Dear Dr Madsen,

Thank you very much for submitting your Research Article entitled 'Close correlation between transcriptomically-inferred stemness and PI3K/AKT/mTOR signalling scores in breast cancer, and a counterintuitive relationship with PIK3CA genotype' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. [Please note that in a revised manuscript it will be essential to adequately address the remaining concerns of reviewer #1].  We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Peter McKinnon

Section Editor: Cancer Genetics

PLOS Genetics

David Kwiatkowski

Section Editor: Cancer Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The revised manuscript from Madsen et al does honestly quite little to address what I still believe to be reasonable concerns about the paper. I do agree with the other two reviewers that the conceptual point, which I understand to be that higher dimensional markers of PI3K activity (“scores”) are likely to be more clinically useful and indicative of response to anti-PI3K drugs, in comparison to the simple single genetic mutation marker, is a useful and important concept. This paper does have analyses that support this idea. Yet, I still contend that an important control is to ask the question of how many other such “scores” show the types of trends that are observed, but by chance? This is not simply looking for another alternative PI3K score as was done by the authors. Perhaps, this kind of rigor is beyond scope in the opinions of the editors and other reviewers, but to me it does seem warranted to understand the fidelity of the finding, particularly given the absence of experimental follow up. Further, I apologize for the lack of clarity in the type of experimental evidence I thought might be needed to make the paper more sufficiently convincing. To me, cell line and cell culture experiments would be perfectly adequate…I was not stating new animal / tissue / human data was needed for this paper. However, I still find a major gap in the paper to be that no phenotypic or signaling markers of PI3K/mTOR/AKT activity (e.g. S473 phosphorylation on AKT) are measured to show the scores have validated meaning, and/or whether cell lines with different mutations in PI3K and scores have differential responses to PI3K inhibitors. Those should be relatively straightforward cell culture experiments. The only cell line data I found in the paper was for stem cell lines that were homo or heterozygous for PI3K H1047R (Fig. 4D), showing they have different PI3K and stemness scores. Thus, while overall it seems that I am in agreement more than not with the other two reviewers, I may still differ in my opinions as to additional analyses that would help make this paper convincing enough to have wide impact. Yet, I am OK if the others disagree with that assessment and find the revised manuscript to be satisfactory.

Reviewer #2: I am happy to recommend acceptance. The authors have responded carefully and thoughtfully to the 3 reviewers' critiques, and have made the appropriate changes to the manuscript. I have no concerns to communicate.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

Dear Dr Madsen,

We are pleased to inform you that your manuscript entitled "Positive correlation between transcriptomic stemness and PI3K/AKT/mTOR signaling scores in breast cancer, and a counterintuitive relationship with PIK3CA genotype" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

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If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Peter McKinnon

Section Editor: Cancer Genetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: It seems the authors revision shows a correlation of PI-3K pathway activation in cell culture models with established GSEA sets (Figure 2). Much of the author's claims in the paper revolve around PI-3K activity score based on GVSA. The distinction between the two is hard to decipher, but I am fine with publication.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

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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

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Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-21-00915R1

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Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

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