Proteomics-Based Study of Potential Emphysema Biomarkers Reveals Systemic Redox System and Extracellular Matrix Component Dysregulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors report a cross-sectional study that included 40 participants divided into patient swith emphysema and COPD, COPD without emphysema, healthy smokers and healthy never-smokers. Plasma samples were analyzed using LC/MS.folllowed by differential expression and pathway overrepresentation analysis. A total of 940 proteins were identified. NADP-ME was candidate circulating biomarker associated with emphysema. Given the sample size, the finding are exploratory and required validation in a a larger, independent  cohort.

Major changes

• The study reports that participants were included in a non-probabilistic manner (line 125), and in the abstract and in line 427, it is detailed that the study used probabilistic sample. Matching-based recruitment is non-probabilistic. Please correct the manuscript.
• The sample is small to perform ROC analysis and sensitivity/specificity. Recommend NADP-ME is a candidate biomarker rather than a biomarker.
• Please address how serum NADP-ME could explain what the mechanism of emphysema in the lung tissue and not a systemic response to a confounder.

Minor changes

• Please report limitations of identifying emphysema qualitatively by radiologist and not CT metrics.  

Author Response

We thank the Reviewer for their remarks which helped us improve the quality of manuscript. Based on the Reviewer's remarks, we have now thoroughly revised our manuscript and taken all their remarks into account. Below, we include a point-by-point response. Please also note that references might be disordered, which is due to the restructuring of the Discussion section and the inability to update them through our citation software.

Comment: The study reports that participants were included in a non-probabilistic manner (line 125), and in the abstract and in line 427, it is detailed that the study used probabilistic sample. Matching-based recruitment is non-probabilistic. Please correct the manuscript.

Response: We thank the Reviewer for noticing this. This was a type-writing error which has now been corrected.

Comment: The sample is small to perform ROC analysis and sensitivity/specificity. Recommend NADP-ME is a candidate biomarker rather than a biomarker.

Response: We thank the Reviewer for their remark. We agree that these results need to be validated in a larger cohort and with commplementary methods. We have added the word „candidate“ in the discussion section and in the conclusion to communicate this more clearly with the readers.

Comment: Please address how serum NADP-ME could explain what the mechanism of emphysema in the lung tissue and not a systemic response to a confounder.

Response: We thank the Reviewer for this question. We have now restructured the discussion section which now reads:

“... the biological roles of NADP-ME may span several interconnected levels relevant to emphysema pathogenesis: intermediary metabolism, redox homeostasis, amplifcation of inflammatory signaling cascades and anoikis [8,34,48,50,51,53]. We hypothesize that the disturbance in these pathogenic axes in the setting of emphysema might lead to a release of NADP-ME to systemic circulation and propose that plasma NADP-ME might be a potential plasma biomarker candidate of emphysema. However, further studies with other methods are required to validate our results.“

 

Comment: Please report limitations of identifying emphysema qualitatively by radiologist and not CT metrics.  

Response: We thank the Reviewer for allowing us to communicate this more clearly. We have now added the underlined text to the previously mentioned limitation: „Additional study limitation is the fact that emphysema was not quantified, but only qualitatively assessed by a radiologist“

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Grgur Salai and colleagues aimed to detect potential emphysema biomarkers and to assess the systemic effects of emphysema in blood plasma. We conducted a small cross-sectional shotgun proteomics study.

I read the manuscript with interest, and in general, it is well written and engaging.

I have some comments and suggestions for the authors to address before making a final decision on its suitability for acceptance.

Methods section:
The authors do not specify the recruitment source for cases and controls. Although they mention approval from two institutions, they should clarify the source of the recruited patients, especially the selection process for the controls. Are the controls also part of a chest CT scan? What criteria were used for their selection?
Please expand the meaning of "CT" to ensure it refers to computed tomography and include the values used.

In the sentence "All subjects in the study signed an informed consent form," please modify it as follows: after being invited to participate and accepting, all participants in the study signed an informed consent form.

Add the catalog number for the RC DC Lowry protein assay (Bio Rad, Hercules, CA, USA). As well as most of the reactives employed, some of the others include only the manufacturers, not catalog numbers or complete manufacturers' details.

Correct "Visaulizer" to Visualizer.

In Table 1 and the corresponding text, please expand the information about smoking variables beyond active smoking status. I mean, the packs/years, years smoking, and tobacco index "TI" (composed of both variables). Please note that a higher TI is associated with the emphysema phenotype.
Also, describe the use of biomass as fuel for cooking and exposure to chronic smoke from biomass combustion, which is a common and known environmental risk factor for COPD and is related to the chronic bronchitis phenotype in COPD.
The FVC and FEV1/FVC ratio should be included in Table 1 and the corresponding text.

In lines 201-202, it is stated: "Proteins with an adjusted p-value<0.05 and a fold change (FC) |FC|>2 were deemed statistically significant," but in Figures 2 and 3, the thresholds indicated by vertical dotted lines show FC= +/-1.0. Please explain or correct this discrepancy and make the necessary modifications.

The Discussion section should be shortened and focused on the main results. Currently, it is verbose and vague. The authors state that "Smoking levels in terms of pack-years measurement was also comparable between smoking groups (CE, CN, HS)," but the related variables are not described.

 

Author Response

We thank the Reviewer for their remarks which have greatly improved the quality of our manuscript and allowed for a more clear communication of our results to the readers. We have now thoroughly revised our manuscript, added new information in Materials and methods, as well as in Table 1 and refocused the Discussion. We hope that with the corrected changes, it will now be suitable for publication. Please also note that references might be disordered, which is due to the restructuring of the Discussion section and the inability to update them through our citation software.

 

Comment: Methods section: The authors do not specify the recruitment source for cases and controls. Although they mention approval from two institutions, they should clarify the source of the recruited patients, especially the selection process for the controls. Are the controls also part of a chest CT scan? What criteria were used for their selection?

Response: We thank the Reviewer for their important remark. We have now added explanation regarding the participant recruitment, crtieria for recruiting healthy participants and the explanation regarding CT to the Materials and methods section.  This section now reads:

Participants were included in the study in a non-probabilistic manner in order to be age, gender, comorbidity and body mass index matched to ensure group homogeneity in these regards. Patients with COPD were recruited from the Department of Pulmonology’s out-patient clinic and healthy individuals were recruited from the University Hospital Dubrava’s Polyclinic after they underwent a health-screening examination. After being invited to participate and accepting, all participants in the study signed an informed consent form.

Patients with COPD (CE and CN groups) were GOLD 2B patients as defined by GOLD 2023 [17], active smokers (>20 pack-years), on dual inhalation therapy (combination of long-acting β₂ agonist (LABA) and long-acting muscarinic antagonist (LAMA)). Healthy participants were people without respiratory symptoms and with normal spirometry. Exclusion criteria for all participants were: reversible airflow limitation, positive bronchodilator test (either by the GINA criteria or by the ERS/ATS technical standard 2022 [17–19]); concomitant malignancy, autoimmune disease or concomitant asthma, as well as alpha-1-antitrypsin deficiency. Participants that consumed tobacco products other than classical cigarettes (including e-cigarettes, heat not-burn tobacco products, vape, cigars and cigarillos) were also excluded from the study. None of the participants were taking glucocorticoids or other immunosuppressive medications. Exclusion criteria for COPD patients was an acute exacerbation of COPD in the last 6 months.

Comorbidities were assessed using the Charlson's comorbidity index and also a modified Charlson's comorbidity index in which “chronic pulmonary disease“ was excluded. The participants underwent spirometry testing and all COPD patients had a chest CT no older than 3 months prior to inclusion. Due to ethical concerns of exposing the healthy participants to radiation without clinical indication, they were not required to have a chest CT in order to be included in the study. Spirometry with a subsequent bronchodilator test (with 400mcg of salbutamol) was performed according to the international standards, using the Global Lung Initiative (GLI) reference values [19–21]. Expiratory airflow limitation was determined using the proposed GOLD criteria with a fixed FEV1/FVC ratio of 0.7 [17].

 

Please expand the meaning of "CT" to ensure it refers to computed tomography and include the values used. We thank the Reviewer for their remark.

We thank the Reviewer for noticing this. In the discussion section we defined the abbreviation of CT: „Nowadays, emphysema is commonly diagnosed using chest computed tomography (CT) scans, visible as areas of low attenuation, usually without visible walls“, but in order to ensure the clear readibility, we have also done so in the first mention of the Materials and methods section. Additionally, we have added the attenuation value used as a cut-off of emphysema in the materials and methods section: „This study included a total of 40 participants, divided into four equal age and gender-matched groups (N=10 participants per group): 1.) patients with COPD and radiologically verified emphysema (with a threshold of -950 HU) on chest computed tomography (CT) (CE group), confirmed by a radiologist“

 

In the sentence "All subjects in the study signed an informed consent form," please modify it as follows: after being invited to participate and accepting, all participants in the study signed an informed consent form.

We thank the Reviewer for allowing us to communicate the ethical aspects of the study more clearly, we have now corrected this.

 

Add the catalog number for the RC DC Lowry protein assay (Bio Rad, Hercules, CA, USA). As well as most of the reactives employed, some of the others include only the manufacturers, not catalog numbers or complete manufacturers' details.

               We thank the Reviewer for noticing our omission. We have now added the catalogue numbers.

 

Correct "Visaulizer" to Visualizer.

Response: We have now corrected this omission.

 

Comment: In Table 1 and the corresponding text, please expand the information about smoking variables beyond active smoking status. I mean, the packs/years, years smoking, and tobacco index "TI" (composed of both variables). Please note that a higher TI is associated with the emphysema phenotype.

Response: We thank the Reviewer,  we have now added the information regarding tobacco-index, as well as years of smoking and average daily cigarette packs into Table 1.

 

Comment: Also, describe the use of biomass as fuel for cooking and exposure to chronic smoke from biomass combustion, which is a common and known environmental risk factor for COPD and is related to the chronic bronchitis phenotype in COPD.

Response:  We thank the Reviewer for their remark. Even though biomass fuel exposure is a significant world-wide COPD exposure, it is not prevalent in Croatia. None of the participants had significant biomass fuel exposure. We have now added this in the materials and methods section: “No participant had a significant exposure to biomass fuels“

Comment: The FVC and FEV1/FVC ratio should be included in Table 1 and the corresponding text.

Response: We thank the Reviewer for pointing this out. We initially omitted this information for the sake of brevity. We have now added the FVC values as percentages, as well as FEV1/FVC values as ratios in Table 1. We also added the following in the materials and methods section:

As expected, participants with COPD (CE and CN) differed from the healthy individuals (HS and HN) in terms of FEV1, FVC and FEV1/FVC values and Charlson's comorbidity index. When a modified score of Charlson's comorbidity index (in which we omitted the category for “chronic pulmonary disease“) was employed, there was no statistically significant difference between the groups. Games-Howell post-hoc test for FEV1 and FVC, as well as DSCF pairwise comparison for FEV1/FVC did not reveal statistically significant differences between CE vs. CN and HS vs. HN.

 

In lines 201-202, it is stated: "Proteins with an adjusted p-value<0.05 and a fold change (FC) |FC|>2 were deemed statistically significant," but in Figures 2 and 3, the thresholds indicated by vertical dotted lines show FC= +/-1.0. Please explain or correct this discrepancy and make the necessary modifications.

We thank the Reviewer for the opporutinity to clarify this. The vertical dotted lines show log2-transformed values (log₂(2)=1). In order to ensure consistency and more clear communication of our results, we have now changed the definition of fold change to reflect the logarithmized values:

Proteins with an adjusted p-value<0.05 and a log₂-transformed fold change (FC) of log₂(FC)>1 or log₂(FC)<-1 were deemed statistically significant

 

Comment: The Discussion section should be shortened and focused on the main results. Currently, it is verbose and vague. The authors state that "Smoking levels in terms of pack-years measurement was also comparable between smoking groups (CE, CN, HS)," but the related variables are not described.

Response: We thank the Reviewer for this important remark. We have now restructured the discussion and omitted the parts which are not relevant in order to refocus it to the main study results. The smoking levels are now stated in Table 1, with accompanying formal analysis between smoking study groups. We hope that it is now more clearly structured and easier to read.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed satisfactorily to my previous concerns.