Development of E-ice-COLD-PCR assay combined with HRM analysis for Nucleophosmin1 gene mutation detection in acute myelogenous leukemia

Rattana Kongta; Noppamas Panyasit; Wuttichote Jansaento; Suwit Duangmano

doi:10.1371/journal.pone.0274034

Peer Review History

Original SubmissionMarch 18, 2022
23 Jun 2022 Decision Letter - Daniel Thomas, Editor PONE-D-22-05079Development of E-ice-COLD-PCR assay combined with HRM analysis for Nucleophosmin1 gene mutation detection in acute myelogenous leukemiaPLOS ONE Dear Dr. Duangmano, 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 Aug 07 2022 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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See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions Additional Editor Comments (if provided): Good method development paper; important test for patient management, any improvements in sensitivity and turn around time celebrated. Will likely be citied. Minor points: Check/provide reference that AML is responsible for most number of deaths from blood cancer in Thailand Abstract: define HRM, reduce number of abbreviations if possible Line 61: poor grammar: results of NPM1 molecular analysis change management Turn around time is perhaps better english than time to result Can the authors provide a discussion of similar methods for other molecular markers/ other cancers in discussion? and brief discussion on cost vs routine methods Discussion: Could emphasis that VAF of NPM1 is also important for prognosis and its order of acquisition: See Figure 4f Nat Commun . 2021 Dec 13;12(1):7244. doi: 10.1038/s41467-021-27472-5. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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: No Reviewer #2: No ****** 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 have attempted to develop and report a method to characterise Nucleophosmin1 gene mutation in AML samples using E-ice-COLD-PCR assay combined with HRM analysis. The study cohort used include 83 samples and they have detected NPM1 mutations in 9 patients with AML using the E-ice-COLD-PCR/HRM analysis assay. The sincere efforts and hard work of the authors in collecting the samples, developing the assay, optimising it and compiling the data are evident in the presented study. However, the manuscript in its current form is not suitable for publication as the presentation of the manuscript does little justice to the presented work. The results, figures and discussion are presented poorly to fully evaluate the findings reported. For instance, in section, Detecting NPM1 gene mutations in patient samples using the E-ice-COLD-PCR assay, opening sentence mentions "NPM1 mutations were detected in a total of 83 patient samples using the E-ice-COLD-PCR assay" which is wrong. Several inaccurate and misleading sentences are present throughout the manuscript. Similarly, the figures and associated legends should be revised as it is difficult to evaluate them in their current form. For example, In Figure 1, the authors could indicate which samples they believe to include double bands as wild type in gel that includes samples 60 to 65 appears to have have a double band and the contrasts are different between gel pictures. It is very difficult to understand clearly and appreciate why and how the E-ice-COLD-PCR assay is better than the other existing assays. In the current manuscript, a one line limitation statement ("However, most of these methods are technically challenging, complicated, and expensive") is provided and it would be beneficial to have a clear comparison with an existing method and highlight how E-ice-COLD-PCR assay is more suitable showing better accuracy. The data shows 9 positive cases and one that has discrepancy between the sequencing and the PCR assay. However, the results sections do not describe the data well. The results include figure legends instead of clear description of the data. Thus, the presentation of the results section makes it very hard to evaluate the figures, data in general and the methods. Therefore, major, rigorous revision of all the sections and complete rewriting of the results section with clear introduction that places the use of NPM1 mutations detection in resource limited clinical settings including Thailand is needed before this can be considered for Publication. The discussion can be revised to actually focus on the data presented and how this compares to other currently available methods and the significance of the assay. The presented study can be useful and may have a potential to contribute towards AML stratification and MRD but the manuscript requires major revision. Reviewer #2: In general, the work is well written, aiming to develop a quick and cost effective method for the detection of NPM1 mutation in acute myeloid leukemia patients. However, I believe that some important points need to be answered by the authors before the work can be considered for publication. My comments are as follows: 1. Abstract, line 25: I think the authors need to use the word "ability" instead of "inability", since it refers to the limitation of the previous detection methods. 2. Abstract, line 26: "HRM" is written for the first time, thus, must be written in full. 3. Abstract, line 27-28: Is the samples from AML patients with normal karyotype? 4. Introduction: I suggest the authors carefully check each of the statement that need to cite the reference. For examples, "AML is the major cause of death among hematological malignancies in Thailand" and "Recently, researchers have suggested that mutations of the nucleophosmin 1 (NPM1) gene represent the most frequent molecular alterations in AML that affect the processes of cellular differentiation and apoptosis, especially in the presence of a normal karyotype." There are other few statements. 5. Result, line 222-223: I suggest the authors write or label the respective patient samples with the mutation. 6. Result, line 239-241: Are you referring to Fig. 2a? 7. Result, line 289-290: The authors state that "The results showed that nine samples contained NPM1 mutations, and the remaining samples were wild-type" in reference to Fig. 5. However, the number of mutant curve in Fig. 5 does not correspond with the number of samples with NPM1 mutation. Please clarify. 8. Result, line 295: I suggest the authors to show the calculation of PPV and NPV. 9. Is the manuscript has been proofread? If not, I suggest that the authors get editing help from someone with full professional proficiency in English. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0274034.r001
Revision 1
18 Aug 2022 Author Response 1. Additional Editor Comments: 1.1 Check/provide reference that AML is responsible for most number of deaths from blood cancer in Thailand Answer: We have already revised the sentence 1.2 Abstract: define HRM, reduce number of abbreviations if possible Answer: We have revised 1.3 Line 61: poor grammar: results of NPM1 molecular analysis change management Answer: We didn’t find this sentence at line 61 1.4 Turn around time is perhaps better english than time to result Answer: We have revised 1.5 Can the authors provide a discussion of similar methods for other molecular markers/ other cancers in discussion? and brief discussion on cost vs routine methods Answer: We have provided a discussion at line 361-383 and 436-441 2. Reviewer #1: 2.1 The results, figures and discussion are presented poorly to fully evaluate the findings reported. For instance, in section, Detecting NPM1 gene mutations in patient samples using the E-ice-COLD-PCR assay, opening sentence mentions "NPM1 mutations were detected in a total of 83 patient samples using the E-ice-COLD-PCR assay" which is wrong. Several inaccurate and misleading sentences are present throughout the manuscript. Similarly, the figures and associated legends should be revised as it is difficult to evaluate them in their current form. For example, In Figure 1, the authors could indicate which samples they believe to include double bands as wild type in gel that includes samples 60 to 65 appears to have have a double band and the contrasts are different between gel pictures. Answer: We have revised the sentence that represent inaccurate or misleading sentence. The contrasts are difference between gel because we analyzed PCR products by acrylamide gel electrophoresis for 5-6 samples per a gel, but we have been tried to adjust the contrast for each gel in similar condition. However, the results are still difficult interpretation. This is a reason that why we develop new method for detecting NPM1 mutations. 2.2 It is very difficult to understand clearly and appreciate why and how the E-ice-COLD-PCR assay is better than the other existing assays. In the current manuscript, a one line limitation statement ("However, most of these methods are technically challenging, complicated, and expensive") is provided and it would be beneficial to have a clear comparison with an existing method and highlight how E-ice-COLD-PCR assay is more suitable showing better accuracy. Answer: We have provided the discussion at line 367-378 and 401-405 2.3 The data shows 9 positive cases and one that has discrepancy between the sequencing and the PCR assay. However, the results sections do not describe the data well. The results include figure legends instead of clear description of the data. Thus, the presentation of the results section makes it very hard to evaluate the figures, data in general and the methods. Therefore, major, rigorous revision of all the sections and complete rewriting of the results section with clear introduction that places the use of NPM1 mutations detection in resource limited clinical settings including Thailand is needed before this can be considered for Publication. The discussion can be revised to actually focus on the data presented and how this compares to other currently available methods and the significance of the assay. The presented study can be useful and may have a potential to contribute towards AML stratification and MRD but the manuscript requires major revision. Answer: We have revised 3. Reviewer #2 3.1 Abstract, line 25: I think the authors need to use the word "ability" instead of "inability", since it refers to the limitation of the previous detection methods. Answer: We have revised at line 26 3.2 Abstract, line 26: "HRM" is written for the first time, thus, must be written in full. Answer: We have revised. 3.3 Abstract, line 27-28: Is the samples from AML patients with normal karyotype? Answer: The samples weren’t examined the karyotype test because in this study we aim to develop the highly specific and sensitive screening method for NPM1 mutation in AML patients. We don’t concern about the karyotype of AML samples using validate the developed method. So, we use all types of AML samples without the identification of normal or abnormal karyotype to validate the developed method. 3.4 Introduction: I suggest the authors carefully check each of the statement that need to cite the reference. For examples, "AML is the major cause of death among hematological malignancies in Thailand" and "Recently, researchers have suggested that mutations of the nucleophosmin 1 (NPM1) gene represent the most frequent molecular alterations in AML that affect the processes of cellular differentiation and apoptosis, especially in the presence of a normal karyotype." There are other few statements. Answer: We have already revised the sentence 3.5 Result, line 222-223: I suggest the authors write or label the respective patient samples with the mutation. Answer: We have revised at line 240-241. 3.6 Result, line 239-241: Are you referring to Fig. 2a? Answer: Yes, we are. We have revised. 3.7 Result, line 289-290: The authors state that "The results showed that nine samples contained NPM1 mutations, and the remaining samples were wild-type" in reference to Fig. 5. However, the number of mutant curve in Fig. 5 does not correspond with the number of samples with NPM1 mutation. Please clarify. Answer: The fig 5, we represented the HRM difference curve from few mutant and wild-type samples because we would like to give some examples of data generated from HRM analysis. In addition, we represented mutant and wild-type results after HRM analysis in the same figure because we would like to show the differences between mutant and wild-type HRM difference curve in the same figure. For HRM analysis of total 83 patient samples, the PCR products were analyzed by HRM analysis, and the data generated in separated HRM difference curve from each other. The separation of HRM analysis in each sample resulting in clearly interpretation of HRM difference curve of patient samples. We show the results of the total 83 patients in supplementary data. 3.8 Result, line 295: I suggest the authors to show the calculation of PPV and NPV. Answer: We have described the calculation of PPV and NPV at line 222-227. 3.9 Is the manuscript has been proofread? If not, I suggest that the authors get editing help from someone with full professional proficiency in English. Answer: Yes, it is. We have proofread the manuscript. Attachments Attachment Submitted filename: response to reviewers.docx https://doi.org/10.1371/journal.pone.0274034.r002
22 Aug 2022 Decision Letter - Daniel Thomas, Editor Development of E-ice-COLD-PCR assay combined with HRM analysis for Nucleophosmin1 gene mutation detection in acute myelogenous leukemia PONE-D-22-05079R1 Dear Dr. Duangmano, 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, Daniel Thomas, MD Academic Editor PLOS ONE Additional Editor Comments (optional): Editor is satisfied that all reviewers' comments have been adequately addressed. Reviewers' comments: https://doi.org/10.1371/journal.pone.0274034.r003
Formally Accepted
5 Sep 2022 Acceptance Letter - Daniel Thomas, Editor PONE-D-22-05079R1 Development of E-ice-COLD-PCR assay combined with HRM analysis for Nucleophosmin1 gene mutation detection in acute myelogenous leukemia Dear Dr. Duangmano: 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. Daniel Thomas Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0274034.r004

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