Multi-Imaging Investigation to Evaluate the Relationship between Serum Cystatin C and Features of Atherosclerosis in Non-ST-Segment Elevation Acute Coronary Syndrome
, Francesco Saia 1, Francesco Buia 3, Giacomo Maria Lima 2, Valeria Marco 4, Antonio Giulio Bruno 1, Francesco Prati 4,5, Stefano Fanti 2 and Claudio Rapezzi 1Round 1
Reviewer 1 Report
Review comments for Applied Sciences 402378 Manuscript - Revision 1
General Comments
The authors have addressed most of the major comments in their response to the reviewers and the revised manuscript. However, certain points remain open and, thus, further edits are highly recommended to increase the readability and value of the manuscript before further consideration can be made. These issues are discussed in the specific comments section below.
The authors are kindly asked to explicitly mention in their response to the reviewers the section of the manuscript they revised (in most cases the page and line numbers they indicated did not match the actual page and line of their revisions in the final pdf manuscript resubmitted).
Specific comments
Authors response to Reviewer #1, Comment 1, “In the revised manuscript these observations have been included along with appropriate references Page 10 line 24”: The suggested pages and lines, where revisions had been issued, do not correspond to the currently available resubmitted pdf manuscript version. In order to make it easier for the reviewers to effortlessly spot the issued revisions, the authors should ensure that their markers point to the actual location of the revisions in the final pdf manuscript, or, otherwise, explicitly refer to/include the paragraph sections they revised.
Authors response to Reviewer #1, Comment 2: The authors should specify which section in the new manuscript has been revised and in which way, such that it addressed the reviewer comment. The current response of the authors is quite vague, not quite useful and thus unacceptable.
Abstract, Lines 30-34, “After univariable… with low level CysC.”: This is a very long sentence that needs to be read multiple times before the correct meaning is understood by an expert reader. The authors are strongly suggested to use more commas or (better) split the sentence into more than one to enhance readability and ensure the correct message is passed to the reader. This is particularly important for the abstract of a manuscript. In addition, the "distance" between the subject "18F-FDG uptake" and the respective verb "was independently associated" is too large.
Figure 2, Caption/Legend, Page 5, Lines 153-155: The authors should include more distinctive markers of the indicated regions of focal uptake. The asterisk is not visible and the arrowhead and arrows are very small. The authors should use indicators of more distinctive shape and larger size.
Figure 3, Caption/Legend, Page 5, Lines 163-167: In Figure 3, the asterisk is visible but still quite small. The indicators here should also be replaced with ones of larger size.
Page 8, Lines 260-261, “Nonetheless, the utilization of… would allow to…”: The authors did include the suggested reference to 18F-NaF studies but did not mention also the possibility of combined 18F-FDG and 18F-NaF imaging such as in the context of dual-tracer 18F-FDG:18F-NaF imaging. Such scan protocol would have benefited from the type of analysis presented by the authors in this manuscript and should be referenced. Previously the following recent abstract was suggested to the authors for information regarding dual-tracer 18F-FDG:18F-NaF cardiovascular imaging:
Karakatsanis, N., Trivieri, M., Dweck, M., Robson, P., Abgral, R., Soler, R., Calcagno, C., Mani, V., Tsoumpas, C., Kovasic, J. and Fayad, Z., 2017. Simultaneous assessment of carotid plaque inflammation and micro-calcification with dual-tracer 18F-FDG: 18F-NaF PET-MR imaging: a clinical feasibility study. Journal of Nuclear Medicine, 58(suppl. 1), p. 446 (http://jnm.snmjournals.org/content/58/supplement_1/446 )
Page 8, Line 262, “intiml”: The authors should correct the typo error here. Perhaps they emant "initima" or "intimal".
Page 8, Conclusions section: I insist on adequately addressing the comment from the previous review cycle regarding the need to refer to the levels of association (if any) between CysC and OCT/CAC scores. This is a major finding in this study and should be clearly presented both in the abstract and the conclusions (not only in the results). It is certainly feasible to include such explanation/clarification without exceeding the word limit. This is an important element that should be clarified in the abstract and conclusions. If necessary, other parts of the manuscript can be revised to save up word space for such an addition in the conclusions and the abstract.
Author Response
Review comments for Applied Sciences 402378 Manuscript - Revision 1
General Comments
The authors have addressed most of the major comments in their response to the reviewers and the revised manuscript. However, certain points remain open and, thus, further edits are highly recommended to increase the readability and value of the manuscript before further consideration can be made. These issues are discussed in the specific comments section below.
The authors are kindly asked to explicitly mention in their response to the reviewers the section of the manuscript they revised (in most cases the page and line numbers they indicated did not match the actual page and line of their revisions in the final pdf manuscript resubmitted).
Specific comments
Authors response to Reviewer #1, Comment 1, “In the revised manuscript these observations have been included along with appropriate references Page 10 line 24”: The suggested pages and lines, where revisions had been issued, do not correspond to the currently available resubmitted pdf manuscript version. In order to make it easier for the reviewers to effortlessly spot the issued revisions, the authors should ensure that their markers point to the actual location of the revisions in the final pdf manuscript, or, otherwise, explicitly refer to/include the paragraph sections they revised.
Re: Sorry for the mistake. In the discussion section the revised manuscript has been changed as follows:
Because of several technical issues such as 18F-FDG -PET low spatial resolution, small size of coronary plaques, cardiac motion, and myocardium avidity for glucose as energetic substrate 18F-FDG -PET to measure inflammation in coronary artery plaques is suboptimal(45). Conversely, 18F-sodium fluoride(45) have been shown to better identify coronary plaques with high-risk features but this was beyond the scope of our study. However, it has been extensively demonstrated that 18F-FDG activity in the great artery wall such the aorta or the carotid arteries correlates with macrophage infiltration along with other features of plaque vulnerability(38,46,47), is highly reproducibile(48) and predicts outcome(49). These characteristics have led to an increasingly use of 18F-FDG -PET as surrogate endpoint in trials of novel therapies endpoint(50) and make this tool particularly suitable for studies like ours aimed at exploring the association between an imputed biomarker of atherosclerosis and the metabolic activity within the atheroma. Nonetheless, the utilization of other PET tracer such as 18F-sodium fluoride would allow to evaluate the association between biomarkers and intimal microcaclification that also have been associated with plaque vulnerability and progression(51). Yet, dual-tracer 18F-FDG-18F-sodium fluoride PET/Magnetic Resonance(52) or CT(53) have been suggested for in-vivo clinical simultaneous evaluation of both inflammation and micro-caclification that might increase understanding of mechanisms of atherosclerosis. Discussion section Page 9 line 24
Authors response to Reviewer #1, Comment 2: The authors should specify which section in the new manuscript has been revised and in which way, such that it addressed the reviewer comment. The current response of the authors is quite vague, not quite useful and thus unacceptable.
Re: Sorry again. In the revised manuscript figure 2 shows representative 18F-FDG-PET/CT images of thoracic aorta from both a patient with low cystatin C level and high cystatin C level. Figure 3 shows that aorta calcifications are not associated with significant FDG uptake.
Abstract, Lines 30-34, “After univariable… with low level CysC.”: This is a very long sentence that needs to be read multiple times before the correct meaning is understood by an expert reader. The authors are strongly suggested to use more commas or (better) split the sentence into more than one to enhance readability and ensure the correct message is passed to the reader. This is particularly important for the abstract of a manuscript. In addition, the "distance" between the subject "18F-FDG uptake" and the respective verb "was independently associated" is too large.
Re: thank you for the suggestion. The revised abstract has been changed as follows:
Results: After univariable and multivariable analyses, 18F-FDG uptake in the descending aorta (DA) was independently associated with low level of CysC [(Odds Ratio=0.02; 95%CI 0.0004-0.89; p=0.044; 18F-FDG uptake measured as averaged maximum target to blood ratio); (Odds Ratio=0.89; 95%CI 0.82-0.98, p=0.025; 18F-FDG uptake measured as number of active slices)]. Page 2 line 12
Figure 2, Caption/Legend, Page 5, Lines 153-155: The authors should include more distinctive markers of the indicated regions of focal uptake. The asterisk is not visible and the arrowhead and arrows are very small. The authors should use indicators of more distinctive shape and larger size.
Re: Modified accordingly
Figure 3, Caption/Legend, Page 5, Lines 163-167: In Figure 3, the asterisk is visible but still quite small. The indicators here should also be replaced with ones of larger size.
Re: Modified accordingly
Page 8, Lines 260-261, “Nonetheless, the utilization of… would allow to…”: The authors did include the suggested reference to 18F-NaF studies but did not mention also the possibility of combined 18F-FDG and 18F-NaF imaging such as in the context of dual-tracer 18F-FDG:18F-NaF imaging. Such scan protocol would have benefited from the type of analysis presented by the authors in this manuscript and should be referenced. Previously the following recent abstract was suggested to the authors for information regarding dual-tracer 18F-FDG:18F-NaF cardiovascular imaging:
Karakatsanis, N., Trivieri, M., Dweck, M., Robson, P., Abgral, R., Soler, R., Calcagno, C., Mani, V., Tsoumpas, C., Kovasic, J. and Fayad, Z., 2017. Simultaneous assessment of carotid plaque inflammation and micro-calcification with dual-tracer 18F-FDG: 18F-NaF PET-MR imaging: a clinical feasibility study. Journal of Nuclear Medicine, 58(suppl. 1), p. 446 (http://jnm.snmjournals.org/content/58/supplement_1/446 )
Re thank you. We have added this reference and modified the manuscript as follows:
Yet, dual-tracer 18F-FDG-18F-sodium fluoride PET/Magnetic Resonance(52) or CT(53) have been suggested for in-vivo clinical simultaneous evaluation of both inflammation and micro-caclification that might increase understanding of mechanisms of atherosclerosis. Discussion section Page 10 line 10
Page 8, Line 262, “intiml”: The authors should correct the typo error here. Perhaps they emant "initima" or "intimal".
Re: Modified accordingly
Page 8, Conclusions section: I insist on adequately addressing the comment from the previous review cycle regarding the need to refer to the levels of association (if any) between CysC and OCT/CAC scores. This is a major finding in this study and should be clearly presented both in the abstract and the conclusions (not only in the results). It is certainly feasible to include such explanation/clarification without exceeding the word limit. This is an important element that should be clarified in the abstract and conclusions. If necessary, other parts of the manuscript can be revised to save up word space for such an addition in the conclusions and the abstract.
Re: Thank you: in the revised manuscript we have change the conclusion sections as follows:
Conclusions: In patients with NSTE-ACS, 18F-FDG uptake in DA is associated with low level of serum CysC. There was no relation between CysC levels and OCT-assessed coronary plaque vulnerability or CAC score. These findings suggest that high levels of CysC may not be considered as independent markers of atherosclerosis.
Reviewer 2 Report
The presented data must be further confirmed qualitatively. The exact conceptual break through of the study seems to be missing.
Author Response
Thank you
During the past 2 decades intense research activities focused on cystatin C.
Initially it was observed that high level of cystatin C are associated with worse outcome in different clinical scenario.
Later it had been demonstrated that this association is not causal
Then, why high level of cystatin C are associated with an increased risk of death and myocardial infarction?
Is Cystatin C a marker of preclinical kidney dysfunction that may promote atherosclerosis and its clinical consequences?
Alternatively, is Cystatin C a marker of extent of coronary artery disease? With this regard studies based on coronary angiography showed conflicting results?
Our study for the first time evaluated the association between cystatin c levels and different aspects of atherosclerosis based on a multi-imaging approach that in particular included functional information as those obtained by means of FDG-PET.
Despite our small sample size we found an inverse relationship between cystatin C and aortic inflammation. There was no relation between cystatin C levels and OCT-assessed features of plaque vulnerability or calcium score.
We do believe that thanks to these findings we might contribute to answer to the latter question and fill a gap of knowledge.
These aspects have been afforded thoroughly in our work
Reviewer 3 Report
I am support the publication of paper based on revised version. Although it may cause some potential technical argument, it is a good result and question for the society to discuss in future.
Author Response
thank you very much
Reviewer 4 Report
In this study authors analyzed the relationship between cystatin C concentration and atherosclerosis indices in 31 patients with acute coronary syndromes not associated with ST segment elevation. Patients were categorized into two groups with cystatin C below and above the median. The results indicate that glucose uptake by descending aorta was higher in patients with low cystatin level. No difference between groups was observed regarding plaque vulnerability indices assessed by optical coherence tomography or coronary artery calcification scores. The results suggest that low cystatin C is associated with vascular wall inflammation.
The topic and the results are of interest. The study is methodologically sound regarding modern imaging techniques.
Specific comments:
1) I regret that cystatin C was treated only as the binary variable (below vs. above the median). The study could benefit from examining quantitative correlation analysis of cystatin vs. atherosclerosis indices.
2) It would be of interest to present classic risk factors such as blood lipid levels for these patients.
Author Response
In this study authors analyzed the relationship between cystatin C concentration and atherosclerosis indices in 31 patients with acute coronary syndromes not associated with ST segment elevation. Patients were categorized into two groups with cystatin C below and above the median. The results indicate that glucose uptake by descending aorta was higher in patients with low cystatin level. No difference between groups was observed regarding plaque vulnerability indices assessed by optical coherence tomography or coronary artery calcification scores. The results suggest that low cystatin C is associated with vascular wall inflammation.
The topic and the results are of interest. The study is methodologically sound regarding modern imaging techniques.
Specific comments:
1) I regret that cystatin C was treated only as the binary variable (below vs. above the median). The study could benefit from examining quantitative correlation analysis of cystatin vs. atherosclerosis indices.
Re: thank you for the suggestion. Accordingly we performed a spearman test of correlation showing an inverse relationship between cystatin C levels and number of active slices in the descending aorta. The revised manuscript has been changed as follows:
Statistical section: Correlations between Cystatin C and features of atherosclerosis modelled as continuous variables were determined by Spearman’s rank correlation test. Page 3 and line 110
Result section: When modelled as continuous variable Cystatin C levels showed a strong trend towards an inverse relationship with the number of active slices in DA (rho = -0.35, p=0.06). Yet, after multivariable analysis levels of cystatin C were independently associated with FGD uptake in DA as measured by either averaged max TBR or number of active slices (table 3). Page 5 line 145
2) It would be of interest to present classic risk factors such as blood lipid levels for these patients.
Re: thank you again. The revised manuscript has been changed accordingly. Table 1 page 4
Author Response File: 
Author Response.pdf
