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You have addressed all concerns in a satisfactory manner.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
Some of the corrections mentioned in the rebuttal letter were not implemented in the manuscript. Please check the annotated version attached and introduce the requested changes before re-submitting.
[# PeerJ Staff Note: Please note that this revision has only been reviewed by the Academic Editor at this point. Once you have addressed their comments, it might still need to be sent out for peer review. #]
The reviewers have several suggestions to improve your manuscript in order to reach the quality necessary and even provide you an annotated manuscript. Reviewer 3 suggests adding a figure after datamining TCGA for EpCAM and CSV expression, which is an additional good idea.
Please let us know if you need more time to revise your manuscript if feasible for you to address the point of tumor origin.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
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Comments:
This is an interesting study investigating the CTC detection rates with EpCAM and CSV capture antibodies in different solid tumors. The findings provide evidence that EpCAM can be used in CRC, LC, GC, BCa, EC, HNSCC, CC and PCa, but not suitable in PDAC, HCC and OC, while CSV can be used in most solid tumors for CTC detection.
The manuscript is well designed and the analyses are sufficiently performed. I have some comments to mention:
Question 1: The method and result part are not clear enough to read and to understand. The authors should revise these parts. It should be easily to read and give the reader the chance to understand.
Question 2: The authors should describe clearly that how many patients were captured CTCs with EpCAM and CSV antibodies separately and both? The numbers in the manuscript are very confusing. Please make it clear.
Question 3: The patients’ clinic-pathological characteristics are described poorly. The authors should give an exact overview of disease histology, grade, stage, the types of surgery, and IHC staining data and disease status.
Question 4: The authors should provide the patients’ status at the time of blood draw, pre- or post-surgery, pre- or post-chemotherapy or immune therapy, first-line or second line therapy, et al.
Question 5: The authors should describe the method of data collection in method and material parts, including clinical data collection and blood sample collection.
Question 6: The authors should provide the inclusion and exclusion criteria of enrolled patients and healthy donors in methods and materials part.
Question 7: Typing error: the authors should double check the number of enrolled patients. In abstract part, the total number is 695, but in method part, the number is 690.
The authors describe and compare detection of CTCs with classical EpCAM-based approach and CSV-based method in order to capture more mesenchymal CTCs.
The use blood of different cancer patients and of healthy persons.
For a small subset they test both antibodies in the the same blood sample. The observe different positivity rates for both approaches in different tumor entities with CSV slightly outperforming EpCAM.
- language good: only small mistakes
- literature: sufficient
- structure: good, tables are large and complicated and may be placed in a supplement, Figures are small and hard to read.
- discussion is a bit redundant to introduction
- please change the abstract. too many details
- is within scope
- question is well defined
- instead of discussing the potential reason of false-positive "CTCs" the authors could have verified tumor origin by single cell aCGH or low pass sequencing. It would anyway be good to have executed this on a few EpCAM and CSV-positive CTCs. In addition, comparison of such cells from the same patient may point towards different origin of EpCAM and CSV-positve cells.
- antibody clones should be provided
- see above (aCGH and low pass)
- there is hardly any statistically significant outcome (it should be marked in the figure)
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I have made a few comments in the PDF.
- please change the abstract. too many details
The English in the manuscript is proficient and professional. It is unambiguous for most part except a few lines (line 74, Available should be replaced with discovered/found, line 87-88) Never use etc. (line 63) in a manuscript. Be definite about what you are stating.
Introduction needs more detail. It focuses about EpCAM and CSV, it would be pertinent for authors to describe other CTC markers that are under study and describe why did they chose to focus on these particular markers.
Last section of introduction focuses more on the CytoSorter and that should be a part of the methods section and not introduction.
Figure quality is adequate. In Fig 1, the merged field should always be on the right (last image) by convention and not the first. In Fig 2, the authors need to mention what does the red line signify in Fig 2A and B. Fig D is too small to appreciate the data!
1] The biggest concern I have is that the authors are using a CytoSorter system and for monitoring studies like this a flow based approach is highly adopted for you run a high number of cells to give a high power for statistical significance. Stating number of CTCs, in each sample lacks context. How many cells were actually analysed, what is the percentage in those cells etc.
2] Staining for CSV and the EpCAM should have been performed on the same aliquot together so that would have answered:
a] Are these markers exclusively expressed on cells or have a concurrent expression? Figure 2D would have made more sense if they would have done a flow and then measured it.
Why flow wasn’t used for this study?
3] Authors should have first established the EpCAM and CSV expression in different cancers through TCGA. ( A figure pertaining to it would be nice). That builds up the base for the manuscript, which it currently lacks apart from the explanation in the discussion section. Is there a correlation with EpCAM and CSV expression ?
Line 159-162 and 173 – 174 does not inspire confidence in me. “Healthy patients have less than 1 and Cancer patients usually have more than 2 CTCs detected”. I infer that many patients have also 0 or 1 CTC detected the same as healthy! There is not much of a difference between the healthy and cancer. Since, the average EpCAM detection rate is ~62% and ~70% for CSV , the authors wouldn’t be able to confidently tell the healthy and patient apart using this methodology if given a sample blind folded. Again, this would have been overcome if a flow based approached would have been used.
Line 167-168 has data not shown! To be open to the scientific community, I urge the authors to share the data.
It is an extensive study with many different tumor types and numbers.
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