Probing Folate-Responsive and Stage-Sensitive Metabolomics and Transcriptional Co-Expression Network Markers to Predict Prognosis of Non-Small Cell Lung Cancer Patients

Round 1

Reviewer 1 Report

This manuscript reports a study on the influence of folate status on metabolite levels in tissues of non-small cell lung cancer (NSCLC) patients. Paired tissue samples from patient’s NSCLC tissue and noncancerous lung tissue were compared, as was the influence of cancer stage. 

 

This manuscript presents a large amount of data, some of which is very interesting and novel. Although much of the data are descriptive and the mechanism behind many of the observed effects remains to be established, the data do point to some potentially very interesting effects of changes in folate status in these tumor cells. The analyses revealed the expected stage-dependent changes in glycolytic intermediates such as lactate in the tumor tissues but also showed that low folate increased indicators of glycolytic index. A novel effect of poorer folate status was an increase in glutamine and related amino acids, which also occurred in a stage-dependent manner. Whether these observations can suggest improved treatment for these cancers remains to be established.

 

Because of the large amount of material presented in the figures, it is quite difficult to follow some of the figures. It might be an improvement if some of the material was increased in size and/or some put in the Appendix.

 

 

Author Response

Comments and Suggestions for Authors

This manuscript reports a study on the influence of folate status on metabolite levels in tissues of non-small cell lung cancer (NSCLC) patients. Paired tissue samples from patient’s NSCLC tissue and noncancerous lung tissue were compared, as was the influence of cancer stage. 

 

This manuscript presents a large amount of data, some of which is very interesting and novel. Although much of the data are descriptive and the mechanism behind many of the observed effects remains to be established, the data do point to some potentially very interesting effects of changes in folate status in these tumor cells. The analyses revealed the expected stage-dependent changes in glycolytic intermediates such as lactate in the tumor tissues but also showed that low folate increased indicators of glycolytic index. A novel effect of poorer folate status was an increase in glutamine and related amino acids, which also occurred in a stage-dependent manner. Whether these observations can suggest improved treatment for these cancers remains to be established.

Because of the large amount of m aterial presented in the figures, it is quite difficult to follow some of the figures. It might be an improvement if some of the material was increased in size and/or some put in the Appendix.

Answer:

Thanks for pointing out the unclear figures; we have fixed the problems. We adjusted the size of the figures to easier forms to read. Moreover, we shifted the results which were metabolites and metabolites particle correlation in Figure 5 to supplemental data (Figure S5) (Line 646), making Figure 5 easy to read. Please check the renewed manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

Please find attached the recommendations to improve the quality of the paper.

Kind regards

Comments for author File: Comments.pdf

Author Response

Dear authors,

The topic is very interesting, the techniques and statistical analyses used in this study are very detailed described and the statistical analyses are robust.

However, please find below some recommendations:

Line 21 what does it mean the abbreviation LC/MS/MS?

Answer:

Thank you for pointing out the missing. We correct the LC/MS/MS to ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) at Line 21.

 

Introduction

Line 72 Again LC/MS/MS

Answer:

Thank you for pointing out the missing, and we fixed the problem as the above. We correct the LC/MS/MS to ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) at Line 72.

 

Methodology

Line 89 What does it mean IRB (abbreviation) protocol

Answer:

Thank you for pointing out the missing. The full name of IRB is institutional review board. This study was conducted according to the guideline of the Declaration of Helsinki, and approved by the ethics committee at Fu Jen Catholic University Hospital (ethic approval code: C105018) and National Taiwan University Hospital (ethic approval code: 201701123RINC). We explain more detailed content from Line 89 to Line 91.

 

Line 96 Please explain how were collected the demographic data by dieticians. How do You calculate the dietary folate? Please specify the BMI calculation formula.

Answer:

The dietary folate intake was calculated by quantitative food frequency questionnaire (qFFQ), specifically designed for the assessment of folate. The reference was provided above.

Lee C.H., W.J., Tzeng M.S., Huang R.F.S. Dietary profile of folate intake in long-term post-stroke patients. NutritionResearch 2005, 25, 465.

The formula of BMI is body weight in kilograms divided by the square of height in meters. We added the detailed information from Line 101 to Line 104.

 

Line 93 Please explain the abbreviation IASLC/ATS/ERS

Answer:

Thanks for pointing out the missing. The IASLC/ATS/ERS is the International Association for the Study of Lung Cancer (IASLC), American Thoracic Society (ATS), and European Respiratory Society (ERS). We added the full name from Line 96 to Line 97.

 

Line 132 Please specify the metabolomics (15) that you identify using the technique

Answer:

In this study, we used targeted liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) to identify the metabolites. Using standards to identify the metabolites and detect concentration. Because targeted UPLC/MS/MS has more accuracy than untargeted UPLC/MS/MS. We added the more information from Line 138 to Line 139.

 

Line 138 MS abbreviation what does it mean?

Answer:

Thanks for pointing out the confusion about MS. We used ‘mass spectrometer’ to replace the MS from Line 146 to Line 147. It is cleaner to descript the machine.

 

Line 139: MS/MS please explain

Answer:

Thanks for pointing out the typing error. We deleted the ‘MS/MS’ from manuscript at Line 149.

 

Line 144 isogradient please correct

Answer:

Thanks for pointing out the typing error. We have corrected the error at Line 152.

 

Line 237 RBC abbreviation. Please add

Answer:

Thanks for pointing out the missing. The first time showed red blood cells (RBC) is at Line 107, and we have added the full name.

 

Line 335 What are suggesting the differences? Figure 3

Answer:

The enrichment analysis results showed that ‘lactate degradation’ and ‘pyrimidine metabolism’ were the top two enrichment pathways in the early stage tumour. It matched the results shown in figure 1D that the concentration of lactate and AMP were lower in the early stage than in the late stage. On the contrary, the ‘amino sugar metabolism’ was the top enrichment pathway, and the concentration of GlcNAc and UDP-GlcNAc were higher in the late stage than in the early stage. Although ‘lactate degradation’ was the second top enrichment pathway in the late stage but the enrichment ratio was less than in the early stage. Therefore, the concentration of lactate was higher in the advance stage than in the early stage.

 

Line 596 In the section on conclusions, please specify what kind of nutrition intervention or drug development can be made

Answer:

Thanks for the good suggestion. This study showed low folate status would promote tumour more malignance. Therefore, we modified dietary folate interventions and anti-folate drug at Line 884 and Line 885 at the manuscript.

 

 

Kind regards

Author Response File: Author Response.pdf