Lung cancer associated autoantibody responses are detectable years before clinical presentation

Jared Allen; Graham Healey; Isabel Macdonald

doi:10.1371/journal.pone.0315220

Peer Review History

Original SubmissionApril 17, 2024
17 Apr 2024 Author Response https://doi.org/10.1371/journal.pone.0315220.r001
15 Jul 2024 Decision Letter - Afsheen Raza, Editor PONE-D-24-11623Lung Cancer Associated Autoantibody Responses are Detectable Years Before Clinical PresentationPLOS ONE Dear Dr. Allen, 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 manuscript lacks novelty but has the capability to build on the foundation knowledge on early CDT. The reviewers 1 and 2 have identified various limitations of the study that need proper justification from the authors. Kindly review each and every comment of reviewers 1 and 2 and address them accordingly. Please submit your revised manuscript by Aug 29 2024 11:59PM. 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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 Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Re: The manuscript entitled “Lung cancer associated autoantibody responses are detected years before clinical presentation by Allen and co-workers. Certainly, the study is interesting but as EarlyCDT lung test is commercial making the paragraphs nos. 5 and 6 below important. As the authors state it is previously described that autoantibodies appear long before clinical symptoms. It makes it necessary to define the new findings of the paper. 1. The objectives of the paper are unclear. 2. The authors state that previous work has shown that autoantibodies precede clinical presentation of lung cancer. Obviously, the current study confirms earlier findings. The Abstract must define what is new in the article. 3. What is the exact meaning of “clinical presentation” of lung cancer? The start of symptoms? The presentation by the family physician? The day of X-ray suspicion of malignant disease, or the day of the PAD diagnostic. Due to doctor’s delay, definitions of “presentation” may differ substantially thereby yielding different results. 4. Row 29. The sentence ending “with 7.2 years before it can be confirmed by imaging” needs a reference. 5. The EarlyCDT lung test is commercial. Obviously, the test is positive long before the appearance of radiology findings. If the surgeon does not have a visible tumour, it is impossible to operate. Thus, it is necessary to explain the clinical consequences of a positive finding. Frequent bronchoscopies? 6. The laboratory kit is for sale and the current findings make it tenable to consider its use for screening purposes. The authors must highlight and discuss its potentials as a screening tool. Which groups are suitable to screen? The number of smokers necessary to screen before finding a new lung cancer case? How to handle false positive samples? 7. The last sentence of the Discussion must be rephrased. 8. Obviously, the samples were run in 2016. For what reason is the publication delayed for almost 8 years? Reviewer #2: I recommend rejecting this article for the following reasons: - The sample size is low and lacks diversity, including only female participants from a single age category. This limitation impacts the generalization of the study's findings. - Several studies showed that autoantibodies could be detected up to 5 years before lung cancer is diagnosed. The presented data is not considered as a novelty. - The statistical methods used for sensitivity and specificity calculation are not correct. - Tables 5, 6, and 7 are presented without adequate explanation. - The authors did not mention the inclusion and exclusion criteria for the participants. In addition, there is no information on whether the patients were screened for the presence of autoimmune diseases. - The discussion is poorly written. Adding other recent articles to support and compare the authors' findings would improve the discussion. - The authors did not validate their funding in another independent dataset. Reviewer #3: Overall a very good study indeed. Registry studies are important to generate evidences and for a long time they are being used in the HDI countries, it would be great if we consider this type of studies in LMICs too to make it more global and inclusive. ****** 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: Sarra Mestiri Reviewer #3: Yes: Suvro Sankha Datta ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. 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Revision 1
13 Sep 2024 Author Response Reviewer #1: Re: The manuscript entitled “Lung cancer associated autoantibody responses are detected years before clinical presentation by Allen and co-workers. Certainly, the study is interesting but as EarlyCDT lung test is commercial making the paragraphs nos. 5 and 6 below important. As the authors state it is previously described that autoantibodies appear long before clinical symptoms. It makes it necessary to define the new findings of the paper. 1. The objectives of the paper are unclear. Response: We appreciate the reviewers feedback, and have added to both the abstract and introduction to try and clarify the study objectives. 2. The authors state that previous work has shown that autoantibodies precede clinical presentation of lung cancer. Obviously, the current study confirms earlier findings. The Abstract must define what is new in the article. Response: While there has been prior research showing elevated autoantibodies preceding cancer diagnoses, this is the first study to show that these autoantibody levels are detectable on a validated commercially available panel using commercially validated cutoff thresholds (against a clinical population of over 1600 subjects), we have now highlighted this novelty in the manuscript. Additionally, the previous studies were of lower sample size, being 49 cases and 54 controls (from an asbestosis population) in the case of Li et al., and 33 cases matched to 45 controls in the Lu et al. study, compared to the cohort of 142 cases and 142 controls explored in this study. 3. What is the exact meaning of “clinical presentation” of lung cancer? The start of symptoms? The presentation by the family physician? The day of X-ray suspicion of malignant disease, or the day of the PAD diagnostic. Due to doctor’s delay, definitions of “presentation” may differ substantially thereby yielding different results. Response: Clinical presentation in this case referred to the date at which cancer diagnosis has been recorded in national cancer registries after histologic, cytologic, or clinical investigation had confirmed the presence of cancer. Manuscript wording has been updated to clarify this. 4. Row 29. The sentence ending “with 7.2 years before it can be confirmed by imaging” needs a reference. Response: The sentence ending “with 7.2 years before it can be confirmed by imaging” is based on mathematical extrapolation of the previously referenced minimum tumour size for imaging detection of 100,000 cells (Fischer et al., 2006) which requires a tumour to double 16.6 times, and the mean doubling time for a malignant lung cancer of 158 days (Kanashiki et al., 2012), this then gives a time of 2622.8 days (16.6 multiplied by 158), or 7.2 years, for a tumour to grow to 100,000 cells. 5. The EarlyCDT lung test is commercial. Obviously, the test is positive long before the appearance of radiology findings. If the surgeon does not have a visible tumour, it is impossible to operate. Thus, it is necessary to explain the clinical consequences of a positive finding. Frequent bronchoscopies? Response: There is indeed a potential issue with an autoantibody testing indicating a malignancy prior to imaging detection. Health economic analysis of results from the prospective phase IV ECLS trial has however shown that enhanced CT screening 6 monthly for 2 years in the case of a positive EarlyCDT test is cost effective, and would allow for the detection of tumours at the earliest point that they become visible. This detail has now been added to the discussion section of the manuscript, along with reference to the Health Economics analysis that has been published since the submission of this manuscript. 6. The laboratory kit is for sale and the current findings make it tenable to consider its use for screening purposes. The authors must highlight and discuss its potentials as a screening tool. Which groups are suitable to screen? The number of smokers necessary to screen before finding a new lung cancer case? How to handle false positive samples? Response: The utility of the EarlyCDT test for screening is examined and discussed in greater depth in the reported results of the ECLS trial, a phase IV prospective trial which recruited high-risk patients, the most recent follow up data on this study has since been released as a preprint on medRxiv (https://www.medrxiv.org/content/10.1101/2024.06.13.24308919v1.full). Additionally details of the intended use populations for both screening and IPN risk stratification are detailed in the EarlyCDT Lung IFU. Greater detail of the ECLS trial and the use of the EarlyCDT test as a screening tool have been added to the discussion, although the principal aim of the reported study was to establish whether autoantibody responses preceded clinical presentation of lung cancer, and if so by how long. 7. The last sentence of the Discussion must be rephrased. Response: While we are unsure as to the rationale behind the request to rephrase the last sentence, we have rephrased. 8. Obviously, the samples were run in 2016. For what reason is the publication delayed for almost 8 years? Response: Freenome Ltd. (Formerly Oncimmune Ltd.) is a relatively small group, and unfortunately commercial priorities frequently impose upon our research goals, in the time since the data was initially made available to us we have undergone various commercial leadership changes, endured a global pandemic, gone through a period of restructure, and been purchased. None of these, however, impact upon the importance of the findings described in this study. Reviewer #2: I recommend rejecting this article for the following reasons: - The sample size is low and lacks diversity, including only female participants from a single age category. This limitation impacts the generalization of the study's findings. Response: These limitations are acknowledged and discussed in the paper, and are a consequence of the difficulty and expense associated with sourcing adequate numbers of longitudinal samples in diseases with low prevalence. The sample size of 142 cases and 142 controls is much larger than the previously published work by Li et al. (49 cases and 54 controls – predominantly male from an asbestosis population) and Lu et al. (33 female breast cancer cases matched to 45 female controls), while the lack of male participants is unfortunate, establishment of diagnostic cutoff thresholds for the EarlyCDT test was completed on age and sex matched cohorts comprised of males and females, therefore we have no reason to expect the results reported here would differ in male subjects. The age range (50-75 year olds) represents a population with a higher risk of developing lung cancer, and is reflective of the intended use population of the EarlyCDT test (https://earlycdt.com/assets/ifus/IFU_EN_ECDTL2_Instructions%20for%20Use%20v14.pdf). The authors felt that in spite of the limitations inherent in the studied dataset, these findings were more robust than previously reported data, and the findings are of importance in demonstrating that elevated autoantibodies are present and measurable years before clinical presentation of lung cancer, and this validates the potential of autoantibody screening tests to identify cancer at the earliest stages when prognosis is vastly improved. - Several studies showed that autoantibodies could be detected up to 5 years before lung cancer is diagnosed. The presented data is not considered as a novelty. Response: We have now clarified the novelty of these findings in the manuscript, in that that this is the first time prediagnostic autoantibodies have been reported using a clinically validated autoantibody panel, using samples obtained from a prospective clinical trial. The reported results also used a larger cohort than previous studies, and sample positivity was determined using commercially used cutoffs defined during clinical validation studies, and not based on comparison of cases and controls within this study. This adds to the robustness of the reported results as no cutoff training was performed within the analysis. - The statistical methods used for sensitivity and specificity calculation are not correct. Response: The authors are unsure as to what statistical methods the reviewer is referring, sensitivity and specificity calculations are based upon standard contingency table calculations (detailed by Altman DG and Bland JM, Statistics Notes: Diagnostic tests 1: sensitivity and specificity. BMJ 1994;308:1552) and the numbers presented in Table 2, whereby: Sensitivity = TP / (TP+FN) = 37 / (37+105) = 37 / 142 = 26.1% Specificity = TN / (TN+FP) = 126 / (126 + 16) = 126 / 142 = 88.7% - Tables 5, 6, and 7 are presented without adequate explanation. Response: Explanation for tables 5, 6, and 7 was previously included in the patient cohort section and discussion, the authors have added contextual explanation to the results section as a preface to the tables. - The authors did not mention the inclusion and exclusion criteria for the participants. In addition, there is no information on whether the patients were screened for the presence of autoimmune diseases. Response: The manuscript states that the source of the samples was the UKCTOCS Study, eligibility and exclusion criteria for which are detailed in the Jacobs et al. paper referenced (Eligibility criteria were 50–74 years of age - aligning with the EarlyCDT intended use population - and postmenopausal status. Exclusion criteria were self-reported previous bilateral oophorectomy or ovarian malignancy, increased risk of familial ovarian cancer, or active non-ovarian malignancy). Smoking status was not part of the inclusion criteria, however only 20% of the cohort reported as never smokers. No screening was undertaken for autoimmune diseases, and autoimmune disease is not currently an exclusion criteria for the EarlyCDT Lung test. - The discussion is poorly written. Adding other recent articles to support and compare the authors' findings would improve the discussion. Response: The manuscript underwent review both externally and internally during it’s finalization, and no concerns were previously raised about the quality of the writing. Studies into prediagnostic cancer biomarkers, especially autoimmune based biomarkers, require large cohorts due to the low disease incidence, with follow up of patients over several years, this makes these studies logistically difficult, extremely expensive, and as a result extremely uncommon, and recent articles that could be used to support or contrast our findings do not exist. - The authors did not validate their funding in another independent dataset. Response: Again, this speaks to the sparsity of similar studies due to the logistical difficulty and expense associated with collecting adequate sample numbers for such longitudinal analyses in low population prevalence disease. As we mentioned before, clinical validation of the EarlyCDT test was performed independently of this cohort using a total of 1153 lung cancer cases and 937 normal controls, confirmed in eight separate patient groups totaling 451 lung cancer patients and 8831 normal subjects as well as an audit cohort of 1613 subjects (https://earlycdt.com/assets/ifus/IFU_EN_ECDTL2_Instructions%20for%20Use%20v14.pdf, Lam et al. 2011, Chapman et al. 2012, Jett et al. 2014), lending to the robustness of this data. Should an independent longitudinal dataset become available we would be extremely interested in validating these results. Reviewer #3: Overall a very good study indeed. Registry studies are important to generate evidences and for a long time they are being used in the HDI countries, it would be great if we consider this type of studies in LMICs too to make it more global and inclusive. Response: We agree that further prospective longitudinal blood collection and disease monitoring studies would be beneficial in LMICs, and would be happy to collaborate with groups undertaking such studies. Attachments Attachment Submitted filename: Reviewer Responses 2024-08-27.docx https://doi.org/10.1371/journal.pone.0315220.r003
22 Nov 2024 Decision Letter - Milad Khorasani, Editor Lung Cancer Associated Autoantibody Responses are Detectable Years Before Clinical Presentation PONE-D-24-11623R1 Dear Dr. Allen, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Milad Khorasani, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #3: Appreciate your effort in responding to the comments made by the reviewers. Overall, I found that it is OK. ****** 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: Yes: Petter Järemo MD, PhD Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0315220.r004
Formally Accepted
Acceptance Letter - Milad Khorasani, Editor PONE-D-24-11623R1 PLOS ONE Dear Dr. Allen, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Milad Khorasani Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0315220.r005

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