Title: Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal
fibrostenotic strictures in patients with Crohn’s disease
Version: 3 Date: 29 Apr 2025
Reviewer's report:
The study investigates the interest to measure a circulating collagen fragment (PRO-C16) for
the diagnosis of intestinal fibrosis in patients with Crohn’s disease. Overall, the study is well-
written and interesting. Please find below some questions, suggestions and minor points:
- Could you please better explain what is PRO-C16 in the introduction? Is it a peptide? Is
it generate by enzymatic digestion? Which enzyme and in which condition it is generated? I
suggest adding an image explaining the process by which PRO-C16 can be measured in blood.
-Could you please state in the method section that disease activity was evaluated with the CDAI?
-Could you please indicate in the legend of the figures which statistical test has been applied?
-“ appropriate cut-off values based on the ROC curves”. Could you please explain what is
appropriate cut-off values?
- The PRO-C16 assay protocol is not well explained (competition ELISA). For instance, without
knowing what is exactly the “biotinylated peptide” (I suppose peptide from PRO-C16) a reader
cannot understand the methodology. “Unbound primary antibodies were washed away”, the
addition of antibodies what not reported, and the target of antibodies was not explained. Could
you please better explain this part?
- Please check Table 1, some percentages for parameters of the cohort 2 are not reported.
-“PRO-C16 did not correlate with disease activity, disease location, gender, or age but PRO-C16
was numerically elevated in CD patients with active disease compared to inactive disease (data
not shown).” Could you please present this data as supplemental? It is surprising that a marker
of fibrosis does not correlate with ileal location. Could it be a question of power? I recommend
not being too definitive with this statement.
-“ PRO-16 did not correlate with disease activity, which could be explained by the disease
activity scores reflecting ongoing inflammation.” I am not well convinced since the CDAI is
based on symptoms, and it does not necessarily reflect the ongoing inflammation. I recommend
not to interpret this result as it could be caused by a lack of power. What is the opinion of the
authors?
- Please consider removing the healthy donor group in Figure 2. The difference of PRO-C16
between healthy donor and Crohn’s disease patients is already shown in Figure 1 (redundant
information).
- “PRO-16”: The C is lacking 2 times.
-Does MMP9 can generate PRO-C16? What is the role of neutrophil enzymes (eg, MMP9) in
generating PRO-C16? If relevant, please discuss this point.
-“Type XVI collagen has been shown to increase the expression and activation of MMP-9 locally
in tissue at the site of insult”. Through the stimulation of neutrophils?
“The two human cohorts included were cross-sectional studies and some discrepancies were
observed e.g. disease activity and diagnosis of fistula” I did not understand this sentence. Could
you please better explain this point?
-There is no treatment for fibrosis. In this context, what could be the interest to monitor the
fibrosis process? Is it for future treatments? Could you please add a small discussion on this
point?
-Could you please discuss rapidly the link between fibrosis and ileal location? Could you please
discuss rapidly the potential link between PRO-C16 and disease location?
Title: Serological assessment of PRO-C16 (type XVI collagen formation) reflects intestinal
fibrostenotic strictures in patients with Crohn’s disease
Version: 3 Date: 29 Apr 2025
Reviewer's report:
The study investigates the interest to measure a circulating collagen fragment (PRO-C16) for
the diagnosis of intestinal fibrosis in patients with Crohn’s disease. Overall, the study is well-
written and interesting. Please find below some questions, suggestions and minor points:
- Could you please better explain what is PRO-C16 in the introduction? Is it a peptide? Is
it generate by enzymatic digestion? Which enzyme and in which condition it is generated? I
suggest adding an image explaining the process by which PRO-C16 can be measured in blood.
-Could you please state in the method section that disease activity was evaluated with the CDAI?
-Could you please indicate in the legend of the figures which statistical test has been applied?
-“ appropriate cut-off values based on the ROC curves”. Could you please explain what is
appropriate cut-off values?
- The PRO-C16 assay protocol is not well explained (competition ELISA). For instance, without
knowing what is exactly the “biotinylated peptide” (I suppose peptide from PRO-C16) a reader
cannot understand the methodology. “Unbound primary antibodies were washed away”, the
addition of antibodies what not reported, and the target of antibodies was not explained. Could
you please better explain this part?
- Please check Table 1, some percentages for parameters of the cohort 2 are not reported.
-“PRO-C16 did not correlate with disease activity, disease location, gender, or age but PRO-C16
was numerically elevated in CD patients with active disease compared to inactive disease (data
not shown).” Could you please present this data as supplemental? It is surprising that a marker
of fibrosis does not correlate with ileal location. Could it be a question of power? I recommend
not being too definitive with this statement.
-“ PRO-16 did not correlate with disease activity, which could be explained by the disease
activity scores reflecting ongoing inflammation.” I am not well convinced since the CDAI is
based on symptoms, and it does not necessarily reflect the ongoing inflammation. I recommend
not to interpret this result as it could be caused by a lack of power. What is the opinion of the
authors?
- Please consider removing the healthy donor group in Figure 2. The difference of PRO-C16
between healthy donor and Crohn’s disease patients is already shown in Figure 1 (redundant
information).
- “PRO-16”: The C is lacking 2 times.
-Does MMP9 can generate PRO-C16? What is the role of neutrophil enzymes (eg, MMP9) in
generating PRO-C16? If relevant, please discuss this point.
-“Type XVI collagen has been shown to increase the expression and activation of MMP-9 locally
in tissue at the site of insult”. Through the stimulation of neutrophils?
“The two human cohorts included were cross-sectional studies and some discrepancies were
observed e.g. disease activity and diagnosis of fistula” I did not understand this sentence. Could
you please better explain this point?
-There is no treatment for fibrosis. In this context, what could be the interest to monitor the
fibrosis process? Is it for future treatments? Could you please add a small discussion on this
point?
-Could you please discuss rapidly the link between fibrosis and ileal location? Could you please
discuss rapidly the potential link between PRO-C16 and disease location?
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: 29 Apr 2025
Reviewer's report:
The study investigates the interest to measure a circulating collagen fragment (PRO-C16) for
the diagnosis of intestinal fibrosis in patients with Crohn’s disease. Overall, the study is well-
written and interesting. Please find below some questions, suggestions and minor points:
- Could you please better explain what is PRO-C16 in the introduction? Is it a peptide? Is
it generate by enzymatic digestion? Which enzyme and in which condition it is generated? I
suggest adding an image explaining the process by which PRO-C16 can be measured in blood.
-Could you please state in the method section that disease activity was evaluated with the CDAI?
-Could you please indicate in the legend of the figures which statistical test has been applied?
-“ appropriate cut-off values based on the ROC curves”. Could you please explain what is
appropriate cut-off values?
- The PRO-C16 assay protocol is not well explained (competition ELISA). For instance, without
knowing what is exactly the “biotinylated peptide” (I suppose peptide from PRO-C16) a reader
cannot understand the methodology. “Unbound primary antibodies were washed away”, the
addition of antibodies what not reported, and the target of antibodies was not explained. Could
you please better explain this part?
- Please check Table 1, some percentages for parameters of the cohort 2 are not reported.
-“PRO-C16 did not correlate with disease activity, disease location, gender, or age but PRO-C16
was numerically elevated in CD patients with active disease compared to inactive disease (data
not shown).” Could you please present this data as supplemental? It is surprising that a marker
of fibrosis does not correlate with ileal location. Could it be a question of power? I recommend
not being too definitive with this statement.
-“ PRO-16 did not correlate with disease activity, which could be explained by the disease
activity scores reflecting ongoing inflammation.” I am not well convinced since the CDAI is
based on symptoms, and it does not necessarily reflect the ongoing inflammation. I recommend
not to interpret this result as it could be caused by a lack of power. What is the opinion of the
authors?
- Please consider removing the healthy donor group in Figure 2. The difference of PRO-C16
between healthy donor and Crohn’s disease patients is already shown in Figure 1 (redundant
information).
- “PRO-16”: The C is lacking 2 times.
-Does MMP9 can generate PRO-C16? What is the role of neutrophil enzymes (eg, MMP9) in
generating PRO-C16? If relevant, please discuss this point.
-“Type XVI collagen has been shown to increase the expression and activation of MMP-9 locally
in tissue at the site of insult”. Through the stimulation of neutrophils?
“The two human cohorts included were cross-sectional studies and some discrepancies were
observed e.g. disease activity and diagnosis of fistula” I did not understand this sentence. Could
you please better explain this point?
-There is no treatment for fibrosis. In this context, what could be the interest to monitor the
fibrosis process? Is it for future treatments? Could you please add a small discussion on this
point?
-Could you please discuss rapidly the link between fibrosis and ileal location? Could you please
discuss rapidly the potential link between PRO-C16 and disease location?
assistant