GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States

Igor Vidal; Qizhi Zheng; Jessica L. Hicks; Jiayu Chen; Elizabeth A. Platz; Bruce J. Trock; Ibrahim Kulac; Javier A. Baena-Del Valle; Karen S. Sfanos; Sarah Ernst; Tracy Jones; Janielle P. Maynard; Stephanie A. Glavaris; William G. Nelson; Srinivasan Yegnasubramanian; Angelo M. De Marzo

doi:10.1371/journal.pone.0241934

Peer Review History

Original SubmissionNovember 23, 2020
22 Mar 2021 Decision Letter - Craig N Robson, Editor PONE-D-20-36882 GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States PLOS ONE Dear Dr. De Marzo, 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 May 03 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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Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests Additional Editor Comments: The reviewers have requested several amendments to the manuscript and to include checking the grammar and spellings. Any information on the methylation status of GSTP1 gene would be helpful to include and a suggestion was made to include African American cell lines for an in vitro model as important to conclude this study. Please could you ensure that all the points raised by both reviewers have been fully addressed - this manuscript will be looked at again by the original reviewers. [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: 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper entitled "GSTP1 positive prostatic adenocarcinomas are more common in Black than Whitemen in the United States" is interesting. However, the authors need to check and present data a little more seriously. There are many typos in the Manuscript, such as "GSPT1" as "GSP1" or White men as Whte men, etc. A little serious presentation of their findings will significantly increase the quality of the paper. I have the following comments Comments: 1. The term "Black" and "Whitemen" are not preferable in scientific journals. I suggest the authors use the term African American and Caucasian in the "Title" and the Manuscript's main body. 2. Expand the term "TMA" in the abstract section. 3. Recently another publication observed that BMI1 expressed prominently among African Americans (PMID=30087142). How do the authors compare their findings with this publication? 4. The data on the methylation status of the GSTP1 gene will surely make the paper more valuable for the readers. Authors may include this data. 5. The number of patients between black and white patients is significantly different. Do the authors think that their difference can influence the final result of their observation? 6. Figure 2: Authors should include one or two cell lines representative of the black population. As the study emphasizes the black population, this population in vitro model is a must for this figure. 7. Figure 3: Authors must explain the background of the "representative case" in the legend. Is it from black or white patients"? Reviewer #2: In this manuscript the authors have analyzed a large collection of prostate adenocarcinoma tissue specimen for the expression of the Glutathione-S-transferase family member GSTP1 by immunohistochemistry. Technically the analysis of the tissue micro arrays is well-performed and statistical analysis adequate. A significant higher number of GSTP1+ cases among Black men compared to White was found. However, while this finding is new and interesting, large parts of this descriptive-only study confirm already published data for GSTP1+ expression in a subset of prostate cancer patients. The authors speculate whether GSTP1 positive cancer might be a distinct molecular subtype of this disease. Molecular subtypes should be defined by genomic aberrations and/or gene expression signatures, expression of a single gene (GSTP1) is – in my opinion – not sufficient to define a molecular subtype. In addition, GSTP1+ cases were found in ERG+ and ERG- cases representing two recognized molecular subtypes of prostate cancer. To get more robust data for the definition of a molecular subtype a gene expression study could be performed on the “recent case” TMA used for RNA in situ hybridization. This would give a first indication whether GSTP1+ cases have a different gene expression signature compared to GSTP1- cases. Minor points: 1) The GSTP1 antibody was well-controlled prior staining the TMAs. However, results of stainings in the cell lines and knock-out tissue should be added to supplementary material. 2) Which GSTP1 antibody was used? Please indicate clone, company and dilution for every antibody used. 3) Methods section: LNCaP instead of LnCAP 4) Higher magnifications of images in Figure 1 should be shown. In Fig 1C it is difficult to interprete whether the area marked with “tumor cells negative” is indeed malignant tissue? 5) Please include a table for the most important clinical parameters (age, BMI, etc) for all patients in this study (differences in mean between Black and White populations?) ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
20 May 2021 Author Response Reviewer #1: The paper entitled "GSTP1 positive prostatic adenocarcinomas are more common in Black than Whitemen in the United States" is interesting. However, the authors need to check and present data a little more seriously. There are many typos in the Manuscript, such as "GSPT1" as "GSP1" or White men as Whte men, etc. A little serious presentation of their findings will significantly increase the quality of the paper. I have the following comments Response: We appreciate the reviewer’s positive comment that the paper is interesting. We acknowledge that there are some typos in the manuscript and we appreciate those being pointed out and have now corrected them. Comments: 1. The term "Black" and "Whitemen" are not preferable in scientific journals. I suggest the authors use the term African American and Caucasian in the "Title" and the Manuscript's main body. Response: We have communicated separately with the editor about this issue, who responded to us after they communicated with the reviewer about this. Since the NCI SEER program currently prefers the terminology we used, it was agreed we would keep these designations as we have them. 2. Expand the term "TMA" in the abstract section. Response: We thank the reviewer for pointing that out. We have expanded the acronym TMA in the abstract. 3. Recently another publication observed that BMI1 expressed prominently among African Americans (PMID=30087142). How do the authors compare their findings with this publication? Response: We would like to thank the reviewer for pointing out this paper and have included a brief discussion of it in the revised submission in the discussion. 4. The data on the methylation status of the GSTP1 gene will surely make the paper more valuable for the readers. Authors may include this data. Response: While we very much appreciate the importance of this question, we have recently begun a relatively large study of this question and plan to publish it separately as it has enough scope and complexity to present separately. The results are currently not available and are beyond the scope of the current manuscript. 5. The number of patients between black and white patients is significantly different. Do the authors think that their difference can influence the final result of their observation? Response: At Johns Hopkins our fraction of patients over the last several decades that are Black that undergo prostatectomy overall is 9.5% and the percentage that is White is 85.5%. In the current study we augmented these numbers by increasing the percentage of Black patients by including TMAs that were enriched for Black patients, such that our percentage of Black men in this study was 20% (see methods section for TMA dataset descriptions). We submit that our estimate found in this study of the percentage of GSTP1-positive cancers in Black men represents a highly statistically significant difference between Black and White men, and a reasonable estimate of the overall prevalence of this phenotype, since we included a relatively large number (N= 336) of Black men. To better refine our estimates, we are currently working toward increasing our overall number of both Black and White men in additional studies from patients both within our institution and beyond, although this will take more than one year at least to complete, and we feel that publishing the current findings are important to allow other researchers to also follow up on these studies 6. Figure 2: Authors should include one or two cell lines representative of the black population. As the study emphasizes the black population, this population in vitro model is a must for this figure. Response: There are relatively few prostate cancer cell line models over all in the field (compared to that for other common cancer types), and even fewer cell lines from African American subjects. Nonetheless, we agree this is an important point and have now included the results of GSTP1 in situ hybridization from MDA-PCa-2 cell line, which was derived from an African American (PMID:9815652). We found this cell line to be negative for both GSTP1 mRNA and protein. We have revised the updated Figure 2 to include these results. 7. Figure 3: Authors must explain the background of the "representative case" in the legend. Is it from black or white patients"? Response: We appreciate the reviewer for pointing out that it would be helpful if provided additional information about this case. We have now added details about this case. The specimen was from a 73 year old Black man with a Gleason score of 4+5=9, and a pathological stage of T2N0Mx with margins negative. This information is now included in the manuscript. Reviewer #2: In this manuscript the authors have analyzed a large collection of prostate adenocarcinoma tissue specimen for the expression of the Glutathione-S-transferase family member GSTP1 by immunohistochemistry. Technically the analysis of the tissue micro arrays is well-performed and statistical analysis adequate. A significant higher number of GSTP1+ cases among Black men compared to White was found. However, while this finding is new and interesting, large parts of this descriptive-only study confirm already published data for GSTP1+ expression in a subset of prostate cancer patients. The authors speculate whether GSTP1 positive cancer might be a distinct molecular subtype of this disease. Molecular subtypes should be defined by genomic aberrations and/or gene expression signatures, expression of a single gene (GSTP1) is – in my opinion – not sufficient to define a molecular subtype. In addition, GSTP1+ cases were found in ERG+ and ERG- cases representing two recognized molecular subtypes of prostate cancer. To get more robust data for the definition of a molecular subtype a gene expression study could be performed on the “recent case” TMA used for RNA in situ hybridization. This would give a first indication whether GSTP1+ cases have a different gene expression signature compared to GSTP1- cases. Response: We appreciate the positive feedback regarding the performance of GSTP1 IHC and analysis on a large collection of adenocarcinoma specimens. In terms of defining a new molecular subtype, we agree with the Reviewer that the expression of a single gene, such as GSTP1, is not sufficient to define a molecular subtype. In a number of locations in the manuscript we indicate that GSTP1 status may help define a novel molecular subtype, not that it indeed does define such a subtype. For example, in the abstract we stated: “This observation should prompt additional studies to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer.” Also, in the discussion we stated: “Given the large breadth of molecular alterations in prostate cancer, additional studies with larger numbers of patients are required to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer.” We further agree that it is important to obtain more robust data for the identification of a molecular subtype by performing a gene expression study. In fact, we are currently performing such a study on a large number of fresh frozen tissues in which we have GSTP1 expression and RNAseq. The data from that study are still being analyzed and will take some time to complete given the substantial scope of that effort. Thus, that analysis is beyond the scope of the current study and we submit that the current study will also prompt others to perform similar investigations. Minor points: 1) The GSTP1 antibody was well-controlled prior staining the TMAs. However, results of stainings in the cell lines and knock-out tissue should be added to supplementary material. Response: We thank the reviewer for this suggestion. We have added a supplemental Figure (supplemental Fig. 1) to show the cell lines for both IHC and in situ hybridization. We have removed the reference to the knockout tissue as we realized those were stained with a different antibody. 2) Which GSTP1 antibody was used? Please indicate clone, company and dilution for every antibody used. Response: We appreciate pointing out this omission and have added the details for this. 3) Methods section: LNCaP instead of LnCAP Response: We have fixed this typo. 4) Higher magnifications of images in Figure 1 should be shown. In Fig 1C it is difficult to interprete whether the area marked with “tumor cells negative” is indeed malignant tissue? Response: We have added a higher power image as a separate panel to the revised Figure 1. 5) Please include a table for the most important clinical parameters (age, BMI, etc) for all patients in this study (differences in mean between Black and White populations?) Response: At present, although we don't have BMI on all of these patients, we will annotate that information for a followup study when we look at patient outcomes with respect to GSTP1 expression status. We did add the mean and SD of the patient ages by race in the results section, first paragraph in the revised manuscript. https://doi.org/10.1371/journal.pone.0241934.r002
2 Jun 2021 Decision Letter - Craig N Robson, Editor GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States PONE-D-20-36882R1 Dear Dr. De Marzo, 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, Craig N Robson Academic Editor PLOS ONE Additional Editor Comments (optional): The authors have fully addressed all the comments raised by both reviewers. The manuscript has been improved by inclusion of additional data and through individual amendments to the main text as suggested by the reviewers. Reviewers' comments: https://doi.org/10.1371/journal.pone.0241934.r003
Formally Accepted
17 Jun 2021 Acceptance Letter - Craig N Robson, Editor PONE-D-20-36882R1 GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States Dear Dr. De Marzo: 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 Prof Craig N Robson Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241934.r004

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