Analysis of the p53 pathway in peripheral blood of retinoblastoma patients; potential biomarkers

Mayra Martínez-Sánchez; Mariana Moctezuma-Dávila; Jesús Hernandez-Monge; Martha Rangel-Charqueño; Vanesa Olivares-Illana

doi:10.1371/journal.pone.0234337

Peer Review History

Original SubmissionMarch 23, 2020
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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: Partly ******** 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: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: 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: In this manuscript, the authors have attempted to monitor the status of the p53 pathway in peripheral blood samples obtained from retinoblastoma patients. They measure the mRNA levels of p53, RB, MDMX and MDM2 which could be further used as biomarkers for early diagnosis. Although it is an interesting premise and could be a good diagnostic approach, I am not able to understand the clear rationale behind utilizing the blood samples of RB patients to analyze the p53 pathway. The authors are requested to elaborate more on this. Moreover, other signaling pathways like the Wnt signaling is also highly functional in RB and could have higher significance as compared to p53. Have the authors looked into these pathways (mRNA analysis of beta-catenin etc)? Depending on the severity of RB, as mentioned, it could be unilateral or bilateral (simultaneously in both eyes) or in cases, it could start off as unilateral and at a later stage it could become bilateral. How could the presented approach address this issue? Also, peripheral blood would have circulating tumor DNA that could be coming from tumors that have metastasized from the retina to other parts of the body? In that case, would the diagnosis mentioned here delineate between the original vs metastasized tumor that could have a totally different p53 signature? The authors are recommended to discuss these in detail. Optional: This may or may not be addressed. What about the more rarer cases of trilateral RB? Have the authors looked into the p53 signaling in these cases? If not, it would be good to include a few points about the same if possible. Minor Changes: 1. Introduction...Line 1...Change "Paediatric" to "Pediatric". 2. The grammar needs to be formatted. There are lots of places where the sentences need corrections e.g. "it has been used to search of early biomarkers (Introduction)...should be changed to "it has been used to search or identify early biomarkers". 3. Proteasome 26 S needs to be written without any space. 4. I do not see the significance of writing about the housekeeping genes in detail. It could be just mentioned in a sentence or two. 5. The authors are requested to include these references: a.https://www.ncbi.nlm.nih.gov/pubmed/16231976 b.https://www.ncbi.nlm.nih.gov/pubmed/15190215 Reviewer #2: In this manuscript authors have analyzed peripheral blood samples from patients suffering from retinoblastoma for mRNA levels of RB, p53, MDM2, MDMX and investigated the involvement of these genes in the development of cancer. There were some minor issues with the manuscript grammar and use of appropriate scientific language. The authors need to provide complete profiling of the RB and p53 genes in the samples through sequencing and the status of mutations in them. The authors also show the comparison in the gene levels between patients, controls and healthy family in the same graph. While the analysis shows some significant correlations between the p53, MDM2 and RB mRNA levels in patients and controls, some of the associations are not novel like that between p53 and MDM2. The sample size and the scope of analysis performed in this manuscript is not sufficient enough at this point to prove the use of these gene signatures as potential biomarkers. ****** 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. 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Revision 1
7 May 2020 Author Response Reviewer #1: In this manuscript, the authors have attempted to monitor the status of the p53 pathway in peripheral blood samples obtained from retinoblastoma patients. They measure the mRNA levels of p53, RB, MDMX and MDM2 which could be further used as biomarkers for early diagnosis. Although it is an interesting premise and could be a good diagnostic approach, I am not able to understand the clear rationale behind utilizing the blood samples of RB patients to analyze the p53 pathway. The authors are requested to elaborate more on this. It has been shown that the p53 pathway is, indeed altered in retinoblastoma tumours, if this could be one of the causes of the diseases as has been proposed for several authors, then we thought that it could have a hereditary component, meaning that could be studied in any cell of the body. That was also the motivation to study the family members. We add more about it in page 4 and page 14 on the MS. Moreover, other signaling pathways like the Wnt signaling is also highly functional in RB and could have higher significance as compared to p53. Have the authors looked into these pathways (mRNA analysis of beta-catenin etc)? The reviewer is right. Other pathways indeed could also be potential biomarkers for retinoblastoma. Has recently been published a review paper about it: Jie Sun , Hui-Yu Xi1,2, Qing Shao and Qing-Huai Liu. Biomarkers in retinoblastoma. Int J Ophthalmol, Vol. 13, No. 2, Feb.18, 2020. In the paper, the authors revised the potential biomarkers for retinoblastoma and both Wnt and p53 pathways, among many others, are mentioned. In this particular work, we focus on p53. Depending on the severity of RB, as mentioned, it could be unilateral or bilateral (simultaneously in both eyes) or in cases, it could start off as unilateral and at a later stage it could become bilateral. How could the presented approach address this issue? This is an interesting question that we have been thinking about. We thought that a hereditary component of the p53 pathways is present. Then we could imagine that the bilateral cases would present higher significant differences in the levels of p53, MDMX or MDM2, more than the unilateral. However, this is difficult to demonstrate since, as the reviewer explains, a patient that at the moment of the study is unilateral, can later become bilateral. However, we found a correlation between the age of diagnosis and the levels of expression of p53 and MDMX m mRNA, and as has been postulated that the age of diagnosis in correlated with the laterality. Also, peripheral blood would have circulating tumor DNA that could be coming from tumors that have metastasized from the retina to other parts of the body? In that case, would the diagnosis mentioned here delineate between the original vs metastasized tumor that could have a totally different p53 signature? The authors are recommended to discuss these in detail. Of course, this is true, and it was our omission because we did not explain the inclusion/exclusion criteria in the MS. Now we add the following information on page 5 of the MS: Inclusion criteria: Children with the diagnosis of retinoblastoma, without a known diagnosis of other cancer. Exclusion criteria: Patients with a diagnosis of other types of cancer. Controls: Healthy children at pediatric age and healthy adults in the same range of age of the family member patients. On the other hand non of the participating patient's in the study developed metastasis. Now we add a table with the characteristics of the patients in the MS. Optional: This may or may not be addressed. What about the more rarer cases of trilateral RB? Have the authors looked into the p53 signaling in these cases? If not, it would be good to include a few points about the same if possible. This is a good suggestion; unfortunately we were unable to obtain the sample of a patient with these characteristics. Minor Changes: 1. Introduction...Line 1...Change "Paediatric" to "Pediatric". Done 2. The grammar needs to be formatted. There are lots of places where the sentences need corrections e.g. "it has been used to search of early biomarkers (Introduction)...should be changed to "it has been used to search or identify early biomarkers". Thank you, the correction is done and the MS has been carefully formatted 3. Proteasome 26 S needs to be written without any space. Done 4. I do not see the significance of writing about the housekeeping genes in detail. It could be just mentioned in a sentence or two. We reduced this part, and sent the Figure 1 (about the housekeeping genes) to Supplemental information. 5. The authors are requested to include these references: a.https://www.ncbi.nlm.nih.gov/pubmed/16231976 b.https://www.ncbi.nlm.nih.gov/pubmed/15190215 Thank you for the suggestions, we added them! Reviewer #2: In this manuscript authors have analyzed peripheral blood samples from patients suffering from retinoblastoma for mRNA levels of RB, p53, MDM2, MDMX and investigated the involvement of these genes in the development of cancer. There were some minor issues with the manuscript grammar and use of appropriate scientific language. Done The authors need to provide complete profiling of the RB and p53 genes in the samples through sequencing and the status of mutations in them. It is a good idea to get the mutation spectrum of RB and p53 in the patients. However, this is not the focus of the present work. In this manuscript we focus on the analysis of the levels of mRNA of the p53 pathways genes and RB. We will consider studying the mutation status of these genes for a future work. The authors also show the comparison in the gene levels between patients, controls and healthy family in the same graph. While the analysis shows some significant correlations between the p53, MDM2 and RB mRNA levels in patients and controls, some of the associations are not novel like that between p53 and MDM2. The reviewer is right, the correlation between p53 and MDM2 it has been very well documented. Our results corroborates that in blood samples of retinoblastoma patients the correlation do not changes due to the disease. The sample size and the scope of analysis performed in this manuscript is not sufficient enough at this point to prove the use of these gene signatures as potential biomarkers. We agreed; however, we hope that our work will stimulate others too and together, we can finally found a genes signature for this childhood cancer. Attachments Attachment Submitted filename: Comments to the Author.docx https://doi.org/10.1371/journal.pone.0234337.r002
26 May 2020 Decision Letter - Sumitra Deb, Editor Analysis of the p53 pathway in peripheral blood of retinoblastoma patients; potential biomarkers PONE-D-20-08362R1 Dear Dr. Olivares-Ilana, 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. 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With kind regards, Sumitra Deb, 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 #2: (No Response) ******** 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: The authors have addressed all the questions in the previous review. However, the figures seem to be pixelated. The authors are requested to check that. 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.0234337.r003
Formally Accepted
28 May 2020 Acceptance Letter - Sumitra Deb, Editor PONE-D-20-08362R1 Analysis of the p53 pathway in peripheral blood of retinoblastoma patients; potential biomarkers Dear Dr. Olivares-Illana: 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. Sumitra Deb Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0234337.r004

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