CKLF as a Prognostic Biomarker and Its Association with Immune Infiltration in Hepatocellular Carcinoma

Round 1

Reviewer 1 Report

The authors uncovered CKLF as a potential prognostic biomarker associated with immune infiltration in hepatocellular carcinoma (HCC)

Points to be addressed:

1) The rationale of why the authors came up with this research is scanty and is related to a lack of novelty: please highlight what this manuscript might add.

2) What is the information that is not exactly available that motivated the authors to come up with this information. What are the current caveats and how do the authors highlight the current research in answering them? If not they need to address in background and infuture directions .

3)State of the art figures are required: scale bar should be provided in high resolution.

4)The authors could provide a little more consideration of genomic directed stratifications in clinical trial design and enrolments. 

 

 

 

 

5)The underlying message here is that more precision and individualized approaches need to be tested in well-designed clinical trials – a challenge, but I would be interested in their perspective of how this might be done. If beyond the scope of the manuscript, this should be highlighted as a limitation

6) The authors need to highlight what new information the review is providing to enhance the research in progress

7) Did the author check for biases and correct in a multivariable model (i.e. cox) the confounding factors for survival?

8)In this frame of thinking, did the author check for hazard's proportionality?

9) By constructing a roc curve, shifting the cut-off along the entire range of possible results, a theoretically infinite number of paired sensitivity and specificity values would be obtained, but choosing a threshold in which all true positives could be identified would certainly result in a high number of false positives. Therefore the most convenient solution derives from the compromise between the recovery of a high number of true positives and an acceptable number of false positives.

By placing the false positives on the abscissas of a Cartesian system and the true positives on the ordinates, a curve called ROC (receiver operating characteristics) is constructed which helps to choose the most appropriate discriminating value between the healthy and the diseased population. Please comment and expand highlighting the study's limitations

9)this reviewer personally misses some insights regarding the fact that hcc is one of most common cancers and the fourth leading cause of death worldwide. Commonly, HCC development occurs in a liver that is severely compromised by chronic injury or inflammation. Liver transplantation, hepatic resection, radiofrequency ablation (RFA), transcatheter arterial chemoembolization (TACE), and targeted therapies based on tyrosine protein kinase inhibitors are the most common treatments. The latter group have been used as the primary choice for a decade. However, tumor microenvironment in HCC is strongly immunosuppressive; thus, new treatment approaches for HCC remain necessary. The great expression of immune checkpoint molecules, such as programmed death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activating gene 3 protein (LAG-3), and mucin domain molecule 3 (TIM-3), on tumor and immune cells and the high levels of immunosuppressive cytokines induce T cell inhibition and represent one of the major mechanisms of HCC immune escape. Recently, immunotherapy based on the use of immune checkpoint inhibitors (ICIs), as single agents or in combination with kinase inhibitors, anti-angiogenic drugs, chemotherapeutic agents, and locoregional therapies, offers great promise in the treatment of HCC (please refer to PMID: 34065489  and PMID: 34146196 and expand).

Author Response

请参阅附件。

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

I've read your paper with great interest and although you may find many comments in the pdf file, I believe your work has potential but a lot of space for improvement.

I believe you could detail a bit more the whole Immune Checkpoint Genes part as it has important clinical value nowadays for tumors and for HCC as well. Are any of those genes prognostic factors as well and associated with response to immune checkpoint inhibitors? Your discussion should talk more about this rather than take all the CMTM family one by one and present a review like story of all of them. I mean you paper gradually focuses on CKLF so keep it focused till the end.

The validation on your HCC cohort is extremely superficial. Please consider extending this part to complete the paper.

Pay attention to the English.

Hope you manage to take into consideration as many of my comments as possible. I look forward to your revised work.

Best of luck!

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have clarified several of the questions I raised in my previous review. Most of the major problems have been addressed by this revision. 

Author Response

Dear Reviewers,

Thank you for your comments on our manuscript! We greatly appreciate the opportunity that we have been given. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our study. And we will continue to improve our study, once again, we express our heartfelt gratitude to you.

Thank you and best regards.

Yours sincerely,

Dan Li

E-mail: [email protected]

Reviewer 2 Report

My dears,

Thank you for taking my suggestions into considerations. However, a proper validation of your results on your cohort of patients is still missing and I believe this is a critical point for this article. An IHC staining unfortunately does not suffice...

Author Response

Dear Reviewer

Thank you for your comments on our manuscript! We greatly appreciate the opportunity that we have been given. Suggestions and comments by you are all valuable, and very helpful for us to revise and improve our manuscript.

We read all comments carefully and revised our manuscript accordingly. All changes are highlighted in revision format in the revised manuscript. Hopefully, this revision will be received positively by you.

Thanks again for your consideration of our manuscript for publication in your journal and look forward to your favorable decision.

Sincerely yours,

Dan Li

E-mail: [email protected]

The following is a response to your comments.

Comment: Thank you for taking my suggestions into considerations. However, a proper validation of your results on your cohort of patients is still missing and I believe this is a critical point for this article. An IHC staining unfortunately does not suffice...

Reply: Thanks for your valuable comments. We analyzed the relationship between CKLF expression and clinicopathological parameters in clinical HCC samples. The results show that CKLF expression levels was significantly correlated with tumor stage (Supplementary Table S2). In addition, univariate multivariate Cox regressions analyses was added in this study. The results showed that high CKLF expression and TNM stage were considered as risk factors affecting the prognosis of patients with HCC (Supplementary Table S3). These results further validate the role of CKLF in clinical HCC samples.

      Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Dear authors,

Thank you! last details:

For consistenty choose if you want to present COX results in table or in a forest plot. For example in fig6 there's a forest plot and in the last analysis you added during revision you have a table. Always state which group is the reference in your analysis. In supplementary table S3 it seems that low expression of CKLF is worse for OS.

Add a Kaplan curve for OS to complete the new data on your patients.

Add the COX analysis in material and methods.

 

Best of luck with your future research and projects!

Author Response

Please see the attachment

Author Response File: Author Response.docx