Title: Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal
fibrostenotic strictures in patients with Crohn’s disease
Version: 3 Date: 16 Apr 2025
Reviewer's report:
A case-control study with a DSS-murine model to investigate if PRO-C16 in the serum (as
surrogate marker of type XVI collagen - part of the FACIT collagens that anchor fibrillar
collagens in the ECM), more than a marker of increased matrix turnover, is also a marker of
fibrostenotic strictures in CD.
MAJOR COMMENTS
My concern is if authors only aim to identify a marker of B2 phenotype, as mentioned in the title,
or if they really aim to find a marker of human intestinal fibrosis, which can only be extrapolated
from the model findings – as it seems to be the case considering L201 of the Discussion.
If so, authors should continue this study using human surgical specimens of both B2 and B3
patients with measuring of fibrosis components (like collagens) and confirm if serum PRO-C16
still holds as a marker of fibrosis. It must be bared in mind that intestinal fibrosis occurs in all
3 CD phenotypes, especially in advanced disease with bowel damage (B2 and B3), not only in
B2. Moreover, in B2, it seems that the muscle hypertrophy (MM and external MP) may be the
major cause of luminal reduction.
In conclusion, a non-invasive marker of intestinal fibrosis is much needed, as the authors
recognize in the Conclusion. Yet herein the authors could only demonstrate an association with
B2 phenotype in a small sample of patients with an underrepresented B3 group, as well as an
association with existing fibrosis in the murine model – where also muscle hypertrophy exists.
The clinical utility of this marker is yet to be demonstrated, but before it can be studied in anti-
fibrotic therapy, it has to be shown that it is really a marker of human intestinal fibrosis, not
only of stricturing CD.
Methodological problem: CD activity was studied through CDAI which is mostly a clinical
index. Herein the biological behaviour of the disease is being studied, hence, the use of
biomarkers of activity is crucial. I would strongly suggest the use of CRP and/or fecal
calprotectin (Fcal) obtained within 1-2 months of the day that the patient serum was collected,
as more reliable measures of biologic disease activity.
L163 – “PRO-C16 was numerically elevated in CD patients with active disease compared to
inactive disease (data not shown)” should be moved to Line 170 (before “PRO-C16 serum
levels…”) and the non-significant p-value should be shown. Although only numerical, it is too
important for the data not to be shown. If authors change the way disease activity was measured,
as requested, this comparison should also figure here.
L173 - In the section “PRO-C16 is associated with fibrostenotic strictures in Crohn’s disease” I
would suggest that authors could additionally adjust for differences in the 2 cohorts and analyse
both as a group, in order to increase subgroup numbers and correct for subgroup differences,
namely that in the cohort 2 the B2 and B3 group are very unbalanced. And in this joint group
then study the true AUC, Sens and spec.
L209-212 – “PRO-16 did not correlate with disease activity, which could be explained by the
disease activity scores reflecting ongoing inflammation. Thus, it further strengthens the notion
that the PRO-C16 biomarker is associated with ECM remodelling and ongoing fibrogenesis”
and down below authors near contradict themselves in L217-219: “Type XVI collagen has
been shown to increase the expression and activation of MMP-9 locally in tissue at the site of
insult30. This could explain the elevated levels of PRO-C16 observed in Crohn’s disease patients
with active disease…” – indeed, although only numerically different (maybe due to the use of
CDAI?), the levels of the biomarker seem to change with activity and this must be accounted for,
as inflammation triggers the ECM turnover. Maybe if results of the analysis are reformulated
preformed with the use of CRP or Fcal these paragraphs may be clarified.
L227-228 – “As a result, luminal CD patients with elevated PRO-C16 serum levels could likely
have increased ECM remodeling and intestinal fibrogenesis with premature and undiagnosed
fibrostenotic stricture.” – Authors show no evidence for this sentence, that is purely speculative.
Please show data on levels of B1 phenotype vs B2 vs. B3, as only data of B2 vs. B1+B3 was
shown.
L230-232 – “While CD patients with the B3 often are presented with concomitant intestinal
fibrosis, we also believe that the PRO-C16 marker measures active fibrogenesis. As such, the
low levels of PRO-C16 in the B3 groups could indicate non-active intestinal fibrogenesis.” As
it has been shown that both phenotypes exhibit fibrosis, these sentences are confusing, please
rephrase.
L272-274 – Based on the murine model…
MINOR COMMENTS
L143 – typo: were purchased
Table 1- % are missing in disease location for Cohort 2
Title: Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal
fibrostenotic strictures in patients with Crohn’s disease
Version: 3 Date: 16 Apr 2025
Reviewer's report:
A case-control study with a DSS-murine model to investigate if PRO-C16 in the serum (as
surrogate marker of type XVI collagen - part of the FACIT collagens that anchor fibrillar
collagens in the ECM), more than a marker of increased matrix turnover, is also a marker of
fibrostenotic strictures in CD.
MAJOR COMMENTS
My concern is if authors only aim to identify a marker of B2 phenotype, as mentioned in the title,
or if they really aim to find a marker of human intestinal fibrosis, which can only be extrapolated
from the model findings – as it seems to be the case considering L201 of the Discussion.
If so, authors should continue this study using human surgical specimens of both B2 and B3
patients with measuring of fibrosis components (like collagens) and confirm if serum PRO-C16
still holds as a marker of fibrosis. It must be bared in mind that intestinal fibrosis occurs in all
3 CD phenotypes, especially in advanced disease with bowel damage (B2 and B3), not only in
B2. Moreover, in B2, it seems that the muscle hypertrophy (MM and external MP) may be the
major cause of luminal reduction.
In conclusion, a non-invasive marker of intestinal fibrosis is much needed, as the authors
recognize in the Conclusion. Yet herein the authors could only demonstrate an association with
B2 phenotype in a small sample of patients with an underrepresented B3 group, as well as an
association with existing fibrosis in the murine model – where also muscle hypertrophy exists.
The clinical utility of this marker is yet to be demonstrated, but before it can be studied in anti-
fibrotic therapy, it has to be shown that it is really a marker of human intestinal fibrosis, not
only of stricturing CD.
Methodological problem: CD activity was studied through CDAI which is mostly a clinical
index. Herein the biological behaviour of the disease is being studied, hence, the use of
biomarkers of activity is crucial. I would strongly suggest the use of CRP and/or fecal
calprotectin (Fcal) obtained within 1-2 months of the day that the patient serum was collected,
as more reliable measures of biologic disease activity.
L163 – “PRO-C16 was numerically elevated in CD patients with active disease compared to
inactive disease (data not shown)” should be moved to Line 170 (before “PRO-C16 serum
levels…”) and the non-significant p-value should be shown. Although only numerical, it is too
important for the data not to be shown. If authors change the way disease activity was measured,
as requested, this comparison should also figure here.
L173 - In the section “PRO-C16 is associated with fibrostenotic strictures in Crohn’s disease” I
would suggest that authors could additionally adjust for differences in the 2 cohorts and analyse
both as a group, in order to increase subgroup numbers and correct for subgroup differences,
namely that in the cohort 2 the B2 and B3 group are very unbalanced. And in this joint group
then study the true AUC, Sens and spec.
L209-212 – “PRO-16 did not correlate with disease activity, which could be explained by the
disease activity scores reflecting ongoing inflammation. Thus, it further strengthens the notion
that the PRO-C16 biomarker is associated with ECM remodelling and ongoing fibrogenesis”
and down below authors near contradict themselves in L217-219: “Type XVI collagen has
been shown to increase the expression and activation of MMP-9 locally in tissue at the site of
insult30. This could explain the elevated levels of PRO-C16 observed in Crohn’s disease patients
with active disease…” – indeed, although only numerically different (maybe due to the use of
CDAI?), the levels of the biomarker seem to change with activity and this must be accounted for,
as inflammation triggers the ECM turnover. Maybe if results of the analysis are reformulated
preformed with the use of CRP or Fcal these paragraphs may be clarified.
L227-228 – “As a result, luminal CD patients with elevated PRO-C16 serum levels could likely
have increased ECM remodeling and intestinal fibrogenesis with premature and undiagnosed
fibrostenotic stricture.” – Authors show no evidence for this sentence, that is purely speculative.
Please show data on levels of B1 phenotype vs B2 vs. B3, as only data of B2 vs. B1+B3 was
shown.
L230-232 – “While CD patients with the B3 often are presented with concomitant intestinal
fibrosis, we also believe that the PRO-C16 marker measures active fibrogenesis. As such, the
low levels of PRO-C16 in the B3 groups could indicate non-active intestinal fibrogenesis.” As
it has been shown that both phenotypes exhibit fibrosis, these sentences are confusing, please
rephrase.
L272-274 – Based on the murine model…
MINOR COMMENTS
L143 – typo: were purchased
Table 1- % are missing in disease location for Cohort 2
Please review this paper.
user
Title: Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal
fibrostenotic strictures in patients with Crohn’s disease
Version: 3 Date: 16 Apr 2025
Reviewer's report:
A case-control study with a DSS-murine model to investigate if PRO-C16 in the serum (as
surrogate marker of type XVI collagen - part of the FACIT collagens that anchor fibrillar
collagens in the ECM), more than a marker of increased matrix turnover, is also a marker of
fibrostenotic strictures in CD.
MAJOR COMMENTS
My concern is if authors only aim to identify a marker of B2 phenotype, as mentioned in the title,
or if they really aim to find a marker of human intestinal fibrosis, which can only be extrapolated
from the model findings – as it seems to be the case considering L201 of the Discussion.
If so, authors should continue this study using human surgical specimens of both B2 and B3
patients with measuring of fibrosis components (like collagens) and confirm if serum PRO-C16
still holds as a marker of fibrosis. It must be bared in mind that intestinal fibrosis occurs in all
3 CD phenotypes, especially in advanced disease with bowel damage (B2 and B3), not only in
B2. Moreover, in B2, it seems that the muscle hypertrophy (MM and external MP) may be the
major cause of luminal reduction.
In conclusion, a non-invasive marker of intestinal fibrosis is much needed, as the authors
recognize in the Conclusion. Yet herein the authors could only demonstrate an association with
B2 phenotype in a small sample of patients with an underrepresented B3 group, as well as an
association with existing fibrosis in the murine model – where also muscle hypertrophy exists.
The clinical utility of this marker is yet to be demonstrated, but before it can be studied in anti-
fibrotic therapy, it has to be shown that it is really a marker of human intestinal fibrosis, not
only of stricturing CD.
Methodological problem: CD activity was studied through CDAI which is mostly a clinical
index. Herein the biological behaviour of the disease is being studied, hence, the use of
biomarkers of activity is crucial. I would strongly suggest the use of CRP and/or fecal
calprotectin (Fcal) obtained within 1-2 months of the day that the patient serum was collected,
as more reliable measures of biologic disease activity.
L163 – “PRO-C16 was numerically elevated in CD patients with active disease compared to
inactive disease (data not shown)” should be moved to Line 170 (before “PRO-C16 serum
levels…”) and the non-significant p-value should be shown. Although only numerical, it is too
important for the data not to be shown. If authors change the way disease activity was measured,
as requested, this comparison should also figure here.
L173 - In the section “PRO-C16 is associated with fibrostenotic strictures in Crohn’s disease” I
would suggest that authors could additionally adjust for differences in the 2 cohorts and analyse
both as a group, in order to increase subgroup numbers and correct for subgroup differences,
namely that in the cohort 2 the B2 and B3 group are very unbalanced. And in this joint group
then study the true AUC, Sens and spec.
L209-212 – “PRO-16 did not correlate with disease activity, which could be explained by the
disease activity scores reflecting ongoing inflammation. Thus, it further strengthens the notion
that the PRO-C16 biomarker is associated with ECM remodelling and ongoing fibrogenesis”
and down below authors near contradict themselves in L217-219: “Type XVI collagen has
been shown to increase the expression and activation of MMP-9 locally in tissue at the site of
insult30. This could explain the elevated levels of PRO-C16 observed in Crohn’s disease patients
with active disease…” – indeed, although only numerically different (maybe due to the use of
CDAI?), the levels of the biomarker seem to change with activity and this must be accounted for,
as inflammation triggers the ECM turnover. Maybe if results of the analysis are reformulated
preformed with the use of CRP or Fcal these paragraphs may be clarified.
L227-228 – “As a result, luminal CD patients with elevated PRO-C16 serum levels could likely
have increased ECM remodeling and intestinal fibrogenesis with premature and undiagnosed
fibrostenotic stricture.” – Authors show no evidence for this sentence, that is purely speculative.
Please show data on levels of B1 phenotype vs B2 vs. B3, as only data of B2 vs. B1+B3 was
shown.
L230-232 – “While CD patients with the B3 often are presented with concomitant intestinal
fibrosis, we also believe that the PRO-C16 marker measures active fibrogenesis. As such, the
low levels of PRO-C16 in the B3 groups could indicate non-active intestinal fibrogenesis.” As
it has been shown that both phenotypes exhibit fibrosis, these sentences are confusing, please
rephrase.
L272-274 – Based on the murine model…
MINOR COMMENTS
L143 – typo: were purchased
Table 1- % are missing in disease location for Cohort 2
assistant