Detection of EGFR mutations in non-small cell lung cancer by droplet digital PCR

Drew F. K. Williamson; Sean R. N. Marris; Vanesa Rojas-Rudilla; Jacqueline L. Bruce; Cloud P. Paweletz; Geoffrey R. Oxnard; Lynette M. Sholl; Fei Dong

doi:10.1371/journal.pone.0264201

Peer Review History

Original SubmissionJune 29, 2021
31 Aug 2021 Decision Letter - Alvaro Galli, Editor PONE-D-21-21322 Detection of EGFR mutations in non-small cell lung cancer by droplet digital PCR PLOS ONE Dear Dr. Dong, 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 Oct 15 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. 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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: No 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: 1, the study was to generate more data to demonstrate potential value of ddPCR for clinical use in detecting tumor mutations especially with ctDNA. The manuscript is in good writen English. 2, more detailed technical information should be provided for readers to better understanding of the potential value and limitation of the ddPCR assays, including 1) brief procedure of the assays; 2) cfDNA/plasma input for the clinical validation; 3) key experimental and performance parameters of the reference NGS-panel assay, such as LOD, sensitivity, specificity. 3, there is a significant difference in terms of technical sensitivity or LOD between the ddPCR assay for L858R and that for E19 deletions, which needs an explanation or interpretation. 4, in the false positive tests, a few reactions reported single false positive event for L858R, and no false posive for E19 deletions, but the positive calling cutoff were set to be 3 positive droplets, which needs an explanation of rational. Considering the value of such assays to to detect ctDNA mutations which is at very limited concentration, maximization of clinical sensitivity with good control of specificity is important. 2 positive droplets from a single reaction or even duplicated reactions might be better. 5, there is a significant difference in terms of analytical and clinical sensitivity between the ddPCR assays and that reported previously such as ref. 15. This should be discussed with sufficiently deepth so that the readers can understand the reasons behind. 6, considering the broad application of NGS for ctDNA in clinic, it would be more informative if there is a head-to-head comparison between the ddPCR assays and a NGS assay for ctDNA detection. Reviewer #2: The manuscript is aimed at validating a dual channel ddPCR system for EGFR L858R and exon 19 deletion detection in plasma and FFPE tissue. Despite the analysis have been conducted in a very rigorous way, the number of mutant samples analysed on FFPE is very low (6 out of 20) and the advantages in terms of specificity and turn around time of a ddPCR are well known. Considering the low sensitivity (only 52%) using plasma samples, I would suggest the authors to accurately consider/discuss the clinical characteristics of patients, since this is one of the major challenge of liquid biopsy. Patients should try to set up their assay on an homogeneous population, to cut out any possible analytical bias to confirm their assay works. ****** 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: Yes:** Guanshan Zhu 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.0264201.r001
Revision 1
28 Jan 2022 Author Response Dear reviewers, Thank you for taking time to review our article and for your helpful comments. To comply with PLOS data availability guidelines, we have provided the minimal data set for all study results in a Supplemental File. Please see the full response to each point of your suggestions below: Reviewer #1: 1, the study was to generate more data to demonstrate potential value of ddPCR for clinical use in detecting tumor mutations especially with ctDNA. The manuscript is in good writen English. Thank you for your comment. 2, more detailed technical information should be provided for readers to better understanding of the potential value and limitation of the ddPCR assays, including 1) brief procedure of the assays; 2) cfDNA/plasma input for the clinical validation; 3) key experimental and performance parameters of the reference NGS-panel assay, such as LOD, sensitivity, specificity. We have updated methods to be more comprehensive. We have expanded the methods section to include more information from our protocol. We have specified the plasma input for cfDNA analysis. The method section for DNA isolation and ddPCR now reads as follows: DNA Isolation DNA was isolated from formalin-fixed, paraffin-embedded tumor tissue. Tumor-enriched areas were macrodissected from ten 4-μm sections. DNA was isolated using QIAamp DNA mini kit (Qiagen; Hilden, Germany) and quantified using Qubit-based dsDNA detection (Qubit 3.0, Life Technologies; Carlsbad, CA). Cell-free genomic DNA (cfDNA) was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit and then quantified using Qubit-based dsDNA detection. DNA was then diluted to a maximum concentration of 3 ng/μL. Droplet Digital PCR DNA was mixed with commercially available master mix (Bio-Rad; Hercules, CA), PCR primers and fluorescently labeled probes (Applied Biosystems; Foster City, CA) for detection of the respective sequences (Table 1). A total volume of 20 μL was used in each reaction, comprised of 12.5 μL master mix, 0.625 μL of 40X Taqman probes, and 6.875 μL of RNase/DNase-free water, and 5 μL of sample DNA or control. For clinical testing, control samples were included with each run: water, a wild type control, as well as low (2% mutant DNA) and high (20% mutant DNA) controls for each mutation. Aqueous droplets containing individual PCR templates were generated in a water-oil emulsion-based droplet generator using 70 μL of oil per reaction. Using 50 μL of the resulting droplets, PCR for 40 cycles of alternating 94 °C and 58 °C was performed for detection of the respective sequences. These droplets were analyzed by flow cytometry (Bio-Rad QX200 ddPCR System), which detected probe-specific fluorescent signals. Flow cytometry event counts were used to detect wild type and mutant signals and quantify variant allele fraction. We have added more details about the NGS panel assay validation, including LOD, sensitivity, and specificity. The following text has been added to the manuscript: On validation, OncoPanel demonstrated 97.8% sensitivity and 100% specificity for single nucleotide variants and 100% sensitivity and 100% specificity for insertion and deletion variants excluding FLT3 and NPM1 variants. The limit of detection was set at 10% variant allele fraction for regions of at least 50x read depth, achieving 98.4% concordance in triplicate runs for variants that meet these criteria.[14] 3, there is a significant difference in terms of technical sensitivity or LOD between the ddPCR assay for L858R and that for E19 deletions, which needs an explanation or interpretation. Thank you for your question. The difference in technical sensitivity/LOD between the L858R and E19 deletion assays is due to assay design (Figure 1 and Figure 2) and has to do with expected results as DNA concentration increases beyond limiting dilution. In the L858R assay, a droplet containing both wild type and mutant alleles will be double-positive for VIC and FAM and will contribute to the calculation for both wild type and mutant alleles. In the E19del assay, a droplet containing both wild type and mutant alleles will also be double-positive for VIC and FAM and will be indistinguishable from wild type only droplets. The end effect is that for the E19del assay, the analytical sensitivity decreases at high loading concentrations and is evident in the experimental data in Table 3. This concept is explored in the section "Calculated Effect of Loading Concentration on Mutation Detection", including in Figure 3. We have added the following text to the relevant section ("DNA Quantification and Limit of Detection") to help clarify this for the reader: The observed difference in limit of detection between the L858R and exon 19 deletion assays was expected based on assay design principles. The analytical sensitivity decreased for the exon 19 deletion reaction at the highest DNA concentration (see "Calculated Effect of Loading Concentration on Mutation Detection"). 4, in the false positive tests, a few reactions reported single false positive event for L858R, and no false posive for E19 deletions, but the positive calling cutoff were set to be 3 positive droplets, which needs an explanation of rational. Considering the value of such assays to to detect ctDNA mutations which is at very limited concentration, maximization of clinical sensitivity with good control of specificity is important. 2 positive droplets from a single reaction or even duplicated reactions might be better. This is an interesting question about how to set thresholds for reporting based on data in analytical validation. For the L858R assay, the overall per-event false positive rate is very low: 5/145019 = 0.003% of any droplet may show a false positive result. However, this risk is cumulative over 15,000 droplets per reaction. The expected number of false positive events is 15,000 x 0.003% = 0.517. This is consistent with the observed data, where 5/10 L858R reactions showed one false positive event each. This data can be further modeled by a Poisson distribution with λ = 0.517. The probability of two or more positive event in a single reaction is 0.0955, and the probability of two replicates each having two or more positive events is 0.0955 x 0.0955 = 0.00911 (0.911%). A false positive rate of almost 1% is not acceptable for a clinical assay. Changing the threshold from 2 to 3 events decreases the probability of false positive events (≥3) from a single reaction to 0.0157 and false positive in duplicate reactions to 0.000247 (0.02%). For the exon 19 deletion assay, there were no false positives observed under experimental conditions. Applying a statistical rule of 3 (if an event does not occur in n observations, then the 95% confidence interval can be approximated by 3/n), changing the threshold from 2 to 3 events will decrease the false positive 95% confidence interval from 0.00304 (0.3%) to <0.001%. You can rationalize setting the exon 19 threshold lower, but we have decided to keep it at 3 in our lab for convenience in clinical interpretation. There is value is having a single threshold for both assays to minimize risk of misinterpretation in a laboratory with many molecular pathologists rotating on service. We have added the following line to the manuscript: With a positive threshold set at 3 events in duplicate reactions, the expected false positive rate in EGFR L858R analysis was 0.02%. 5, there is a significant difference in terms of analytical and clinical sensitivity between the ddPCR assays and that reported previously such as ref. 15. This should be discussed with sufficiently deepth so that the readers can understand the reasons behind. We performed additional analysis, reporting the demographic and clinical characteristics of patients in our cohort. These are addressed in detail in the response to reviewer #2. We noted that most patients in our cohort had been pretreated. Sensitivity among patients with Stage IV disease was 59% and for patients who had not received prior therapy was 71%. During our reanalysis, we identified a data entry error. The final sensitivity for plasma ddPCR was 49/92 = 53%. It was erroneously reported as 48/93 in the prior version. All cases are shown in the minimal data set. 6, considering the broad application of NGS for ctDNA in clinic, it would be more informative if there is a head-to-head comparison between the ddPCR assays and a NGS assay for ctDNA detection. We agree that a direct comparison between ddPCR assay and NGS assay for ctDNA detection would be informative. Unfortunately, our lab has not optimized our NGS assay to the depth of sequencing required for ctDNA detection, and this topic is beyond the scope of the current report. Reviewer #2: The manuscript is aimed at validating a dual channel ddPCR system for EGFR L858R and exon 19 deletion detection in plasma and FFPE tissue. Despite the analysis have been conducted in a very rigorous way, the number of mutant samples analysed on FFPE is very low (6 out of 20) and the advantages in terms of specificity and turn around time of a ddPCR are well known. Thank you for your comment. Considering the low sensitivity (only 52%) using plasma samples, I would suggest the authors to accurately consider/discuss the clinical characteristics of patients, since this is one of the major challenge of liquid biopsy. Patients should try to set up their assay on an homogeneous population, to cut out any possible analytical bias to confirm their assay works. We have reviewed the clinical characteristics of our cohort and reexamined each case. During our reanalysis, we identified a data entry error. The final sensitivity for plasma ddPCR was 49/92 = 53%. The clinical characteristics of the patients are included in the updated results and provided here: In this cohort of patients, the median age was 64 years (range 31 to 97 years) with 64 (70%) female patients. New diagnoses accounted for 28 of 92 (30%) patients with EGFR-mutated cancers with 82 (89%) being at stage IV and 89 (97%) being at stage III or IV. Most patients had received some form of treatment prior to their first plasma ddPCR assay, with 52 (57%) receiving a tyrosine kinase inhibitor, 21 (23%) receiving chemotherapy, and 36 (39%) receiving localized therapy to their primary tumor either in the form of surgery (20, 22%) or radiotherapy (16, 17%). Only 28 (30%) had received no pre-testing therapy for their primary tumor at all. Sensitivity among patients with Stage IV disease was 59%, while for patients with Stage I-II disease, it was only 17%. Sensitivity also varied by treatment received, with sensitivity for patients receiving no treatment being 71%, for those receiving a tyrosine kinase inhibitor being 44%, and for those receiving any systemic therapy (tyrosine kinase inhibitor, chemotherapy, or checkpoint inhibitor) being 37%. We have also updated the discussion: Our cohort has a slightly lower proportion of stage IV patients than most of those previously published. Additionally, more than half of our patients (56%) had already been treated with one or more tyrosine kinase inhibitors at the time of testing which may reduce the ability of our assay to detect mutations, as evidenced by the markedly decreased sensitivity of our assay for patients who has received systemic therapy (37%) versus those who had not received any treatment at all (71%). Thank you for taking time to review our manuscript. We appreciate your input in improving the article. Sincerely, Fei Dong, M.D. Brigham and Women's Hospital Boston, MA Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0264201.r002
7 Feb 2022 Decision Letter - Alvaro Galli, Editor Detection of EGFR mutations in non-small cell lung cancer by droplet digital PCR PONE-D-21-21322R1 Dear Dr. Dong, 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, Alvaro Galli 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 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: Yes 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 #1: Yes 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 #1: All my previous comments have been well addressed. I have no further comments and am happy to see the manuscript to be accepted for publication on the journal. Reviewer #2: (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 Reviewer #2: No https://doi.org/10.1371/journal.pone.0264201.r003
Formally Accepted
15 Feb 2022 Acceptance Letter - Alvaro Galli, Editor PONE-D-21-21322R1 Detection of EGFR mutations in non-small cell lung cancer by droplet digital PCR Dear Dr. Dong: 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. Alvaro Galli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0264201.r004

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