The Role of Serum Calprotectin in Defining Disease Outcomes in Non-Systemic Juvenile Idiopathic Arthritis: A Pilot Study

Round 1

Reviewer 1 Report

I read your article with interest. I suggest that the article requires major revision.

 

1. Please remove abbreviations from the title. Use full words

 

2. MeSH keywords are recommended, and it is better to avoid abbreviations in keywords

 

3. Define all abbreviations when used for the first time (MTX, CRP, ESR) in the article

 

4. Please organise your introduction by reporting globally, regionally and locally. As you do this, try to use a logical flow of ideas between sections using some transition sentences.

 

5. The introduction ends abruptly. Before stating the aim, please note the study rationale. What research gap will this study fill, and why is this study significant to be conducted there?

 

6. In methods, please describe the study settings, the sampling methods, and the data collection procedure. 

 

7. How did you come up with this 111 sJIA number? Where is the sampling calculation?

 

8. www.siedp.it. I do not think this is the correct referencing style. 

 

9. All tables should be self-explanatory. Please add all abbreviations in the footnotes of the tables

 

10. Your figures need to be clarified. What do coloured lines indicate?

11 Your figures need to be clarified. What do coloured lines indicate?

12 The discussion is insufficient. Please discuss your study’s clinical significance and how it will help patients, clinicians and health officials while you try to compare your findings with other studies. Contrast with some studies with opposing results and develop arguments of reasons for your conclusions and their findings. Develop your hypotheses and recommendations, adding supportive scientific evidence. 

 

Author Response

Answers to Reviewer #1

1. Please remove abbreviations from the title. Use full words

Thank you. Abbreviations have been removed from the title.

 

2. MeSH keywords are recommended, and it is better to avoid abbreviations in keywords

Thank you for your advice. Abbreviations were removed from keywords.

 

3. Define all abbreviations when used for the first time (MTX, CRP, ESR) in the article

As suggested, we made this correction in the text.

 

4. Please organise your introduction by reporting globally, regionally and locally. As you do this, try to use a logical flow of ideas between sections using some transition sentences.

Thank you. We introduced a more logical flow in the text, as you suggested.

 

5. The introduction ends abruptly. Before stating the aim, please note the study rationale. What research gap will this study fill, and why is this study significant to be conducted there?

Thank you for your suggestion. We explained the importance of our study.

 

6. In methods, please describe the study settings, the sampling methods, and the data collection procedure.

Thank you for your suggestion. We better clarified these concepts in the text.

 

7. How did you come up with this 111 sJIA number? Where is the sampling calculation?

The whole study group is composed of 56 inactive patients and 55 active patients.

 

8. www.siedp.it. I do not think this is the correct referencing style.

Thank you. We removed this reference in the text.

 

9. All tables should be self-explanatory. Please add all abbreviations in the footnotes of the tables

All tables have been provided with abbreviations

 

10. Your figures need to be clarified. What do coloured lines indicate?

We made this correction in the figure legend.

 

11 Your figures need to be clarified. What do coloured lines indicate?

We made this correction in the figure legend.

 

12 The discussion is insufficient. Please discuss your study’s clinical significance and how it will help patients, clinicians and health officials while you try to compare your findings with other studies. Contrast with some studies with opposing results and develop arguments of reasons for your conclusions and their findings. Develop your hypotheses and recommendations, adding supportive scientific evidence.

Thank you for your suggestion. We added some informations to the discussion.

Reviewer 2 Report

The manuscript submitted by d'Angelo et al. describes a longitudinal study of a group of non-systemic JIA patients which aims to define the potential role of serum calprotectin as a biomarker of disease activity and flare/remission during a period of 18 months. Despite the heterogeneity of the nsJIA group regarding previous treatment, treatment at baseline and during follow-up, as well as the reduction of patient number during follow-up, the study shows interesting results. However, the manuscript needs thorough revision before publication. The following points should be addressed:

1) In the title: replace the abbreviation (MRP8/MRP14) with calprotectin;

2) The abstract is too long. Revise the abstract in order to present a clear summary of the study.

2.1. L20: albumin levels are not shown in the results.

2.2. L14, L94 - iterations: remove "potential" before role or biomarker.

3) Use one type of abbreviation for non-systemic JIA - either non-sJIA or nsJIA.

4) Materials and methods: subsection 2.3. should be revised because it does not include essential description of the methods for CRP, ferritin and calprotectin measurement.

5) Figure 2: indicate n=? for each group, define the statistical test used for determination of p values. It is interesting to see the data from the four timepoints (3, 6, 12, 18 months).

Include a graph showing calprotectin levels during relapse (compared to baseline).

6) Figure 3, B shows Cum survival instead of Log as it is in the other graphs in the figure. Why?

7) Figures 3, 4 and 5 should include a legend that specifies what red and blue baseline indicate.

8) Define the units for calprotectin concentration - ng/mL or ng/dL! The figures (fig. 3, 4, 5) indicate cut off concentrations in ng/dL but according to table 4 cut off values are in ng/mL?

9) L342-344: the correlations are not shown in the results section. These data should be included.

10) Format the references according to the journal guidelines. Ref. # 37 is incomplete.

Author Response

Answers to Reviewer #2

Thank you for your report. We responded point by point to the referee’s comments:

 

1) In the title: replace the abbreviation (MRP8/MRP14) with calprotectin;

We modified the title.

 

2) The abstract is too long. Revise the abstract in order to present a clear summary of the study.

As suggested, we revised and shortened the abstract

2.1. L20: albumin levels are not shown in the results.

Sorry for the mistake; anyhow, as suggested we added albumin level in Table 1

 

2.2. L14, L94 - iterations: remove "potential" before role or biomarker.

We made this correction in the text

 

3) Use one type of abbreviation for non-systemic JIA - either non-sJIA or nsJIA.

Thanks for the suggestion, we standardized this abbreviation in the text

                                           

4) Materials and methods: subsection 2.3. should be revised because it does not include essential description of the methods for CRP, ferritin and calprotectin measurement.

Thank you for your useful tip. The description of CRP, ferritin and calprotectine measurement tests have been added to the work, as you suggested.

 

 

5) Figure 2: indicate n=? for each group, define the statistical test used for determination of p values. It is interesting to see the data from the four timepoints (3, 6, 12, 18 months).

Include a graph showing calprotectin levels during relapse (compared to baseline).

We thank the referee for his/her suggestion. We followed it. In addition, we added a new figure to show calprotectin levels during relapse at every time points compared to baseline

 

6) Figure 3, B shows Cum survival instead of Log as it is in the other graphs in the figure. Why?

We did a mistake and put now the correct graph.

 

7) Figures 3, 4 and 5 should include a legend that specifies what red and blue baseline indicate.

Thank you for suggestion. We added this information in each legend.

 

8) Define the units for calprotectin concentration - ng/mL or ng/dL! The figures (fig. 3, 4, 5) indicate cut off concentrations in ng/dL but according to table 4 cut off values are in ng/mL?

Sorry for these mistakes. We correct all cut off in the text and tables in ng/ml

 

9) L342-344: the correlations are not shown in the results section. These data should be included.

Thank you for suggestion. We added this information in the results section.

 

10) Format the references according to the journal guidelines. Ref. # 37 is incomplete.

Sorry for these mistakes. We corrected this mistake in the references

Reviewer 3 Report

see file

Comments for author File: Comments.pdf

Author Response

Answers to Reviewer #3

Many thanks. We answer point-by-point.

 

1. Abstract

Too expansive and needs condensing – eg all HR in last results line are superfluous

As suggested, we revised and shortened the abstract

 

2. Introduction

The reference for the Wallace criteria is incorrect - this refers to a letter only

As suggested, we changed this reference

the authors state that ESR and CRP cannot detect residual inflammation – please explain, provide rationale and references

As suggested, we changwe  added a brief explanation and the related reference

the paragraph on CP is too long and short be shortened- its intracellular role is not relevant here

As suggested, we shortened this paragraph

 

3. Methods

Patients were recruited from a population-based registry, but only patients referred to the authors department were included? Thus, it is unclear if there was any selection bias.

We recruited patients, who met the inclusion criteria, referred to our department. For clarity we  deleted, since it can actually be confusing, the reference to the polutation based registry in the text,

 

Please clarify. b. The authors mention and use both Wallace criteria (not referenced) and JADAS criteria to determine disease (in-) activity. This is confusing as these are not similar.

We used Wallace's criteria only to define clinically inactive disease/on-off therapy remission. The JADAS has been only used, as routinely in pediatric rheumatology pediatric practice, to assess disease activity at each visit.

Patients using steroids were excluded – please explain why as use of other antiinflammatory medication was allowed.

Thank you for your question. We decided to exclude from the analysis patients receiving steroids, since they are the most potent anti-inflammatory agents and, therefore, they could influence the secretion of acute phase reactants and calprotectin.

Clinical assessment and JADAS description paragraphs are too expansive – most readers would be familiar with these.

As suggested, we shortened this paragraph.

Details of the CP assay need to be provided eg sensitivity, specificity, range , curve analysis and coefficient of variation. Are CP levels stable in frozen serum ? Were CP measurements done in duplex ?

-We thank the referee for his/her comment. We used a standardized kit already used by other research studies, although in adults with Rheumatic disease. We reported sensitivity, specificity and range of the kit. We had no information about curve analysis and coefficient of variation because those were beyond our study aim. We confirmed that CP levels are stable in frozen serum (-20° C) for two months, according to the technical information of the manufacturer’s instructions. So we measured the levels of serum calprotectin within two months from the storage of the sample for all patients in order to keep stable the calprotectin.

Data were collected annually, but only most recent data included. Did the authors check for consistency across different survey responses for each patient ? this would give an indication of the amount of recall bias.

The calprotectin data were not collected annually but only during the course of the study, which is still ongoing due to extension of the follow-up.

 

4. Results

Figure 1 is useful and means the study design paragraph can be deleted or very much condensed.

As suggested, we shortened this paragraph

Table 1 is too expansive and contain irrelevant data eg on data at onset JIA , time to initiation of therapy and the text regarding this should be deleted. Readers only need to know what proportion of patients were on which medication at time of study. The table needs explanation of variables, abbreviations and normal values for lab data.

Thank you for your point.

The table has been modified as you suggested.

The range of CRP and ESR in the inactive group included abnormal high values, which would mean the relevant patient does not fulfil Wallace criteria.

Thank you for this consideration. As regards the value of ERS we had already excluded that upper value (the patient repeated the evaluation 2 days later with a negative value) but we made a mistake in filling in the table, sorry. As regards the CRP, we consider a value at least higher than 10 mg/L as positive.

 Survival analysis line 258-260: please show actual data on CP levels in relapsers vs non relapsers in inactive group. A p value is not sufficient info. It is unclear how the survival analysis in active patients was performed and what the endpoint was. Is that remission (in contrats to flares in inactive group) or is it flare after becoming inactive.

We added actual CP levels in relapsers vs no relapsers.

The end point in suvival analysis in active patients was the achievement of remission, as specified in the lines of the text : “Similarly, in active JIA patients Kaplan-Meier curves were constructed from baseline to disease remission to illustrate the predictive performance of calprotectin”.

To better make easier the interpretation of our findings we also added a clarification at the beginning of the paragraph.

 Line 262-273: this is descriptive data on active group that should be under clinical data in Table 1 , while same data for inactive group should be presented. Again, show actual data on CP levels (baseline presumably or at time of followup?) in remission /non-remission. A p value is not sufficient info.

In table 1 we presented the data of the 3 groups (active and inactive patients and controls) and the p value in the comparison between the 3 groups (Anova and Kruskall-Wallis tests). Therefore also the descriptive data of inactive as well as active patients are present. As for the second part of the tip, we added the CP values ​​in addition to the p value, as suggested

Please replace surviving by “not relapsing” in tables 2 and 3.

In table 2 and 3 “surviving” means “non-relapse” for the active group and “non-remission” for the active JIA patients group, since in the two groups, as we have now specified at the beginning of the paragraph, we evaluated two different outcomes.

Table 2 is unclear- is this about patients divided by active and inactive or by pos/neg CP ?

Table 2 describes the percentage of patients who did not achieve the outcome (i.e. remission in follow-up in active patients at baseline and relapse in inactive patients at baseline), regardless of calprotectin values

 Table 3 is also unclear as heading refers to inactive patients, but active patients are included as well ? All survival curves are poor quality and should preferably become supplemental data g.

We really thank the referee for his/her important comments.

The table 3 referred to the cumulative proportion of the patients, both active and inactive, who did not reach the outcome (remission and relapse, respectively) at each time point considering the levels of serum calprotectin as positive when were higher than 3 ng/ml. However, we decided to delete this table because it cointained no significant information.

We apologize for the poor quality of the survival curves. We believe that the figures are very esplicative for our results, therefore we would prefer to leave them in the text. However,  we could move them to the supplementary section if you suggest it is better at that way.

Table 4 : the first line makes no sense- what is meant with different cut-off values and importantly, HR is 1.003 which is outside 95 CI % !? I would disagree with including time taking MTX or BD, more important to know if concurrent use was relevant for relapse. This is mainly relevant for inactive patients and I cannot see how/why inactive patients were/needed to be included in this analysis. Again, the text and table double up significantly, please condense one of the two.

We thank the referee for his/her important suggestion. We modified correctly the model.

In addition, the confounder model was also adjusted for therapy at baseline, as the referee suggested. However, our findings didn’t changed.

5. Discussion

The authors state that CP levels correlated strongly with JADAS, but not CRP or ESR,- however no correlation data were presented.

Thank you for suggestion. We added this information in the results section.

“The first finding”.. would suggest that CP levels indicate subclinical inflammation (synovial/other) ? in patients with inactive disease. The authors do however not discuss in any detail how this would occur.

As suggested, we revised the paragraph in order to make the possible explanation of this concept more exhaustive.

“The second finding” of CP levels as predictor of flare – it is unclear why the authors landed on the 12 ng/dl levels as the best option – is that based on HR ? This would again come back to discussion of how subclinical inflammation can be progressing to clinical evident arthritis?

We focused on the 12 ng/dl levels based on significant HR value .

Briefly, our findings from survival analysis also confirmed that higher calprotectin values ​​were associated with higher risk of relapse in inactive patient. This aspect might be probably related to a more important immunological subclinical inflammation which could progress becoming clinically evident.

“the third finding” of CP levels as a predictor of treatment response with higher CP levels associated with higher chance of remission is hard to understand. This reasoning is in complete contrast with the findings in inactive patients, where higher levels predict flare. The authors do not offer a staisfactory explanation for this contradictory finding.

Thanks for the suggestion, we added a brief possible explanation of this data.

Limitations should include the fact that CP is an ubiquitous protein as neutrophils may have been activated for other reasons than JIA and at other sites than joints. Infections, gastroenteritis etc come to mind as confounders.

Thanks for the suggestion, we've added this factor among the limitations.

 

Round 2

Reviewer 2 Report

The authors have  reviewed the manuscript according to my comments and recommendations. The article has been significantly improved and suitable for publication.

Minor points for further revision:

1) Figure 1: revise the abbreviation "non sJIA";

2) Figure 2 and 3: include number of individuals in each group (n=? for Clinically active, Clinically inactive, Healthy controls, Basal whole inactive group, Relapse and No relapse for each time-period);

3) Description of albumin measurements shoul be included in section Materials and methods.

Author Response

The authors have  reviewed the manuscript according to my comments and recommendations. The article has been significantly improved and suitable for publication.

 

Minor points for further revision:

 

1) Figure 1: revise the abbreviation "non sJIA"

Thank you for your suggestion. The abbreviation has been revised in the figure 1.

 

2) Figure 2 and 3: include number of individuals in each group (n=? for Clinically active, Clinically inactive, Healthy controls, Basal whole inactive group, Relapse and No relapse for each time-period)

Thank you for your tip. These informations have been added in both figures.

 

3) Description of albumin measurements shoul be included in section Materials and methods.

We thank the referee for the suggestion. We included albumin measurement method in the Materials and methods section.

Reviewer 3 Report

The online abstract has not changed. The conclusion in abstract remains vague and should be  more specific eg ".. a dual role for CP in predicting relapse and treatment response in non-sJIA" 

CP should be mentioned throughout text  and tables instead of MRP8/14

Still no data presented on JIA treatment that active/inactive patients received at the time of enrollment  and no data on new/other treatment prescribed for active patients - were steroids now allowed as it was an exclusion criterion before ? Given the importance the authors put on CP as a predictor of therapy response, these data should be provided to the readership .

Table 1 is still too busy and inaccurate with  4.1 active joints at enrollment in inactive group ??  Weight and height can be deleted as BMI is given, RF and HLA B27 can be deleted as non relevant

Table 2 and Figure 4 can be deleted as the negative results are presented in text 

Tables 4 and 5 can probably be combined into one table if the authors clearly indicate what each HR represents for different groups.  It is very unclear what is actually included within the crude and confounder models - confounders were taken form literature but only a selection was included ? Actual treatment is hopefully in there - this remains a very unclear analysis/presentation 

The discussion of how CP can have a dual role (prediction of relapse and prediction of treatment response) is still unsatisfactory. The newly inserted  reference found higher CP levels in non responders to MTX/Eta which is in contrast with presented results.  

 

Author Response

The online abstract has not changed. The conclusion in abstract remains vague and should be  more specific eg ".. a dual role for CP in predicting relapse and treatment response in non-sJIA"

We thank the referee for the suggestion. We tried to change the online abstract, but we were not able to make it as on journal website there was not the option to change it. Please could you let us know how we should make it? In the meanwhile, all requested changes have been made in the text.

 

CP should be mentioned throughout text and tables instead of MRP8/14

Thank you. Calprotectin has been mentioned instead of MRP8/14 throughout text and tables.

 

Still no data presented on JIA treatment that active/inactive patients received at the time of enrollment  and no data on new/other treatment prescribed for active patients - were steroids now allowed as it was an exclusion criterion before? Given the importance the authors put on CP as a predictor of therapy response, these data should be provided to the readership.

Thank you for your suggestion. These data have been added in the text and in the table. Steroids were not allowed, as it was an exclusion criterion.

 

Table 1 is still too busy and inaccurate with  4.1 active joints at enrollment in inactive group ??  Weight and height can be deleted as BMI is given, RF and HLA B27 can be deleted as non relevant

Thank you for your tips. The requested changes have been made in the table.

 

Table 2 and Figure 4 can be deleted as the negative results are presented in text

Thank you. The table and figure have been deleted as you suggested.

 

Tables 4 and 5 can probably be combined into one table if the authors clearly indicate what each HR represents for different groups.  It is very unclear what is actually included within the crude and confounder models - confounders were taken form literature but only a selection was included ? Actual treatment is hopefully in there - this remains a very unclear analysis/presentation.

 

 

 

 

-We thank the referee for his/her important suggestion. We combined table 4 and 5 into one table. Additionally, we added further information to better clarify what we analyzed.We specified that we ran cox-regression model to evaluate the association of serum calprotectin (at different cut-off values) with the risk of relapse or remission in inactive ad active JIA patients, respectively. We also specified what was the reference category considered. In particular, reference category was represented by JIA patients with serum calprotectin <3, < 10, <12, <15, or <20 for different cut-off levels for both groups and then data were presented as change in HR.

In addition, we adjusted our model for confounders  which we selected based on literature and  on change of the unadjusted effect estimates of 10% when added to the univariate model.

Finally, we would underline that serum calprotectin was associated with relapse or remission in inactive and active JIA patients, respectively, independently of methotrexate and bilogic drug treatments, and mostly independently of the type of therapy at baseline.

 

The discussion of how CP can have a dual role (prediction of relapse and prediction of treatment response) is still unsatisfactory. The newly inserted  reference found higher CP levels in non responders to MTX/Eta which is in contrast with presented results. 

Thank you for your suggestion. We have expanded the discussion a little further. However, unfortunately, the mechanisms by which calprotectin may play a dual role in remission and relapse in patients with JIA are currently unclear in the literature. The studies available for calprotectin in JIA patients are still few and, in the only meta-analysis available, Altobelli et al. selected only 10 studies, which were very heterogeneous and inconclusive.

As regards ref. 47, in these work the authors proved that both S100A8 and S100A9 subunits are significantly accumulated in peripheral bloon mononuclear cells from responders to therapy. Only the S100A9 protein is described to be significantly overexpressed in the serum of responder patients, even if global calprotectin levels seem to be not different between responders and non responders. Furthermore, in this study, there is not evidence that non responders present higher calprotectin levels.

Round 3

Reviewer 3 Report

No further comments