The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: A systematic review and meta-analysis

Peng Ye; Peiling Cai; Jing Xie; Yuanyuan Wei

doi:10.1371/journal.pone.0248775

Peer Review History

Original SubmissionOctober 5, 2020
8 Jan 2021 Decision Letter - Anthony F. Shields, Editor PONE-D-20-31082 The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: a systematic review and meta-analysis PLOS ONE Dear Dr. Ye, 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. As noted by the reviewer, the authors should better describe the assay systems used and the differences in technical approaches. They should also include the full names, not just the abbreviations such as for ARMS (Amplification Refractory Mutation System) and what assay versions were used, such as the new SuperARMS. While some of the studies they discussed may have used KRAS alone, others included NRAS and this should be noted in the review. Finally, as suggested the article is written in English, but this is not the authors’ first language and a through review of the grammar is needed. Please submit your revised manuscript by Feb 13 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 We look forward to receiving your revised manuscript. Kind regards, Anthony F. Shields, M.D., Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1.) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2.) Thank you for including the statement that 'Literature research was performed independently by PY and PC in June 2020'. Please revise this statement to clarify whether all databases were searched from inception, or if there were any limits placed on the publication dates in your search. 3.) At this time, we ask that you please provide the full search strategy and search terms for at least one database used as Supplementary Information. 4.) In the Methods section, please provide the methodology used for data extraction, including the specific reporting items that were extracted from the included studies. [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: I Don't Know 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: 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 investigators performed literature review to identify studies that have examined KRAS status using cell free DNA. They performed meta-analysis of these data and present the results here. The cited references appear to be during or after 2016, shortly after a time when expansion of standard tissue-based platforms adapted to the concept of All-RAS sequencing (more isoforms and testing of NRAS as well). Please clarify confirmation that the papers using meta-analysis adhered to expanded testing, and also clarify whether NRAS (while much less prevalent, 5-10% in CRC) was also assessed in any or all the studies. Also, it would be helpful to clarify whether there were notable differences in testing approach in relation to accuracy by region/country of testing, and whether any potential differences could be accounted for by significant differences in testing approaches. One of the proposed advantages of cell free DNA is more accurate identification of tumor cell heterogeneity, including potential mixed populations of KRAS mutant and KRAS wild type cells. Not all the studies divided examined here, reported what percentage of samples yielded positive results for both within the same specimens. The authors pool results across many different studies, using many different approaches, and the final pulled result does not mean much considering the wide range. For presentation of statistical analysis, it would be preferable to emphasize more the wide range and median values, with a subset analyses to report common themes and approaches that appear to be reproducible and precise (and also hopefully providing accuracy ). There are many disparate results reported by a number of these papers, including a wide range of sensitivity of assays, even when using the same method (eg NGS), and differences between cell free DNA-based assessment versus tissue-based assessment. The latter point is well documented, and more in-depth comments about the wide range of disparities should be included in the discussion section. Concordance rates are poor, yet many oncologists worldwide depend heavily on liquid-based biopsy at assessment for determination of using EGFR inhibitor treatments. What is the authors’ perspective on how this practice should change, and how, based on the results of this meta-analysis? I encourage the investigators to leverage this manuscript as a platform to propose change in the field for better and more accurate use of these tests. The use of serial assessment of KRAS and other markers over time in colorectal cancer has been promoted without strong evidence that this changes management on a large scale, nor results in improvement of overall survival. Can the authors comment on perspective on this angle, noting also that there are multiple prospective trials examining the use of cell free DNA in general in the postoperative setting for earlier stage/resectable colorectal cancers (in the U.S.). On page 18, it is mentioned that some studies assessing KRAS were performed in patients with primary colorectal cancer; please clarify why this was done in the studies, as this type of assessment is not routinely indicated in nonmetastatic patients. As the data are there, it would also be helpful to discuss any notable differences in sensitivity, and/or concordance, between primary and metastatic tumors that were assessed in relation to cell free DNA, and early-stage versus later stage patients. Reviewer #2: In this review/meta-analysis, Peng Ye tried to compare different methods used in detecting KRAS mutations in plasma of CRC patients. Although the authors put a lot of efforts into, there remain some issues/changes to be done: 1.) Improve English! There are several grammer errors! 2.) In this study, the authors are comparing PCR, NGS and ARMS. However, they are describing these methods only superficial. Thus, in the introduction section the authors should describe these techniques (what is better in one to another method? costs issues? ecc...). 3.) The authors are describing that only KRAS is important in the selection for anti-EGFR treatment. This is not correct! The authors should focus, or at least discuss, that next to KRAS mutations also NRAS mutations are very important to select the best treatment for our CRC patients. 4.) The authors are concluding that liquid biopsy may be only important in the metastatic stage, however, also in limited stages liquid biopys may be helpful in monitoring disease or detecting early relapse. Please, discuss this fact. ****** 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.0248775.r001
Revision 1
1 Feb 2021 Author Response Comments from Editor: 1) As noted by the reviewer, the authors should better describe the assay systems used and the differences in technical approaches. Response: Many thanks for the suggestions. We have revised the manuscript and included more descriptions on the three assay systems and their advantages and disadvantages. Please find the revision in the 3rd paragraph of the Introduction section (page 5 of the Revised Manuscript with Track Changes). 2) They should also include the full names, not just the abbreviations such as for ARMS (Amplification Refractory Mutation System) and what assay versions were used, such as the new SuperARMS. Response: Many thanks for the suggestions. We have revised the manuscript and included the detailed type/version of techniques used in the studies. Please find the revision in Table 1 on page 10-17 in the Revised Manuscript with Track Changes. In addition, we also revised the names of techniques and avoided usage of abbreviations. Please find the revisions in the Revised Manuscript with Track Changes. 3) While some of the studies they discussed may have used KRAS alone, others included NRAS and this should be noted in the review. Response: Thanks very much for the suggestion. In the 33 eligible studies, there were in all 20 studies which targeted All-RAS, or expanded isoforms of KRAS. Most of those studies reported KRAS sequencing results separately from NRAS/HRAS, while 6 studies reported only All-RAS results. In this case, the NRAS testing results were included in the systematic review and meta-analysis. We have revised the manuscript accordingly. Please find the revisions in the 1st paragraph of “Review of eligible publications” section (page 9 of the Revised Manuscript with Track Changes). 4) Finally, as suggested the article is written in English, but this is not the authors’ first language and a through review of the grammar is needed. Response: Very sorry for the grammar errors. We have carefully proofread the manuscript and tried our best to remove the grammar errors. Please find the revisions in the Revised Manuscript with Track Changes. Additional requirements: 1.) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: Many thanks for your comments. We have carefully checked and ensured that our manuscript meets PLOS ONE’s style requirement. 2.) Thank you for including the statement that 'Literature research was performed independently by PY and PC in June 2020'. Please revise this statement to clarify whether all databases were searched from inception, or if there were any limits placed on the publication dates in your search. Response: Thanks very much for your suggestion. We did not place any limitation on the publication dates in the literature search. Please find the revised statement in the 1st paragraph in “Literature searching and selection of publication” section (page 6 of the Revised Manuscript with Track Changes). 3.) At this time, we ask that you please provide the full search strategy and search terms for at least one database used as Supplementary Information. Response: Thanks very much for your suggestion. We have included the detailed search strategy as S1 Table. Please find it in the supporting information. 4.) In the Methods section, please provide the methodology used for data extraction, including the specific reporting items that were extracted from the included studies. Response: Thanks very much for your comments. We have revised the manuscript and included all the reporting items used in our data extraction. Please find the revisions in the 2nd paragraph of “Literature searching and selection of publication” section (page 6-8 of the Revised Manuscript with Track Changes). Comments from Review #1: The investigators performed literature review to identify studies that have examined KRAS status using cell free DNA. They performed meta-analysis of these data and present the results here. 1) The cited references appear to be during or after 2016, shortly after a time when expansion of standard tissue-based platforms adapted to the concept of All-RAS sequencing (more isoforms and testing of NRAS as well). Please clarify confirmation that the papers using meta-analysis adhered to expanded testing, and also clarify whether NRAS (while much less prevalent, 5-10% in CRC) was also assessed in any or all the studies. Response: Thanks very much for your suggestions. In our review, 20 eligible studies performed All-RAS sequencing with expanded isoforms of the genes. Most of the studies reported KRAS testing results separately, or ignored NRAS/HRAS testing results. The rest 6 studies reported All-RAS sequencing results only, and in this case, the NRAS testing results were included in the subsequent systematic review and meta-analysis. We also revised the manuscript to describe those details. Please find them in the 1st paragraph of “Review of eligible publications” section (page 9 of Revised Manuscript with Track Changes). 2) Also, it would be helpful to clarify whether there were notable differences in testing approach in relation to accuracy by region/country of testing, and whether any potential differences could be accounted for by significant differences in testing approaches. Response: Thanks very much for the helpful suggestions. We performed meta-analysis across different regions in studies using next-generation sequencing or digital polymerase chain reaction, and the sensitivity was similar across different regions for next-generation sequencing, and sensitivity and diagnostic odds ratio were similar between Europe and Asia, indicating that the differences in accuracy among different regions could be partially explained by significant difference in techniques used in those regions. We also inserted those results and discussions in the 5th paragraph of “Meta-analysis of the accuracy of KRAS mutation detection using cfDNA samples” section (page 29-30 of the Revised Manuscript with Track Changes). 3) One of the proposed advantages of cell free DNA is more accurate identification of tumor cell heterogeneity, including potential mixed populations of KRAS mutant and KRAS wild type cells. Not all the studies divided examined here, reported what percentage of samples yielded positive results for both within the same specimens. Response: Thanks very much for the comments. We agree that the tumor heterogeneity is not a major focus of this meta-analysis or the references of this manuscript. The expression was removed accordingly. Please find the revision in the 2nd paragraph of Introduction section (page 5 of the Revised Manuscript with Track Changes). 4) The authors pool results across many different studies, using many different approaches, and the final pulled result does not mean much considering the wide range. For presentation of statistical analysis, it would be preferable to emphasize more the wide range and median values, with a subset analyses to report common themes and approaches that appear to be reproducible and precise (and also hopefully providing accuracy ). Response: Thanks very much for your suggestions. We have put more effort on the subgroup analysis. In addition to the comparison among different regions in studies using next-generation sequencing or digital polymerase chain reaction, we also investigated the accuracy of subtypes of next-generation sequencing (Guardant360 panel) and digital polymerase chain reaction (Beads, Emulsion, Amplification and Magnetics, and digital droplet polymerase chain reaction). In addition, we emphasized more on the heterogeneity among the studies and also addressed that we focus more on the possible sources of heterogeneity and subgroup analysis. Please find the revisions in the 2nd and 4th paragraph of “Meta-analysis of the accuracy of KRAS mutation detection using cfDNA samples” section (page 25, 29 of the Revised Manuscript with Track Changes), and Table 3 (page 26-28 of the Revised Manuscript with Track Changes). 5) There are many disparate results reported by a number of these papers, including a wide range of sensitivity of assays, even when using the same method (eg NGS), and differences between cell free DNA-based assessment versus tissue-based assessment. The latter point is well documented, and more in-depth comments about the wide range of disparities should be included in the discussion section. Response: Thanks very much for your helpful suggestions. We have looked into the wide range of disparities among the studies, and inserted comments in the discussion section. Please find the revision in the 4th paragraph of Discussion section (page 34 of the Revised Manuscript with Track Changes). 6) Concordance rates are poor, yet many oncologists worldwide depend heavily on liquid-based biopsy at assessment for determination of using EGFR inhibitor treatments. What is the authors’ perspective on how this practice should change, and how, based on the results of this meta-analysis? I encourage the investigators to leverage this manuscript as a platform to propose change in the field for better and more accurate use of these tests. Response: Thanks very much for your helpful suggestions. Accordingly, we also commented on the poor concordance rate and proposed possible ways to limit the risk of misdiagnosis. Please find the comments in the 4th paragraph of Discussion section, after the comments for wide range of disparities among the studies (page 34-35 of the Revised Manuscript with Track Changes). 7) The use of serial assessment of KRAS and other markers over time in colorectal cancer has been promoted without strong evidence that this changes management on a large scale, nor results in improvement of overall survival. Can the authors comment on perspective on this angle, noting also that there are multiple prospective trials examining the use of cell free DNA in general in the postoperative setting for earlier stage/resectable colorectal cancers (in the U.S.). Response: Thanks very much for your helpful suggestions. We have looked into the serial assessment of KRAS using liquid biopsy samples. Please find our comments in the 5th paragraph of Discussion section in the revised manuscript (page 35-36 of the Revised Manuscript with Track Changes). 8) On page 18, it is mentioned that some studies assessing KRAS were performed in patients with primary colorectal cancer; please clarify why this was done in the studies, as this type of assessment is not routinely indicated in nonmetastatic patients. Response: Thanks very much for your suggestions. We have looked into the reasons for the involvement of nonmetastatic patients in those studies, and inserted them in the start of the 3rd paragraph in “Meta-analysis of the accuracy of KRAS mutation detection using cfDNA samples” in Results section (page 26 of the Revised Manuscript with Track Changes). 9) As the data are there, it would also be helpful to discuss any notable differences in sensitivity, and/or concordance, between primary and metastatic tumors that were assessed in relation to cell free DNA, and early-stage versus later stage patients. Response: Thanks very much for your suggestions. We compared the accuracy data between primary and metastatic tumors in relation to cell free DNA, and the results could be found in the 3rd paragraph in “Meta-analysis of the accuracy of KRAS mutation detection using cfDNA samples” in Results section (page 26 of the Revised Manuscript with Track Changes), and the 3rd paragraph (page 32 of the Revised Manuscript with Track Changes) and 5th paragraph of Discussion section (line 3-5 on page 36 of the Revised Manuscript with Track Changes). In addition, following your advices, we tried to extract the accuracy data from early-stage CRC patients. But unfortunately, data could only be extracted from 3 studies, including 1 study which lacked true positive cases and was therefore excluded by the statistical software. The number of the rest studies (2 studies) was too small for meta-analysis. Therefore, we did not continue the comparison between early-stage and late-stage patients. Those results were also described at the end of 3rd paragraph in “Meta-analysis of the accuracy of KRAS mutation detection using cfDNA samples” in Results section (page 26 of the Revised Manuscript with Track Changes). Comments from Review #2: In this review/meta-analysis, Peng Ye tried to compare different methods used in detecting KRAS mutations in plasma of CRC patients. Although the authors put a lot of efforts into, there remain some issues/changes to be done: 1.) Improve English! There are several grammer errors! Response: Very sorry for the grammar errors. We have carefully proofread the manuscript and tried our best to remove the grammar errors. Please find the revisions in the Revised Manuscript with Track Changes. 2.) In this study, the authors are comparing PCR, NGS and ARMS. However, they are describing these methods only superficial. Thus, in the introduction section the authors should describe these techniques (what is better in one to another method? costs issues? ecc...). Response: Thanks very much for the suggestions. We have revised the introduction section and described more advantages and disadvantages of those techniques. Please find the revision in the 3rd paragraph of Introduction section (page 5-6 of the Revised Manuscript with Track Changes). 3.) The authors are describing that only KRAS is important in the selection for anti-EGFR treatment. This is not correct! The authors should focus, or at least discuss, that next to KRAS mutations also NRAS mutations are very important to select the best treatment for our CRC patients. Response: Thanks very much for your comments. We have revised the introduction section and emphasized the importance of NRAS in the anti-EGFR treatment for CRC patients. Please find the revision in the 1st paragraph of Introduction section (page 4 of the Revised Manuscript with Track Changes). 4.) The authors are concluding that liquid biopsy may be only important in the metastatic stage, however, also in limited stages liquid biopys may be helpful in monitoring disease or detecting early relapse. Please, discuss this fact. Response: Many thanks for your comments. We discussed more about the use of liquid biopsy in early detection and monitoring of the disease and prediction of patients prognosis. Please find the revision in the 5th paragraph of Discussion section (page 35-36 of the Revised Manuscript with Track Changes), and in Conclusion section (at end of page 36 of the Revised Manuscript with Track Changes). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0248775.r002
18 Feb 2021 Decision Letter - Anthony F. Shields, Editor PONE-D-20-31082R1 The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: a systematic review and meta-analysis PLOS ONE Dear Dr. Ye, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== The authors misinterpreted the editor's suggestion to "include the full names, not just the abbreviations such as for ARMS" etc. Terms such as ARMS, PCR, and NGS need to be defined at the start, but abbreviations can be used after that. Please correct that. Otherwise, the manuscript is much improved and acceptable for publication. ============================== Please submit your revised manuscript by Apr 04 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 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 We look forward to receiving your revised manuscript. Kind regards, Anthony F. Shields, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (if provided): The authors misinterpreted the editor's suggestion to "include the full names, not just the abbreviations such as for ARMS" etc. Terms such as ARMS, PCR, and NGS need to be defined at the start, but abbreviations can be used after that. Please correct that. Otherwise, the manuscript is much improved and acceptable for publication. [Note: HTML markup is below. Please do not edit.] [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.0248775.r003
Revision 2
18 Feb 2021 Author Response Dear Editor, Thank you very much for your decision letter and advice on our manuscript entitled “The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: a systematic review and meta-analysis” (PONE-D-20-31082R1) to be considered for publication in PLOS ONE. Please see our response to your comments below. 1. The authors misinterpreted the editor's suggestion to "include the full names, not just the abbreviations such as for ARMS" etc. Terms such as ARMS, PCR, and NGS need to be defined at the start, but abbreviations can be used after that. Please correct that. Otherwise, the manuscript is much improved and acceptable for publication. Response: Very sorry for the misinterpretation and troubles made. Following your suggestions, we have changed the full names of ARMS, PCR, NGS, and BEAMing into abbreviations after defining them at first use. Please find the corrections in the Revised Manuscript with Track Changes. Look forward to hearing from you soon. Sincerely yours, Peng Ye Chengdu University, Chengdu 610106, P.R.China Tel: (86)18602885572 E-mail: yepeng@cdu.edu.cn https://doi.org/10.1371/journal.pone.0248775.r004
5 Mar 2021 Decision Letter - Anthony F. Shields, Editor The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: a systematic review and meta-analysis PONE-D-20-31082R2 Dear Dr. Ye, 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, Anthony F. Shields, M.D., Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248775.r005
Formally Accepted
17 Mar 2021 Acceptance Letter - Anthony F. Shields, Editor PONE-D-20-31082R2 The diagnostic accuracy of digital PCR, ARMS and NGS for detecting KRAS mutation in cell-free DNA of patients with colorectal cancer: a systematic review and meta-analysis Dear Dr. Ye: 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. Anthony F. Shields Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248775.r006

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