Prostate-specific membrane antigen in circulating tumor cells is a new poor prognostic marker for castration-resistant prostate cancer

Naoya Nagaya; Masayoshi Nagata; Yan Lu; Mayuko Kanayama; Qi Hou; Zen-u Hotta; Toshiyuki China; Kosuke Kitamura; Kazuhito Matsushita; Shuji Isotani; Satoru Muto; Yoshiro Sakamoto; Shigeo Horie

doi:10.1371/journal.pone.0226219

Peer Review History

Original SubmissionAugust 8, 2019
7 Oct 2019 Decision Letter - Isaac Yi Kim, Editor PONE-D-19-22397 Prostate-specific membrane antigen in circulating tumor cells is a new poor prognostic marker for castration-resistant prostate cancer PLOS ONE Dear Prof Horie, 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. We would appreciate receiving your revised manuscript by Nov 21 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: 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: his manuscript is studies on PSMA expression in CTCs as a poor prognostic marker for CRPC. Overall, this paper is well written. However, I have only several minor comments. Minor comments: 1) Please indicate the number of patients instead of the number of samples to the abstract part. Please indicate the number of patients instead of the number of CTC expression samples to the abstract. Line 9 of page 2, 127/203 samples → ?/79 patients 2) Line 9 of page 2, 62% → 63% ? 3) It would be better to add a general description of CTCs to the introduction part. 4) Page 6, Please present PCR conditions and primer sequences for genes (PSMA, PSA, AR or EGFR) other than AR-V7. If possible, I would like to see representative electrophoresis pictures of PCR products. 5) Line 12 of page 9, Thirty-six → Thirty-nine ? 6) Line 14 of page 14, Three of the five → Three of the six ? 7) When some abbreviations are shown in the text and figure at, please clarify the whole term. For example: EP, LH-RH, ALP, LDH, ENZ, ABI, DOC, CBZ, PE Reviewer #2: In this manuscript, Nagaya et al. first report a clinical significance of PSMA expression statuses of circulating tumor cells as a novel marker to predict the prognosis in castration resistant prostate cancer patients. They investigate the sensitivity of PSMA (62%) in 203 CTCs and revealed an inverse correlation with PSA expression and clearly showed that PSMA-positive patients with poor progression-free survival. These results suggest potential of clinical application to detect PSMA in CTCs. Although there are some remaining points before acceptance for publication in PLoS One, I am recommending that the editor request several minor improvements of the manuscript that I am outlining below before acceptance. 1. The description of treatment-switch (TS) cohorts is not adequate. What “Switch” means in this study? Please put a Supplementary Figure to specify and illustrate their time course. 2. Using the method of AdnaTest in this work, is it possible to detect spliced variants of PSMA? 3. I could not access the cBioPortal for Cancer Genomics from the URL described in the manuscript. Could you make sure whether this URL works properly? 4. In Figure 2C, CTC detection is a good predictor of PSA progression-free survival but not of overall survival. Please mention and discuss this discrepancy. 5. Color Keys of CTC Gene Expression in Figure 4 is so complicated. Please make them simpler such as to change to the gradation pattern. 6. In Figure 6, the authors divided all patients equally into two groups by expression level. Is it statistically valid considering the distribution of expression levels? ****** 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0226219.r001
Revision 1
21 Nov 2019 Author Response Dear Prof. Isaac Yi Kim, We would like to thank you for inviting us to submit a revised draft of our manuscript entitled, “Prostate-specific membrane antigen in circulating tumor cells is a new poor prognostic marker for castration-resistant prostate cancer” to PLOS ONE. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted. To facilitate your review of our revisions, the following is a point-by-point response to the questions and comments delivered in your letter. Responses to Reviewer 1’s Comments: Comments to the Author his manuscript is studies on PSMA expression in CTCs as a poor prognostic marker for CRPC. Overall, this paper is well written. However, I have only several minor comments. Response: We thank for carefully reading our manuscript and for giving useful comments. We have revised our manuscript on the basis of your comments. #1 Please indicate the number of patients instead of the number of samples to the abstract part. Please indicate the number of patients instead of the number of CTC expression samples to the abstract. Line 9 of page 2, 127/203 samples → ?/79 patients Response: Thank you for your suggestion. We have revised the text to “CTCs were detected in 55/79 patients and median serum PSA in CTC-positive patients was 67.0 ng/ml.” at line 9-10 on page 2. #2 Line 9 of page 2, 62% → 63% ? Response: Thank you for pointing it out. I corrected that sentence. #3 It would be better to add a general description of CTCs to the introduction part. Response: Thank you for your valuable opinion. I have added the following sentence, “Since tissue biopsy is invasive and cannot be performed for all tumors, liquid biopsy is drawing attention as a new testing option to overcome these shortcomings. Liquid biopsies are the blood-based analyses of non-solid biological tissues such as exosomes, cell-free DNA and circulating tumor cells (CTCs).　CTCs are characterized as cancer cells that intravasate into the circulatory system from primary locations.” in introduction section at line 3-7 on page 3. #4 Page 6, Please present PCR conditions and primer sequences for genes (PSMA, PSA, AR or EGFR) other than AR-V7. If possible, I would like to see representative electrophoresis pictures of PCR products. Response: Thank you for your suggestion. I have added the following sentence, “The AdnaTest PrimerMix ProstateDetect was used for amplification of PSA, PSMA, and EGFR (PCR condition for PSA, PSMA, and EGFR: 95 ℃ for 15 min, 42 cycles of 94 ℃ for 30 sec, 61 ℃ for 30 sec, 72 ℃ for 30 sec, followed by 10 min of extension). The AdnaTest PrimerMix AR-Detect was used for amplification of AR (PCR condition for AR: 95 ℃ for 15 min, 35 cycles of 94 ℃ for 30 sec, 60 ℃ for 30 sec, 72 ℃ for 60 sec, followed by 10 min of extension).” in methods section at line 3-9 on page 6. The representative electrophoresis images are listed below. (The attached file) #5 Line 12 of page 9, Thirty-six → Thirty-nine ? Response: Thank you for pointing it out. I corrected that sentence. #6 Line 14 of page 14, Three of the five → Three of the six ? Response: Thank you for pointing it out. I corrected that sentence. #7 When some abbreviations are shown in the text and figure at, please clarify the whole term. For example: EP, LH-RH, ALP, LDH, ENZ, ABI, DOC, CBZ, PE Response: Thank you for pointing it out. I added original names of each abbreviation. Responses to Reviewer 2’s Comments: Comments to the Author In this manuscript, Nagaya et al. first report a clinical significance of PSMA expression statuses of circulating tumor cells as a novel marker to predict the prognosis in castration resistant prostate cancer patients. They investigate the sensitivity of PSMA (62%) in 203 CTCs and revealed an inverse correlation with PSA expression and clearly showed that PSMA-positive patients with poor progression-free survival. These results suggest potential of clinical application to detect PSMA in CTCs. Although there are some remaining points before acceptance for publication in PLoS One, I am recommending that the editor request several minor improvements of the manuscript that I am outlining below before acceptance. Response: Thank you for your time and effort in reviewing the paper. I would like to revise and answer your comments. #1 The description of treatment-switch (TS) cohorts is not adequate. What “Switch” means in this study? Please put a Supplementary Figure to specify and illustrate their time course. Response: Thank you for your precious opinion. I have added the Supplementary Figure 1 at line 8 on page 4 to illustrate what “all samples analysis” and “treatment-switch (TS) cohorts analysis” mean in this study. #2 Using the method of AdnaTest in this work, is it possible to detect spliced variants of PSMA? Response: You have raised an important question. We think it would be interesting to identify PSMA splicing variants. Although primers for PSMA splice variants are not included in AdnaTest kit itself, given that primers for AR-V7 (splice variant of AR) are custom-ordered, it is possible to perform RT-PCR for gene of interest with any arbitrary primer sets. However, because cDNA synthesized with this procedure was rather scarce, we focused on PSMA, PSA, AR, EGFR and AR-V7 in this study. Meanwhile, to further dissect CTC expression profile, we are currently trying to perform RNA sequencing with mRNA extracted by Adnatest. This method allows for not only detection of PSMA splice variants but also comprehensive expression profiling of CTCs. Thus, it is our future assignment to investigate the important agenda raised by the reviewer. #3 I could not access the cBioPortal for Cancer Genomics from the URL described in the manuscript. Could you make sure whether this URL works properly? Response: Thank you for pointing it out. I corrected that URL. #4 In Figure 2C, CTC detection is a good predictor of PSA progression-free survival but not of overall survival. Please mention and discuss this discrepancy. Response: Thank you for your suggestion. I have added the following sentence, “Although previous reports have shown that CTC-positive patients have significantly shorter overall survival, the presence of CTC was not a good indicator of overall survival in our study. Other reports using AdnaTest define the presence of CTC as the expression of any of PSA, PSMA, EGFR, or AR. Meanwhile, in our study, prostate cancer CTCs were categorized into only two types: PSA + / PSMA + or PSA + / PSMA-. This means that our CTC stratification based on PSMA is more detailed than other inclusive definition of CTC positivity. Thus, a possible explanation for this discrepancy is that CTC stratification based on PSMA better predicts overall survival than CTC positivity alone in a relatively small cohort like this study. at line 2-10 on page 21. #5 Color Keys of CTC Gene Expression in Figure 4 is so complicated. Please make them simpler such as to change to the gradation pattern. Response: Thank you for your suggestion. I made them simpler, using 2 colors. #6 In Figure 6, the authors divided all patients equally into two groups by expression level. Is it statistically valid considering the distribution of expression levels? Response: Thank you for your valuable opinion. Median was used to generate a cutoff point to divide mRNA expressions into positive and negative. This method is often used when analyzing molecules with no established cutoff points for their expression to predict prostate cancer progression. Again, we would like to thank you all for giving us the opportunity to further improve our manuscript with your informative comments and queries. We have worked hard to incorporate your feedbacks and hope that these revisions persuade you to accept our submission. Sincerely, Shigeo Horie Professor and Chairman, Department of Urology Juntendo University, Graduate School of Medicine 2-1-1 Hongo Bunkyo-ku, Tokyo, Japan 1138431 Tel and Fax: +81-3-5802-1227 E-mail: shorie@juntendo.ac.jp Attachments Attachment Submitted filename: A rebuttal letter.docx https://doi.org/10.1371/journal.pone.0226219.r002
22 Nov 2019 Decision Letter - Isaac Yi Kim, Editor Prostate-specific membrane antigen in circulating tumor cells is a new poor prognostic marker for castration-resistant prostate cancer PONE-D-19-22397R1 Dear Dr. Horie, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Isaac Yi Kim, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0226219.r003
Formally Accepted
27 Nov 2019 Acceptance Letter - Isaac Yi Kim, Editor PONE-D-19-22397R1 Prostate-specific membrane antigen in circulating tumor cells is a new poor prognostic marker for castration-resistant prostate cancer Dear Dr. Horie: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Isaac Yi Kim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226219.r004

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