PONE-D-22-26128The Impact of Variance in Carnitine Palmitoyltransferase-1 Expression on Breast Cancer Prognosis is Stratified by Clinical and Anthropometric FactorsPLOS ONE

Dear Dr. Perry,

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: Ryan Liu and authors in the manuscript “The Impact of Variance in Carnitine Palmitoyltransferase-1 Expression on Breast Cancer Prognosis is Stratified by Clinical and Anthropometric Factors” addressed the connection between breast cancer prognosis and fatty acid metabolism in cancer patients. CPT1A transports long-chain fatty acid to mitochondria for beta-oxidation and gene expression of this enzyme in the breast cancer tissue was used as a measurement of fatty acid metabolism rate within the cancer. To study this relationship, authors worked with available public databases and archive such as RNA-seq data and 18F-fluorothymidine PET-CT images and analyzed the data in relation to obesity and menopausal status using appropriate statistical methods. It is very interesting study, and their findings have significant impacts in the cancer metabolism field. In addition, the novelty of work is that authors skillfully combined research tools such as Fiji ImageJ, Xena Functional Genomics Explorer, MARRVEL, and GTEx Portal.

Despite the above, there are some major points that I would like to point out:

1. 10.83 FPKM is used as cut off level for CPT1A gene expression. How did authors determine this cut off level? It was not explained in the Method section.

2. Figures need to be referred in the Results, not in the Discussion.

3. In the sentence, the results on Figure 1A seems to be described in opposite way than the actual data.

4. Figure 4 on Page 6 and Figure 5 on Page 7 need to be described better and correct way. For these data set, could age rather than menopausal status play a role?

5. In Figure 6, lower CPT1A expression in brain parts is not surprising, because CPT1C is the major isoform in brain. However, the highest expression of CPT1A is observed in GI-tract (colon, duodenum, and small intestine). Can authors add these tissue in the Figure 6 in other to compare with breast tissue?

6. On page 9, the statement “these results are consistent with the fact that CPT1A is a major regulator of breast cancer” needs to be re-written. To make this statement, CPT1A activity assay or at least protein expression need to be determined. Authors for their interpretation, used only RNA-seq data and these data or actual gene expression of CPT1A is not confirmed by quantitative PCR in the tissue. Since metabolism in breast cancer cells not fully understood yet, a statement such as “a major regulator” should be avoided.

Minor points

1. On page 2, reference 3 was cited as “we demonstrated…” However, these authors were not listed as authors in the #3 manuscript. Please clarify this reference.

2. On page 2, “understanding” should be replaced by “understand”.

3. In the Figure 2, should it be simpler and easier to understand if words “normal healthy, overweight and obese” included instead of 2,3,4 numbers?

4. Number of participants (n) should be added in the legends for Figure 2 and 3.

5. Tissue names in Figure 6 on Page 8 are too small and not readable. Can authors replace this with higher resolution figure?

Reviewer #2: The authors analyzed the role of CPT1A differentially expression on Breast Cancer Prognosis concrning Clinical and Anthropometric Factors. The method is approperiatly designed and the manuscript is well presented. The results were validated using different measurement including CT scan and RNA-seq analysis. The work may gain so many interests, however, I have minor concerns:

- The figures are fuzzy. vector-based or high quality figures are required for the final version.

- The authors may refer to recent breast cancer progression works in the literature such as PMID: 35205681 and PMID: 34997055.

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Reviewer #1: No

Reviewer #2: No

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The Impact of Variance in Carnitine Palmitoyltransferase-1 Expression on Breast Cancer Prognosis is Stratified by Clinical and Anthropometric Factors

PONE-D-22-26128R1

Dear Dr. Perry,

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.

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Kind regards,

Divyansh Agarwal

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: All comments have been addressed

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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: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

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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: Yes

**********

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: Ryan Liu and authors have addressed most of the comments in the revision of their manuscript “The Impact of Variance in Carnitine Palmitoyltransferase-1 Expression on Breast Cancer Prognosis is Stratified by Clinical and Anthropometric Factors”.

Though, a few more minor concerns I would like to point out:

1. In the Discussion, instead of citing just Figure 1b, I like the actual sentence that has been removed in the revised version. In continuation of the sentence, how about this idea: “These results are consistent with the idea that breast cancer metabolism could be (might be?) more dependent on CPT1a-mediated mitochondrial fatty acid oxidation”?

2. In the end of the Discussion, the last sentence “Breast tissue is revealed as the tissue type with the highest CPT1A expression level, a feature not previously identified”, are the authors talking about normal breast tissue or breast cancer tissue? Since Figure 6 is so blurry, I really cannot read that 3 words. At least, based on the updated version of Figure 6, they should say “one of the highest…”.

3. Does number of participants n=33 in the legend for Figure 3 mean 33 for each groups or total for 2 groups? It needs to be clarified.

4. Tissue names in Figure 6 are still blurry and difficult to read. Can authors include tissue names in the figure legend (for example name them in order from left to right)?

Reviewer #2: I think the reviewers have done fair efforts addressing reviewers comments. I was wish for more literature. but overall 21 papers in the reference is fine.

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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: Yes: ABEDALRHMAN ALKHATEEB

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