. It is important to evaluate effects of mutations while accounting for the cytogenetic abnormalities, i.e., cytogenetic abnormalities should be included as covariate in the regression analysis. Lines 302-304: “MRD negativity was significantly associated with prolonged PFS (P Person < 0.001) and nearly so for OS (P Fisher =0.065)” Are the data shown in the result? Why using a Chi-square test in the analysis instead of a survival model? Logistic regression should be used to evaluate the association with MRD status so that contributions from multiple mutations can be evaluated jointly and covariates such as cytogenetic abnormalities can be adjusted for. For the Cox regression is the number of mutations per individuals associated with survival? What about the type of genetic alterations (missense, truncating, deletion) of the mutations? Table 3, was age not significant in the multivariate analysis? As mentioned previously cytogenetic abnormalities needs to be adjusted for. Line 250, 49/71 seems to be a typo. Shouldn’t the number of patients with at least one mutation be 54 (23+31)? Reviewer #2: The manuscript by González-Rincón and colleagues reports the results of a deep targeted sequencing approach of the most frequently mutated genes carried out in patients with CLL enrolled in the REM clinical trial where they have been treated with FCR followed by 36 months of rituximab maintenance (bimonthly). They conclude that only TP53 mutations affect the outcome in the study. I have few suggestions: - On lines 247-252, where describing the occurrence of mutations it is reported that 69% of patients harbored at least one mutation, while only 23.9% did not. The numbers do not add up to 100. Indeed, it is also reported that 32.4% had one mutations and 43.7% had more than one, for a total of 76.1% which seems more correct. Please double check the numbers or the wording. - It is interesting to note that SF3B1 is the most frequently mutated gene and much higher than expected in contrast to many other series. How reliable was the VAF in the call for mutations? Any explanation? - It is not clear which conclusions the authors want to draw. As they state in the abstract "The mutational status of our CLL patients, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front line FCR treatment" though in the last sentence of the paper, they mention a possible use of these analyses to decide between "a continuous versus a time-limited therapy". this does not seem to be a conclusion from this study as they do confirm that the analysis for TP53 status is enough to predict poor response to FCR (despite maintenance). Please revise the wording and reconcile the conclusions. Minor comments - Please replace CLL patients with "patients with CLL" throughout the whole text - Please do not capitalize "del(17p) or del(11q)" not even at the beginning of the sentence or in a table (see e.g. table 1) - Please on line 135, replace the sentence "....cut-off of a 2% mismatch from germline IGHV sequences." with " cut-off of 98% identity with the closest germline IGHV gene" - Typically for unmutated IGHV, UM (or U) IGHV is used and not IGHV-U. Consider to align with previous works. - On line 292, as the authors are planning to explore the predictive value of these gene mutations, the authors might simplify the wording by replacing the sentence "we analyzed the clinical significance of these genetic alterations by studying their effect on the therapeutic response" with "we analyzed the predictive value of these genetic alterations in terms of clinical response, PFS, etc". - from line 299 onward, please replace "MRD negative/negativity" with "undetectable MRD" or "detectable MRD" instead of "positivity". Reviewer #3: The manuscript reports on an ancillary biological study of a phase 2 trial where patients with treatment naive CLL received FCR, followed by rituximab maintenance every two months for three years. The study includes 71 patients, whose pre-treatment leukemia samples were profiled for CLL biomarkers including IGHV and targeted gene mutations, and cytogenetics. The study cohort is also provided by longitudinal MRD data. The results further validate previous findings that TP53 abnormalities are the sole lesions associated with outcome. Accordingly, in its current form, the manuscript does not add any further novel finding compared to what already known in the field of prognostic biomarkers of CLL treated with chemoimmunotherapy. In addition, from a conceptual standpoint, FCR has been largely abandoned thanks to the transition to pathway inhibitor therapies in CLL. Thus the results are of eventual historical interest, but do not have actual implications. Finally maintenance with rituximab is not a standard approach in CLL and the study does not help in signaling those patients whose molecular profile may benefit or not from maintenance. Novelty of the manuscript can be substantially improved in the authors can validate or further develop upon AI methods, that leveraging on big data, frameworks for the integration of patient biomarker data over time to improve prognostic accuracy and personalized therapy selection (PMID: 31280963). ****** 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 Reviewer #3: 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.0257353.r001
Revision 1
7 Jul 2021 Author Response Reviewer #1: The authors sequenced the coding exons and UTR regions of over 20 candidate genes of chronic lymphocytic leukemia (CLL) in 71 CLL patients. They evaluated association between the mutations identified and cytogenetic, phenotypic features and survival rates of the patients. I have some questions about the data analysis: Age is a strong player of survival and a dichotomized age variable as a covariate may not be enough to account for age effects. What if age is used as the time scale in Cox regression? R: We considered including the variable “Age” as a continuous variable in the Cox regression analysis since we agree with the reviewer that it is more accurate. However, we eventually decided to dichotomize this variable to use it in the multivariate analysis with other dichotomic variables and ease the comparison with other studies, where “Age” is used as a categoric variable. The threshold at 65 years is the usual standard in most multivariate analysis published in recent years. However, we did the analysis using age as a continuous variable but did not reach significance (P = 0.072). Many of these patients have cytogenetic abnormalities. It is important to evaluate effects of mutations while accounting for the cytogenetic abnormalities, i.e., cytogenetic abnormalities should be included as covariate in the regression analysis. R: Cytogenetic abnormalities were included in the analyses, but none were significant in the regression analysis except when TP53 mutations and del17q were joined as a single variable. Lines 302-304: “MRD negativity was significantly associated with prolonged PFS (P Person < 0.001) and nearly so for OS (P Fisher =0.065)” Are the data shown in the result? Why using a Chi-square test in the analysis instead of a survival model? R: Thank you for the suggestion. We have done the survival models (Cox and Kaplan-Meier) and included them in the manuscript (Page 14, line 306, and Figure S1). Logistic regression should be used to evaluate the association with MRD status so that contributions from multiple mutations can be evaluated jointly and covariates such as cytogenetic abnormalities can be adjusted for. R: Thank you for the suggestion. We have used logistic regression and included the results in page 14, line 310. For the Cox regression is the number of mutations per individuals associated with survival? What about the type of genetic alterations (missense, truncating, deletion) of the mutations? R: Yes, we considered the number of mutations in the survival analysis, but there was no association with OS or PFS. Due to the low number of samples, we considered that we would not obtain relevant results by splitting the type of mutations. The appropriate analysis would be done for each gene independently, and our series does not allow us to do this study properly. Table 3, was age not significant in the multivariate analysis? As mentioned previously cytogenetic abnormalities needs to be adjusted for. R: Age was significant in the univariate analysis, as indicated in now table 2, but loses significance in the multivariate analyses. None of the cytogenetic alterations was significant, except for TP53/del17q, as shown in the table. Line 250, 49/71 seems to be a typo. Shouldn’t the number of patients with at least one mutation be 54 (23+31)? R: Thank you for the observation. We have corrected this and other errors. Reviewer #2: The manuscript by González-Rincón and colleagues reports the results of a deep targeted sequencing approach of the most frequently mutated genes carried out in patients with CLL enrolled in the REM clinical trial where they have been treated with FCR followed by 36 months of rituximab maintenance (bimonthly). They conclude that only TP53 mutations affect the outcome in the study. I have few suggestions: - On lines 247-252, where describing the occurrence of mutations it is reported that 69% of patients harbored at least one mutation, while only 23.9% did not. The numbers do not add up to 100. Indeed, it is also reported that 32.4% had one mutations and 43.7% had more than one, for a total of 76.1% which seems more correct. Please double check the numbers or the wording. R: We apologize for these mistakes; the numbers have been revised and corrected. - It is interesting to note that SF3B1 is the most frequently mutated gene and much higher than expected in contrast to many other series. How reliable was the VAF in the call for mutations? Any explanation? R: According to our experience, data in previous publications, and sequencing depth, we think that VAF is reliable, as we described in the Methods section (page 9, line 200). One possible explanation might be the characteristics of the series, composed by progressive cases in need of treatment compared to others that considered any kind of patient (see page 16, line 347). - It is not clear which conclusions the authors want to draw. As they state in the abstract “The mutational status of our CLL patients, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front line FCR treatment” though in the last sentence of the paper, they mention a possible use of these analyses to decide between “a continuous versus a time-limited therapy”. This does not seem to be a conclusion from this study as they do confirm that the analysis for TP53 status is enough to predict poor response to FCR (despite maintenance). Please revise the wording and reconcile the conclusions. R: Thank you for the suggestion. We have changed the last sentence of the discussion. We hope it would help to clarify our conclusion. “Mutations of most recurrent driver genes in CLL, except for the TP53 gene, do not seem to affect the sustained clinical response obtained with front-line FCR treatment followed by Rituximab maintenance for three years in our cohort of patients”. Minor comments - Please replace CLL patients with “patients with CLL” throughout the whole text - Please do not capitalize “del(17p) or del(11q)” not even at the beginning of the sentence or in a table (see e.g. table 1) - Please on line 135, replace the sentence “....cut-off of a 2% mismatch from germline IGHV sequences.” with “cut-off of 98% identity with the closest germline IGHV gene” - Typically for unmutated IGHV, UM (or U) IGHV is used and not IGHV-U. Consider to align with previous works. - On line 292, as the authors are planning to explore the predictive value of these gene mutations, the authors might simplify the wording by replacing the sentence “we analyzed the clinical significance of these genetic alterations by studying their effect on the therapeutic response” with “we analyzed the predictive value of these genetic alterations in terms of clinical response, PFS, etc”. - from line 299 onward, please replace “MRD negative/negativity” with “undetectable MRD” or “detectable MRD” instead of “positivity”. R: Thank you for the suggestions. We have modified them in this new version and corrected this and other mistakes. Reviewer #3: The manuscript reports on an ancillary biological study of a phase 2 trial where patients with treatment naive CLL received FCR, followed by rituximab maintenance every two months for three years. The study includes 71 patients, whose pre-treatment leukemia samples were profiled for CLL biomarkers including IGHV and targeted gene mutations, and cytogenetics. The study cohort is also provided by longitudinal MRD data. The results further validate previous findings that TP53 abnormalities are the sole lesions associated with outcome. Accordingly, in its current form, the manuscript does not add any further novel finding compared to what already known in the field of prognostic biomarkers of CLL treated with chemoimmunotherapy. In addition, from a conceptual standpoint, FCR has been largely abandoned thanks to the transition to pathway inhibitor therapies in CLL. Thus the results are of eventual historical interest, but do not have actual implications. Finally maintenance with rituximab is not a standard approach in CLL and the study does not help in signaling those patients whose molecular profile may benefit or not from maintenance. Novelty of the manuscript can be substantially improved in the authors can validate or further develop upon AI methods, that leveraging on big data, frameworks for the integration of patient biomarker data over time to improve prognostic accuracy and personalized therapy selection (PMID: 31280963). R: A weakness of this trial is that over the last few years, since its design, new drugs targeting aberrant signaling pathways and newer monoclonal antibodies became available, and the interest in CIT, such as FCR, has weakened. However, it is still accepted as front-line therapy by some clinical guidelines (German, Spanish-CLL, ESMO, etc.) in a subgroup of patients younger than 65 years without comorbidities and absence of cytogenetic abnormalities of worse prognosis (del11q, del17p, complex karyotypes or unmutated IGHV). Unfortunately, REM clinical trial series has not enough data to analyze them using complex algorithms. Moreover, we only have one-point data (from treatment-naïve samples), not serial samples, precluding us from using analyses like those performed in the suggested paper that integrated risk assessments throughout patients’ disease evolution. Attachments Attachment Submitted filename: Rebuttal letter PONE-D-21-13505.docx https://doi.org/10.1371/journal.pone.0257353.r002
28 Jul 2021 Decision Letter - Francesco Bertolini, Editor PONE-D-21-13505R1 Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM) PLOS ONE Dear Dr. Sanchez-Beato, 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 by Reviewer #1. Please submit your revised manuscript by Sep 11 2021 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. 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, Francesco Bertolini, MD, PhD 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 #1: All comments have been addressed Reviewer #3: 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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: 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: (No Response) 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 #1: 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 #1: I only have a minor comment, please specify what variables/covariates were included in the logistic regression. Reviewer #3: The authors have addressed all the reviewer comments and the manuscript is improved in this current version. I have no further issues. ****** 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: No Reviewer #3: 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.0257353.r003
Revision 2
5 Aug 2021 Author Response Reviewer 1: I only have a minor comment, please specify what variables/covariates were included in the logistic regression. Thank you for drawing to our attention this ommision. We have included a sentence indicating the varibles included in the logistic regression analyis (Page 14, lines 312-316). Attachments Attachment Submitted filename: Rebuttal letter_PONE-D-21-13505R1.docx https://doi.org/10.1371/journal.pone.0257353.r004
31 Aug 2021 Decision Letter - Francesco Bertolini, Editor Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM) PONE-D-21-13505R2 Dear Dr. Sanchez-Beato, 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, Francesco Bertolini, MD, PhD 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 #1: 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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) ****** 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: No https://doi.org/10.1371/journal.pone.0257353.r005
Formally Accepted
3 Sep 2021 Acceptance Letter - Francesco Bertolini, Editor PONE-D-21-13505R2 Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM) Dear Dr. Sánchez-Beato: 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. Francesco Bertolini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0257353.r006

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