Impact of TP53 mutations in acute myeloid leukemia patients treated with azacitidine

Pierre Bories; Naïs Prade; Stéphanie Lagarde; Bastien Cabarrou; Laetitia Largeaud; Julien Plenecassagnes; Isabelle Luquet; Véronique De Mas; Thomas Filleron; Manon Cassou; Audrey Sarry; Luc-Matthieu Fornecker; Célestine Simand; Sarah Bertoli; Christian Recher; Eric Delabesse

doi:10.1371/journal.pone.0238795

Peer Review History

Original SubmissionApril 27, 2020
9 Jun 2020 Decision Letter - Francesco Bertolini, Editor PONE-D-20-11937 Impact of TP53 mutations in acute myeloid leukemia patients treated with azacitidine PLOS ONE Dear Dr. Bories, 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 both Reviewers, experts in the AML field. Please submit your revised manuscript by Jul 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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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: Bories et al submit a retrospective analysis of elderly AML patients treated with azacitidine who were considered unfit for induction chemotherapy. They identify 55 patients with TP53 mutations and 68 patients with mutations and/or deletions. The mutations are predominantly missense, organize around hotspots in the DNA-binding domain, are associated with near universal adverse risk karyotypes, all expected phenotypes for TP53 variants in AML. Of note, patients with TP53 mutations had worse performance status. Survival outcomes were worse with TP53 mutations or alterations. Overall, this is a well-written post-hoc analysis of a fairly large dataset. The results are interesting and the discussion is germane. Concerns Performance status > 2 is more common in TP53 mutated/altered cases. Poor performance status is commonly associated with poor survival in older AML studies. In the multivariate analysis, PS < 2 is used. It is unclear why the threshold is changed. The authors should repeat the multivariate analysis using > 2 as the cut off because this is associated with a phenotype among TP53 mutated patients and it would be important to know if poor performance status contributes to the adverse survival in the TP53 mutated cases. Do the authors have access to the number of cycles of therapy received? Is this related to TP53 status, response, or survival? Reviewer #2: This is an interesting retrospective study focusing on the impact of TP53 gene mutations in patients with AML treated with azacitidine in a quite large series of patients prospectively enrolled in the French regional healthcare network between 2007 and 2016 (n=279). The manuscript is well written and the data are clearly and straightforwardly presented, with well-designed tables and pictures. However, in its present version, I have a few major concern requiring some clarifications and possible refined analysis to be performed: 1. The main finding of this analysis is the association between the presence of any TP53 mutation and worse overall survival, with no impact on the treatment outcome. As it has been correctly stated and discussed also by the authors, this latter finding is in countertrend compared to the previously published results by Welch et al with decitabine. However, among the 224 patients who have been here retrospectively screened (by exome sequencing in 49 and NGS multiplex PCR in 179), TP53 mutations have been detected in 55 patients of whom 96.4% were also harbouring high-risk cytogenetics. In light of this strong correlation (adverse cytogenetics were found in only 33% of patients with TP53 WT), the inclusion of a possible interaction factor between the two variables in the multivariate model is highly recommended to verify their real independence. Should a significant interaction exist, it would be very important to verify its effect on the two variables taken individually. 2. Since the number of administered azacitidine cycles has been extremely heterogeneous (ranging from 1 to 67 with a median of 6), it would be also important to investigate the impact of the TP53 mutation on the treatment duration. In other words, what was the median number (and ranges) of administered cycles in the TP53wt, TP53mut and TP53unknown subgroups? Furthermore, were the azacitidine cycles all administered according to the 5-2-2 schedule, or there were also patients who received the 7 days in a raw schedule? If this is the case, any difference in the percentage of TP53 mutation among possible differently treated patients? 3. What was the median response duration among the twelve patients harboring a TP53 mutation and classified as responders? How does this median duration compare to that of responders in the TP53wt and TP53unknown groups? Minor comments/questions: 1. Has been any post-treatment NGS analysis performed among responders to investigate on possible VAF variation for TP53mutations? 2. Tables 1 and 2 include “tumour” among enlisted variables. What does this mean? Patients with tumour are those with a previous/concomitant malignancy other than AML? Please, clarify. ****** 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: Yes: Francesco Onida [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.0238795.r001
Revision 1
31 Jul 2020 Author Response We warmly thank the two reviewers for their comments. Whenever possible, the manuscript has been revised according to their comments and detailed explanations and answers are given in this cover letter. Additionally, an ancillary biomarker study of a phase II trial testing frontline decitabine in elderly AML patients have been recently published with similar results to ours (Becker et al, Annals of Hematology, June 2020). This was added in the discussion (line 279, reference 41). Responses to reviewer 1: 1 Performance status > 2 is more common in TP53 mutated/altered cases. Poor performance status is commonly associated with poor survival in older AML studies. In the multivariate analysis, PS < 2 is used. It is unclear why the threshold is changed. The authors should repeat the multivariate analysis using > 2 as the cut off because this is associated with a phenotype among TP53 mutated patients and it would be important to know if poor performance status contributes to the adverse survival in the TP53 mutated cases. We agree that AML patients with TP53 mutation commonly present with altered performance status (PS) which may be associated with shorter overall survival. In our cohort, among 55 TP53mut AML patients, 21 patients (42%) presented with baseline ECOG PS >1 but only 4 patients (7%) with baseline ECOG PS >2. In order not to unbalanced the groups, we decided to use the >1 cut-off. To avoid any ambiguity between the > and ≥ symbols we present results in table 1 and 2, with ECOG 0-1 and 2-4 groups. As you suggested, we included ECOG PS in the multivariate analysis for OS which is relevant regarding its classical impact on OS and also justified by differences in term of baseline ECOG observed between TP53wt and TP53mut patients. The inclusion of ECOG PS in the multivariable analysis for OS slightly modified the results of the model, notably with the association between TP53 mutation and shorter OS being just below the threshold of significance at 0.081, while the hazard ratio remained at 1.49. Accordingly, we modified the text in the result section (line195) and in the discussion (line 276) 2 Do the authors have access to the number of cycles of therapy received? Is this related to TP53 status, response, or survival? We included the median number of azacitidine cycle received in table 1 for each cohort (global cohort, TP53wt, TP53mut, and TP53 unknown subgroups). The number of treatment cycle is significantly lower in the TP53mut group compared to the TP53wt group, which could be explained by a shorter duration of response in this group. Median duration of response has also been added in table 1, alongside with response rate and median overall survival in the outcome section. Responses to reviewer 2: 1. The main finding of this analysis is the association between the presence of any TP53 mutation and worse overall survival, with no impact on the treatment outcome. As it has been correctly stated and discussed also by the authors, this latter finding is in countertrend compared to the previously published results by Welch et al with decitabine. However, among the 224 patients who have been here retrospectively screened (by exome sequencing in 49 and NGS multiplex PCR in 179), TP53 mutations have been detected in 55 patients of whom 96.4% were also harbouring high-risk cytogenetics. In light of this strong correlation (adverse cytogenetics were found in only 33% of patients with TP53 WT), the inclusion of a possible interaction factor between the two variables in the multivariate model is highly recommended to verify their real independence. Should a significant interaction exist, it would be very important to verify its effect on the two variables taken individually. The required sample size to test an interaction with an acceptable statistical power is usually very large (at least four times more than when no interaction is assumed) (Brookes et al., Journal of Clinical Epidemiology 2004; Schmoor et al., Statistics in Medicine 2000; Peterson et al., Controlled Clinical Trials 1993). Given the size of our study and the number of patients in the subgroup with TP53 mutation and non-adverse cytogenetics (n=2), it was not appropriate to investigate the interaction between TP53 status and cytogenetics risk. As an exploratory purpose, a subgroup analysis was then performed to assess the impact of TP53 status on overall survival in patients with adverse cytogenetics (median OS 7.9 months (mut) vs. 9.6 months (WT); HR=1.61; 95% CI=[1.08;2.41]; p=0.019, Fig 1C). 2) Since the number of administered azacitidine cycles has been extremely heterogeneous (ranging from 1 to 67 with a median of 6), it would be also important to investigate the impact of the TP53 mutation on the treatment duration. In other words, what was the median number (and ranges) of administered cycles in the TP53wt, TP53mut and TP53unknown subgroups? Furthermore, were the azacitidine cycles all administered according to the 5-2-2 schedule, or there were also patients who received the 7 days in a raw schedule? If this is the case, any difference in the percentage of TP53 mutation among possible differently treated patients? We included the median number of azacitidine cycle received in table 1 in each cohort (global cohort, TP53wt, TP53mut, and TP53 unknown subgroups). The number of treatment cycle is significantly lower in the TP53mut group compared to the TP53wt group, which could be explained by a shorter duration of response in this group. Duration of response has also been added in table 1, alongside with response rate and median overall survival In our daily practice azacitidine is mainly administered using the so-called 5-2-2 schedule. Some patients may also have received the 7 days in a raw schedule, in particular for the first cycle frequently performed as inpatient. Unfortunately, this information is not collected in the Toulouse AML database and we cannot assess the impact of azacitidine schedule on outcome of TP53wt versus TP53mut patients. 3) What was the median response duration among the twelve patients harboring a TP53 mutation and classified as responders? How does this median duration compare to that of responders in the TP53wt and TP53unknown groups? Definition of duration of response was added in the method section (line 148). We add this information in table 1 (page 7). Median duration of response (DOR) was 9.3 months in the global cohort of responders (n=54), 9.9 months in patients with TP53wt (n=30), 6,5 months in patients with a TP53 mutation (n=12) and 13.3 months in patient with unknown TP53 status (n=12). Univariable comparison of the DOR between TP53wt and TP53mut patient was not statistically different (9.9 months versus 6.5 months, respectively, p=0.303), presumably due to the limited number of patients in each cohort. Minor comments/questions: 1) Has been any post-treatment NGS analysis performed among responders to investigate on possible VAF variation for TP53mutations? We acknowledge that TP53 mutation clearance would have add valuable information, unfortunately, such samples were not banked in daily practice. 2) 2. Tables 1 and 2 include “tumour” among enlisted variables. What does this mean? Patients with tumour are those with a previous/concomitant malignancy other than AML? Please, clarify In table 1 and 2 “Tumour” in fact designates clinical extra medullary disease such as splenomegaly, hepatomegaly, lymph nodes or gingival hypertrophy. We acknowledge this designation is ambiguous, and changed it in the manuscript and tables to “extramedullary disease” Journal requirements 1. Our manuscript was entirely checked regarding PLOS ONE's style requirements and we made the following changes: - We used level 1 heading for all major sections (abstract, introduction…) with bold type 18pt, and sentence case, level 2 heading for sub-sections with 16pt bold type police, and level 3 heading with bold type 14pt police fur sub-section within section 2. -The term “Figure XX” was replaced by “Fig XX” in the whole manuscript and “Table S1” by “S1 Table”. 2. We add in the material section of the manuscript the following sentence: “The French Commission Nationale de l’Informatique et des Libertés (CNIL) authorised the use of patient data analyzed in our study” (line 95) 3. An ethics statement section was added at the end of the manuscript (line 478) and the Edit Submission. 4. The sentence “This work was supported by the French government under the "Investissement d'avenir" program (ANR-11-PHUC-001).” (line 318) was removed from the acknowledgment section of the manuscript and this information was provided in the Funding Statement. 5. We add the sentence “This does not alter our adherence to PLOS ONE policies on sharing data and materials.” after "Christian Recher has received research funding from Celgene and served as consultant for Celgene" in the Competing Interests section form of the Edit Submission. 6. The manuscript was amended via Edit Submission to include author Stéphanie Lagarde (married name Stéphanie Dufrechou) 7. The manuscript file includes Stéphanie Lagarde as an author instead of Stéphanie Dufréchou 8. Reference to figure 2 was added in the manuscript file (line 241). 9. The section “supplementary material” was renamed “supporting information” and reformat according to PLOS One Supporting Information guidelines. Addendum to the journal requirements on 07/31/ 2020: 1. A minimal data set has been uploaded on Figshare repository. The following sentence has been added in the manuscript line 139 “All the data used for the analyses were deposited in Figshare: https://figshare.com/s/b86087d20fbd9634e156”. Separate captions of the supplementary files are included at the end of the manuscript 2. Please update the funding statement as follow: “This work was supported by the French government under the "Investissement d'avenir" program (ANR-11-PHUC-001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. During the period of the study Stéphanie Lagarde has received salary from the "Investissement d'avenir" program (ANR-11-PHUC-001). 3/4. Copy of S1 table, S2 table and S1 Fig have been uploaded in the Edit submission. https://doi.org/10.1371/journal.pone.0238795.r002
25 Aug 2020 Decision Letter - Francesco Bertolini, Editor Impact of TP53 mutations in acute myeloid leukemia patients treated with azacitidine PONE-D-20-11937R1 Dear Dr. Bories, 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 #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: All previous comments and suggestions have been adequately addressed in this revised version of the manuscript. 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: Yes:** Prof. Francesco Onida https://doi.org/10.1371/journal.pone.0238795.r003
Formally Accepted
21 Sep 2020 Acceptance Letter - Francesco Bertolini, Editor PONE-D-20-11937R1 Impact of TP53 mutations in acute myeloid leukemia patients treated with azacitidine Dear Dr. Bories: 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.0238795.r004

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