Journal of Crohn's and Colitis (2014) 8, 1166–1178

Available online at www.sciencedirect.com

ScienceDirect

REVIEW ARTICLE

Results of the 4th Scientific Workshop of the

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ECCO (Group II): Markers of intestinal fibrosis
in inflammatory bowel disease
Florian Rieder a,b,⁎, Jessica R. de Bruyn c , Bao Tung Pham d ,
Konstantinos Katsanos e , Vito Annese f , Peter D.R. Higgins g ,
Fernando Magro h,j , Iris Dotan i,k,⁎⁎
a
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
b
Department of Gastroenterology & Hepatology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland,
OH, USA
c
Academic Medical Center Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
d
Division of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen,
The Netherlands
e
Department of Gastroenterology, University Hospital of Ioannina, Medical School of Ioannina, Greece
f
Division of Gastroenterology, University Hospital Careggi, Florence, Italy
g
Division of Gastroenterology, University of Michigan, Ann Arbor, MI, USA
h
Department of Pharmacology & Therapeutics, Institute for Molecular and Cell Biology, Faculty of Medicine University of Porto,
Porto, Portugal
i
IBD Center, Department of Gastroenterology and Liver Diseases, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

Abbreviations: ASCA, anti-Saccharomyces cerevisiae antibody; ATG16L1, autophagy-related protein 16-1; BAL, bronchoalveolar lavage; bFGF,
basic fibroblast growth factor; BNP, brain natriuretic peptide; CD, Crohn's disease; CF, cystic fibrosis; CCL, chemokine (C–C motif) ligand; CKD,
chronic kidney disease; COMP, cartilage oligomeric protein; CT, computed tomography; CTE, computed tomographic enterography; CTLA4,
cytotoxic T-lymphocyte antigen 4; CTGF, connective tissue growth factor; DLG5, disks large homolog 5; DSS, dextran sulfate sodium; ECCO,
European Crohn's and Colitis Organization; ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblast growth factor; HBV,
hepatitis B virus; HSP47, heat shock protein 47; IBD, inflammatory bowel disease; ICAM, intercellular adhesion molecule 1; ICTP, carboxy
terminal cross-linked telopeptide of type I collagen; IL, interleukin; INR, international normalized ratio; IP, interferon gamma-induced protein;
IPF, idiopathic pulmonary fibrosis; KL-6, Krebs von den Lungen-6; MALDI–TOF-MS, matrix-assisted laser desorption-ionization time-of-flight mass
spectrometry; MC, mesenchymal cell; miRNA, microRNA; MMP, matrix metalloproteinase; MR, magnetic resonance; MRI, magnetic resonance
imaging; mRSS, modified Rodnan skin score; MT, magnetization transfer; MUC, mucin; NOD, nucleotide-binding oligomerization domain
containing; OMUS, obliterative muscularization of the submucosa; PAI-1, plasminogen activator inhibitor-1; PDGF, platelet-derived growth factor;
PNP, amino terminal procollagen; PG-PS, peptidoglycan-polysaccharide; SMA, smooth muscle actin; SP, surfactant protein; SSc, serum of systemic
scleroderma; TERT, telomerase reverse transcriptase; TGF, transforming growth factor; TIMP, tissue inhibitor of matrix metalloproteinase; TNBS,
trinitro-benzene sulfonic acid; TNF, tumor necrosis factor; UC, ulcerative colitis; UEI, ultrasound elasticity imaging; US, ultrasonography; VCAM,
vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor.
⁎ Correspondence to: F. Rieder, The Cleveland Clinic Foundation, Lerner Research Institute, Department of Pathobiology/NC22, 9500 Euclid
Avenue, Cleveland, OH, 44195, USA. Tel.: + 1 216 445 4916; fax: + 1 216 636 0104.
⁎⁎ Correspondence to: I. Dotan, IBD Center, Department of Gastroenterology and Liver Diseases, Tel Aviv Sourasky Medical Center, 6
Weizmann Street, Tel Aviv 64239, Israel. Tel.: + 972 3 6947305; fax: + 972 3 6974184.
E-mail addresses: riederf@ccf.org (F. Rieder), j.r.debruyn@amc.uva.nl (J.R. de Bruyn), b.t.pham@rug.nl (B.T. Pham),
khkostas@hotmail.com (K. Katsanos), annesev@aou-careggi.toscana.it (V. Annese), phiggins@med.umich.edu (P.D.R. Higgins),
fm@med.up.pt (F. Magro), irisd@tasmc.health.gov.il (I. Dotan).

http://dx.doi.org/10.1016/j.crohns.2014.03.009
1873-9946/© 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
Fourth ECCO Scientific Workshop – Markers of Intestinal Fibrosis 1167

j
Department of Gastroenterology, Hospital de Sao Joao, Porto, Portugal
k
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Received 5 February 2014; received in revised form 12 March 2014; accepted 15 March 2014

KEYWORDS Abstract
Inflammatory bowel
diseases; The fourth scientific workshop of the European Crohn's and Colitis Organization (ECCO) focused
Crohn's disease; on intestinal fibrosis in inflammatory bowel disease (IBD). The objective was to better
Ulcerative colitis; understand basic mechanisms and markers of intestinal fibrosis as well as to suggest new

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Fibrosis; therapeutic targets to prevent or treat fibrosis. The results of this workshop are presented in
Biomarkers three separate manuscripts. This section describes markers of fibrosis in IBD, identifies
unanswered questions in the field and provides a framework for future studies addressing the
unmet needs in the field of intestinal fibrosis.
© 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1167
2. Currently available markers of intestinal fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1168
3. Markers of fibrosis from other fibrotic diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1168
3.1. Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169
3.2. Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1170
3.3. Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1170
3.4. Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1170
4. Clinical Situations Where Markers of Fibrosis Should be Used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1171
5. Approaches for Identifying Markers of Fibrosis in Inflammatory Bowel Disease Patients . . . . . . . . . . . . . . . . 1171
5.1. Which Body Compartments/Fluids Should be Investigated in the Quest for Novel Fibrosis Markers? . . . . 1171
5.2. Which Techniques Could be Used to Identify Novel Fibrosis Markers? . . . . . . . . . . . . . . . . . . . . . 1171
5.3. MicroRNA (miRNA) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1171
5.4. Proteome Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1171
5.5. Transcriptomic Screening of Mucosal Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
5.6. How to Find New Markers of Fibrosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
6. Potential Trial Endpoints for Intestinal Fibrosis Marker Evaluations . . . . . . . . . . . . . . . . . . . . . . . . . 1172
6.1. Endoscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
6.2. Histopathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
6.3. Functional Cell Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1173
6.4. Radiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1173
6.5. Defining a Gold Standard for Use in Intestinal Fibrosis Research . . . . . . . . . . . . . . . . . . . . . . . . 1174
7. Summary and Outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174

1. Introduction stricture formation is a frequent indication for surgery in CD4

The natural history of inflammatory bowel disease (IBD) is
highly heterogeneous and frequently complicated by intestinal
fibrosis and stricture formation. This appears to be the case for
both entities of IBD, ulcerative colitis (UC) and Crohn's disease
(CD).1,2 More than 30% of CD and about 5% of UC patients
develop a distinct fibrostenosing phenotype with progressive
narrowing and potential for intestinal obstruction.2,3 Intestinal
and strictures frequently recur leading to repeated surgeries.5
It appears that both entities of IBD and in particular CD exhibit
a progressive nature with changes in disease behavior
throughout the disease course. In CD, which is a transmural
disease, chronic mucosal inflammation induces remodeling of
the entire intestinal wall. This process is a cascade of events
that includes epithelial cell and intestinal damage and repair,
angiogenesis and lymphangiogenesis and activation of immune
1168
cells and mesenchymal cells (MCs). MCs include fibroblasts,
myofibroblasts and smooth muscle cells and are the major
source of extracellular matrix (ECM) components.6
It is difficult to predict which patients will develop a
fibrostenosing phenotype (though a majority will do so
eventually) and how rapidly they will progress. No specific
therapy to prevent or treat intestinal fibrosis is known. To
enable progress in this area, it is essential to identify
markers of intestinal fibrosis, in order to (1) stratify patients
into different levels of risk before the development of
fibrosis, and (2) detect early stages of fibrosis before clinical
symptoms have occurred. An optimal fibrosis marker should
detect early stages of fibrosis, identify trajectory of fibrosis
development, be predictive of future fibrosis, be predictive
of and responsive to the effect of anti-fibrotic therapies and
be predictive of non-responsiveness to anti-inflammatory
therapies. Accomplishing these goals will open the door for
targeted anti-fibrotic therapy, and the ability to test
candidate anti-fibrotic therapies in clinical trials.
This review first summarizes briefly the current status of
markers for intestinal fibrosis, evaluating three distinct areas:
clinical phenotypes, serologic markers, and genetic markers.
Next, available markers from other fibrotic diseases will be
discussed. The main component of the manuscript focuses on
novel approaches to identify and develop markers of fibrosis,
what gold standard to use for trial endpoints, and to which
clinical situations these can be applied to.
2. Currently available markers of
intestinal fibrosis
No specific and accurate predictors or diagnostic tools for
intestinal fibrosis exist and to date no marker of fibrosis is in
routine clinical use. Several targets have been tested for this
purpose (Table 1). Genetic signatures are attractive as they
are stable, present long before the disease onset and are not
affected by alterations in the disease course. Several genes
have been evaluated for their association with fibrostenosing
CD. Alternations in the nucleotide-binding oligomerization
domain containing 2 (NOD2) gene, the first discovered and
best explored genetic variant, are weakly associated not
only with CD fibrostenosis, but also with ileal disease
location and fistulizing disease7 and hence lack specificity.
Other genetic variants have been described as being linked
to fibrostenosis, such as those in the matrix metallopro-
teinase (MMP)-3 gene8 or in the rs1363670 locus near the
interleukin (IL)12B gene.9 Interestingly, an increasing
number of risk alleles, including NOD2, IBD5, disks large
homolog (DLG)5, autophagy-related protein 16-1
(ATG16L1), and IL23 receptor (IL23R), confer an increasing
risk for intestinal resections in CD.10 Gene variants are
promising markers, but their population frequency is low
and they exhibit incomplete penetrance. The major benefit
of genetic biomarkers is their stability over time and
independence from environmental factors, though epige-
netic modifications at the sites of fibrogenesis might prove
important as well.
The most widely used criteria to predict fibrostenosing CD
are clinical factors. These include the need for corticoste-
roids, early disease onset, perianal fistulizing disease or
small bowel disease location.3,11 These factors however are
F. Rieder et al.
Table 1 Currently available markers of intestinal fibrosis.
Reference
Genetic
NOD2 7
MMP-3 8
rs1363670 9
Increasing amount of risk alleles for NOD2, IBD5, 10
DLG5, ATG16L1, and IL23R
Clinical
Need for corticosteroids during first flare 11
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Early disease onset 11
Perianal fistulizing disease 11
Small bowel disease location 3
Serologic
Anti-microbial antibodies 14,15
ECM molecules (Fibronectin, collagen 16–19
propeptides, laminin)
Growth factors (YKL-40, bFGF) 17,20
NOD-2: nucleotide-binding oligomerization domain containing
2, MMP: matrix metalloproteinase, IBD: inflammatory bowel
disease, DLG: disks large, ATG: autophagy, IL23R: interleukin 23
receptor, ECM: extracellular matrix, YKL-40: tyrosine lysine
leucine-40, and bFGF: basic fibroblast growth factor.
encompassing many different disease phenotypes. On the
other hand, the Montreal classification12 merely identifies
fibrosis after it has become clinically apparent and can only
be used as a descriptor rather than a predictor. Thus,
alternative, noninvasive predictive tools are required. One
predictive tool that might be of use as a biomarker is a panel
of serologic markers.
Circulating antibodies against microbial products are
found in some patients with IBD, such as anti-Saccharomyces
cerevisiae (ASCA) among others. These are believed to arise
from aberrant immune responses towards the luminal
microbiota.13 These antibodies are qualitatively and quanti-
tatively associated with, and predictive of a more complicat-
ed disease phenotype, including fibrostenosis.14,15 However,
they are not specific for this phenotype, but rather predict
complicated CD, including fistulizing disease and the need
for surgery. Extracellular matrix molecules and growth
factors, such as laminin, collagens, collagen propeptides or
telopeptides,16–18 basement membrane components or fibro-
nectin,19 YKL-40 (also known as human cartilage glycoprotein
39, a chitinase-like protein),17 basic fibroblast growth factor
(bFGF)20 and others have been investigated as biomarkers of
fibrosis, with inconclusive or negative results.
3. Markers of fibrosis from other
fibrotic diseases
As no existing marker of fibrosis showed specific promise in
IBD in our systematic literature review, we reviewed
markers in fibrotic diseases of extra-intestinal organs,
evaluating whether they could be relevant for further
study in IBD, focusing on markers found in fibrosis of the
liver, lung, kidney, and skin (Table 2).
Fourth ECCO Scientific Workshop – Markers of Intestinal Fibrosis 1169

Table 2 Examples for markers of fibrosis from other fibrotic diseases.

Reference
Liver
PIIINP, hyaluronic acid, and TIMP1 Serum 23,24
Cystatin C Serum 26
Transient elastography ultrasound Liver 27
Magnetic resonance elastography Liver 30
N-cadherin, inter-alpha-trypsin inhibitor heavy chain H4, haptoglobin and Urine 103
serotransferrin
Enolase-1 (α-enolase) and thrombospondin-1 (TSP-1) Serum 76

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Lung
Krebs von den Lungen-6 Serum and BAL 104,105
Surfactant protein-A and -D Serum 105,106
CCL18 Serum and BAL 35
YKL-40 Serum and BAL 36
Osteopontin Plasma and BAL 38,39
Periostin Serum 40
Napsin A Serum 107
Connective tissue growth factor Plasma 41
HSP-47 Serum 42
MMP1 & 7 Serum and BAL 33,34
MMP7, ICAM1, IL8, VCAM1, and S100A12 Plasma 77
Fibrocytes Blood 44
Peripheral T cell subsets Blood 45,46

Skin
miRNAs Serum and tissue 108
ICTP Serum 53
PINP and PIIINP Serum 54,55
Cytokines/chemokines Serum 57–59
CTGF Serum 48
Cartilage oligomeric protein Serum 49
Thrombospondin Serum 52
Osteopontin Plasma 51
MMP9 Serum 50
Number of myofibroblasts Skin biopsy 61
COMP, TSP-1, IFI44, and SIG1 gene expression Skin biopsy 62
Ultrasound Skin 65
Magnetic resonance imaging Skin 64

Kidney
TGFβ1 Urine 71
CTGF Urine 69
PAI-1 Urine 72
Collagen IV Urine 70
PIIINP: N-terminal propeptide of type III collagen, TIMP: tissue inhibitor of matrix metalloproteinase, CCL18: CC chemokine ligand 18, YKL-40:
tyrosine lysine leucine-40, HSP: heat shock protein, MMP: matrix metalloproteinase, ICAM: intercellular adhesion molecule, IL: interleukin,
VCAM: vascular cell adhesion molecule, S100A12: S100 calcium binding protein A12, miR: microRNA, ICTP: carboxy terminal telopeptide of
type I collagen, PINP: N-terminal propeptide of type I procollagen, CTGF: connective tissue growth factor, COMP: cartilage oligomeric matrix
protein, TSP: thrombospondin, IFI44: interferon-induced protein 44, SIG: small inducible gene, TGF: transforming growth factor, and
PAI: plasminogen activator inhibitor.

3.1. Liver markers have been mainly used in hepatitis B or C, and

Liver biopsy remains the gold standard to evaluate fibrosis,
but is an expensive, invasive procedure with potential side
effects and is limited by sampling bias.21 Non-invasive
methods for indirect determination of liver fibrosis are
already established and in clinical use, making this arena the
most advanced in the field of markers of fibrosis. These
scores have been proposed, sometimes in combination with
other markers of liver disease, like transaminases, albumin,
bilirubin, and international normalized ratio (INR).22 Exam-
ples include the amino terminal procollagen III (PIIINP), a
cleavage product of a collagen precursor which is signifi-
cantly correlated with the histological stage of liver fibrosis;
hyaluronic acid, a high molecular weight glycosaminoglycan,
1170
which is an essential component of ECM in virtually every
tissue in the body and substantially increased in hepatic
fibrosis; and tissue inhibitor of metalloproteinase 1 (TIMP-1)
which inhibits interstitial collagenases and metalloprotein-
ases that are capable of degrading ECM.23,24 Liver and serum
TIMP-1 increase in parallel with the progression of the liver
disease. Therefore TIMP-1 has been considered useful in
hepatic fibrosis.25 Another endogenous inhibitor of prote-
ases, serum cystatin C, also correlates with the stage of liver
fibrosis in chronic liver disorders.26
In addition to these molecular approaches, liver fibrosis has
been studied with markers of tissue structure using ultrasound
and magnetic resonance (MR). Transient elastography, an
ultrasound based technique, has the ability to measure tissue
stiffness in an area of 3 cm3.27 It is an easy to perform, widely
operator independent, easy to learn approach.27 This tech-
nique has shown high accuracy in determining fibrosis and
cirrhosis of the liver.28 MR elastography uses the same
principles as transient elastography, but can assess the whole
liver.29,30 However, MR elastography is more costly, takes
longer, and has limitations in certain patients, including those
with iron overload. Imaging and serologic markers have been
combined as well producing increased accuracy.31 A detailed
overview of the state of the field of liver fibrosis markers has
been published by Duarte-Rojo and colleagues.22
3.2. Lung
Given the poor outcomes and lack of effective therapies for
patients with lung fibrosis, an intense effort to identify
markers of fibrosis has been undertaken to facilitate the
development of novel treatment approaches. These markers
could act as surrogates for clinically meaningful outcomes.
Imaging via high resolution CT scan is sufficient for diagnosis
of pulmonary fibrosis32 without employing specific imaging
sequences for fibrosis. Most markers of fibrosis are experi-
mental and from the serum or blood, but the accessibility of
the lung offers the option to evaluate direct disease
processes by obtaining a bronchoscopy with bronchoalveolar
lavage (BAL).
Several markers of fibrosis with direct relevance and
mechanistic plausibility for fibrosis in general have also been
investigated in pulmonary fibrosis: MMPs 1 and 7 were
increased in idiopathic pulmonary fibrosis.33,34 Chemokine
(C–C motif) ligand 18 (CCL18) is chemotactic for fibroblasts,
stimulating their collagen production. In idiopathic pulmo-
nary fibrosis the CCL18 serum level was elevated.35 YKL-40,
which appears to have mitogenic effects on fibroblasts, was
increased in the serum and bronchial lavage fluid of patients
with pulmonary fibrosis and was associated with survival
time in IPF.36,37 Osteopontin is a glycoprotein involved in
tissue repair through profibrogenic activity of fibroblasts and
is elevated in BAL and plasma of patients with IPF compared
to controls.38,39 Periostin, an ECM protein, was significantly
increased in the serum of patients with idiopathic pulmonary
fibrosis compared to healthy controls, and was inversely
correlated with patients' pulmonary function.40 Connective
tissue growth factor (CTGF) exerts profibrotic effects on
fibroblasts and is elevated in IPF plasma.41 Heat shock
protein 47 (HSP47), a collagen-specific molecular chaperone
that mainly functions in biosynthesis and the secretion of
F. Rieder et al.
collagen was significantly higher in the serum of patients
with acute exacerbations of pulmonary fibrosis compared to
those with stable disease.42 Multiple genetic variants are
linked to susceptibility for IPF, such as polymorphisms within
telomerase reverse transcriptase (TERT), IL-1, IL12p40,
MMP1, NOD2 and others.43
An additional novel conceptual approach has been
pursued in pulmonary fibrosis, by identifying circulating
fibroblast precursors, the so-called fibrocytes, as markers of
disease.44 Fibrocytes are believed to be a minor but
significant component of the pathogenesis of pulmonary
fibrosis. Circulating fibrocytes are elevated compared to
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controls in IPF and were increased during exacerbations. In
addition the relative percentages of T-cell subsets (CD4/
CD28 and Tregs) may have utility as a prognostic markers of
fibrosis.45,46
Detailed reviews of the state of the field of pulmonary
fibrosis markers have been published by Huang et al. and Vij
and Noth.42,47
3.3. Skin
The same principles applied in the liver and lung also apply to
the skin in scleroderma. Multiple proteins related to
transforming growth factor β (TGF-β) activity have been
examined in the serum or plasma of patients with scleroder-
ma. Cartilage oligomeric protein (COMP), thrombospondin,
MMP-9, CTGF, and osteopontin are increased in serum or
plasma of systemic scleroderma (SSc) patients and some of
these markers change with disease severity.48–52 Matrix
components and matrix turnover products have been evalu-
ated as well, such as the carboxy terminal telopeptide of type
I collagen,53 amino terminal procollagen I (PINP),54 PIIINP55 or
TIMP1.56 Inflammatory mediators, such as cytokines,
chemokines and markers associated with adaptive immune
cell activation, including IL-6, IL-8, IL-10, IL-13, CCL2, CCL3,
CCL4, soluble CD30 (sCD30) or cytotoxic T-lymphocyte
antigen 4 (CTLA4)57–60 are elevated in SSc, indicating a link
between inflammation and fibrosis in SSc.
SSc offers unusual accessibility of tissue for marker
measurement directly in skin biopsies. Examples include
the determination of the number of myofibroblasts61 or
different TGF-β related gene expression markers.62 Clinical
scores, such as the modified Rodnan skin score (mRSS),63
ultrasound, or skin MRI to measure dermal thickness64,65
have been developed to evaluate the severity of scleroder-
ma, and if responsive to anti-fibrotic therapies, could be
used in clinical trials.
Detailed reviews of the state of the field of scleroderma
fibrosis have been recently published by Lafyatis and
Moinzadeh et al.66,67
3.4. Kidney
While the inciting agents in renal fibrosis are often different
from the liver, lung, and skin, the fibrotic process appears to
be shared. While lab markers of renal function can inform
about progression of renal disease they do not necessarily
reflect fibrotic burden and they can be influenced by a wide
variety of factors.
Fourth ECCO Scientific Workshop – Markers of Intestinal Fibrosis
Unique to the kidney is the accessibility of urine as a
direct, kidney-specific read-out for renal fibrosis, allowing
the identification of local markers of fibrosis. Urine levels of
TGF-β1, CTGF and collagen IV increase with progression of
chronic kidney disease (CKD).68–71 Urine plasminogen acti-
vator inhibitor-1 (PAI-1) has also been shown to correlate
with renal fibrosis in patients with diabetic nephropathy.72
Ultrasound imaging has also entered the field of kidney
fibrosis. Using the doppler ultrasound technique to calculate
the ‘resistive index’ and ‘atrophic index’ has shown promise
in predicting time to dialysis and survival in chronic kidney
disease.73
One of the challenges of universal fibrosis markers is that
they may be nonspecific, so that a patient with Crohn's
disease may appear to have elevated serum markers of
intestinal fibrosis when they have fibrosis in a different
organ (skin, liver, lung, kidney, etc.). There may be benefit
in obtaining markers of intestinal fibrosis from the gut
(biopsies) or its output (stool) to increase the specificity of
markers for intestinal fibrosis.
In summary, we can learn from fibrotic diseases of the
liver, lung, kidney and skin. Multiple potential biologic and
imaging markers are already in clinical use. While
organ-specific markers, such as creatinine for renal disease
or alveolar epithelial cell specific proteins in the lung, likely
will not be helpful markers of intestinal fibrosis, multiple
markers with a direct link with fibrogenesis have been
identified. As fibrotic mechanisms are shared across organs,
these provide possible candidates for use in the intestine as
well.
4. Clinical Situations Where Markers of Fibrosis
Should be Used
After defining an optimal marker of fibrosis, and discussing
existing and potential markers of intestinal fibrosis, we
evaluated the specific clinical situations in which future
markers could be used in intestinal fibrogenesis (Table 3).
5. Approaches for Identifying Markers of Fibrosis
in Inflammatory Bowel Disease Patients
All existing markers carry limitations and no current strategy
for the development of novel markers of fibrosis is
established. As none of the current markers will likely fulfill
all needs of a perfect marker of fibrosis a quest for additional
targets should continue. Markers of fibrosis can be considered
the most critical missing link in the development of novel
therapeutics of intestinal fibrosis. We next evaluate strate-
gies to develop novel markers in IBD-associated fibrosis and
provide examples from investigations in other organs.
5.1. Which Body Compartments/Fluids Should be
Investigated in the Quest for Novel Fibrosis Markers?
Most of the currently examined markers for intestinal
fibrosis focused on genes, clinical factors or serology.
However, reviewing the literature from IBD as well as other
fibrotic diseases outside of the gastrointestinal tract reveals
other potential sampling sources that could be useful for
1171
markers of fibrosis research, and screening tools that may be
helpful in identifying new markers. Potential body products
that may be useful are stool, saliva, breath, or urine
samples. Those may reflect the mediators and cells that
are active in fibrosis, without the need for invasive
procedures such as endoscopy and biopsies, or surgery.
5.2. Which Techniques Could be Used to Identify
Novel Fibrosis Markers?
Using single marker candidates remains a viable option.
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However, this might not reflect the complexity of the
underlying disease process and hence likely lacks accuracy.
Available screening tools, using whole classes of molecules,
instead of just single markers or a small panel of biomarkers,
are virtually endless, but emphasis has been put on the
‘omics’ arena, specifically: proteomics, genomics, metabo-
lomics and transcriptomics. Examples of screening tools and
body compartments explored can be found below.
5.3. MicroRNA (miRNA) Analysis
miRNAs are important post-transcriptional regulators and
are aberrantly expressed in several fibrotic diseases, such as
systemic sclerosis. miRNAs with pro- or antifibrotic proper-
ties were found to be dysregulated in skin fibrosis and were
associated with disease activity and severity.74 Chen et al.
reported that miR-200b is overexpressed in the serum of
CD patients with fibrosis, and conversely administration of
miR-200b could partially protect from fibrogenesis in
vitro.75
5.4. Proteome Analysis
The proteome is the entire set of expressed proteins in a
given type of cell, tissue, or organism, at a given time, under
defined conditions. One study of urine from methotrexate-
induced hepatic fibrosis patients revealed multiple proteins
associated with hepatic fibrosis, including N-cadherin, inter-
alpha-trypsin inhibitor heavy chain H4, haptoglobin and
serotransferrin. Proteomic methods were also used to screen
serum samples from patients with hepatitis B virus infection.
Two-dimensional electrophoresis (2-DE) and matrix-assisted
laser desorption-ionization time-of-flight mass spectrometry
(MALDI–TOF-MS) identified 27 differentially expressed pro-
teins in serum from hepatic fibrosis compared to hepatitis B
virus (HBV) carriers.76 Examining the concentration of 92
proteins in IPF five candidates (MMP7, intercellular adhesion
molecule 1 (ICAM1), IL8, vascular cell adhesion molecule 1
(VCAM1), S100A12) showed that they were associated with
disease progression and mortality.77 A study of the saliva of
asthma and cystic fibrosis patients found that biomarkers
including human VEGF, interferon gamma-induced protein 10
(IP-10), IL-8, epidermal growth factor (EGF), MMP-9, and
IL-1β could be identified in subpicomolar range. It was found
that four of the six proteins were significantly elevated in
asthma and cystic fibrosis (CF) patients compared with
healthy controls.78
1172 F. Rieder et al.

Table 3 Clinical situations where markers of fibrosis should be used.

Clinical situation
Diagnosis of fibrostenosis and
discrimination of fibrotic
from inflammatory stenosis
Assessment of ‘fibrogenic
activity’
Prediction of disease
progression or regression
Rationale
Decision about optimal therapy
(medical versus endoscopic
versus surgical)
Decision about optimal therapy
(medical versus endoscopic versus
surgical)
Prevention of progression in
intestinal fibrosis
References for examples from other organs
MMP1 and MMP7 in IPF34; hyaluronic acid, PIIINP
or TIMP1 in liver fibrosis23,24; cross-linked carboxy
terminal telopeptides of type I collagen (ICTP) and
TIMP-2 in SSc109,110
Mucin 1, YKL-40, SP-A, CCL18 or MMP-7 in
IPF35,37,77,111,112

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No therapy for spontaneously
regressing strictures
Stratification of patient
populations for clinical trials
Early response to therapy Screening of investigational Markers of TGF-β1 activation (e.g. SMAD
anti-fibrotic compounds phosphorylation) in IPF (e.g. clinical trial113);
Reduction in costs for clinical downstream markers of kinase inhibition
trials (VEGF, FGF, PDGF receptor) in a trial involving
receptor tyrosine kinases (e.g. clinical trial114)
Amount of fibrosis/fibrotic Prediction of disease courses Serum TGF-β1 in early SSc115; N-terminal CTGF in
burden Studies on fibrogenic late SSc48; PIIINP and TIMP-2 in SSc110,116; TIMP-1
mechanisms in humans in liver fibrosis117
Prognosis after resection Prevention of re-stenosis Re-fibrosis after liver118–120 or kidney
Prevention of surgical recurrence transplantation121
MMP: matrix metalloproteinase, IPF: idiopathic pulmonary fibrosis, PIIINP: N-terminal propeptide of type III collagen, TIMP: tissue inhibitor
of matrix metalloproteinase, ICTP: carboxy terminal telopeptide of type I collagen, SSc: systemic scleroderma, YKL-40: tyrosine lysine
leucine-40, SP-A: surfactant protein A, TGF: transforming growth factor, VEGF: vascular endothelial growth factor, FGF: fibroblast growth factor,
PDGF: platelet derived growth factor, and CTGF: connective tissue growth factor.

5.5. Transcriptomic Screening of Mucosal Samples
The transcriptome is the set of all RNA molecules, including
mRNA, rRNA, tRNA, and other non-coding RNA produced in
one or a population of cells. This technique was performed in
a study of chronic kidney disease progression. Among the
targets identified, periostin, an extracellular matrix pro-
tein, presented a significantly higher mRNA expression in
more advanced renal fibrosis.79
5.6. How to Find New Markers of Fibrosis?
Regardless of etiology, various biological factors are
involved and interact in the process of chronic remodeling
and fibrosis in the intestine. Thus these may be used as
markers in Crohn's disease in a single candidate approach. In
line with a systems biology approach, however, we suggest
that it could be necessary to quantitatively analyze the
interactions of all components of a biological system in order
to define fibrotic processes in Crohn's disease. The future
could well lie in the creation of a biological “profile” of the
pathology in intestinal fibrosis, integrating all the above
techniques in distinct body compartments.
6. Potential Trial Endpoints for Intestinal
Fibrosis Marker Evaluations
The leap in technologies now available allows the quantifi-
cation of a wide variety of mediators in body compartments
with high accuracy. However the lack of a clear definition of
fibrosis useable for trial endpoints has limited progress in the
field. Currently all available endpoints rely on clinically
apparent strictures that are confirmed by cross-sectional
imaging.
6.1. Endoscopy
Full thickness histopathological sections to assess fibrosis
generally require surgical specimens. While this is an
endpoint of the fibrotic process, it is not a helpful fibrosis
marker. Endoscopy, typically performed in order to assess
mucosal healing, could in the future enable follow-up of
fibrosis. This could be achieved by determination of luminal
diameter — visually or with devices allowing quantification.
Novel optical techniques including confocal endomicroscopy
might also be adapted to measure components of intestinal
fibrosis. Thus, subepithelial myofibroblasts, ECM compo-
nents, and other cell types may be visualized with or without
the use of specific stains. As more insights are gained,
endoscopy could become a useful tool in the assessment and
the follow-up of fibrosis.
6.2. Histopathology
Endoscopic biopsies are accessible during endoscopies and
could serve as potential endpoints for marker trials. The
predominant connective tissue protein in the intestine is
collagen. Type I is the most abundant in the body and its
Fourth ECCO Scientific Workshop – Markers of Intestinal Fibrosis
function is to give tensile strength. Type III is associated with
tissues that require motile structure, type IV is the major
component of epithelial basement membranes and type V is
pericellular and can be produced by smooth muscle cells.80
The presence and composition of collagens have been
investigated in intestinal fibrosis. Intestinal strictures in CD
are characterized by an increase in type V collagen.81
Collagen types IV and V are elevated in the muscularis
propria and around ganglia, while collagen type III is
extensively present in ulcerations.80 Moreover, in CD, a
significant increase in submucosal type III collagen fiber
content has been shown in stenosed intestine, with a
particular increase in the outer aspect of the submucosa.82
Collagen composition may also allow clues as of the
temporal relation of wound healing. When collagen deposi-
tion is rapid, the ratio of type III collagen to type I collagen is
increased. This may be defined as the early stage of fibrosis,
characterized by an increase in the accumulation of collagen
type III in relation to collagen type I. In contrast, during the
late stage of fibrosis, when active collagen deposition
diminishes, the ratio of type III collagen to type I collagen
decreases.83 While collagens predominate, their increase is
not exclusive as other ECM components (i.e. tenascin) are
highly increased in inactive CD and UC.84
Accumulation of myofibroblasts and alterations of the
enteric nerves are associated with fibromuscular oblitera-
tion of the submucosa, and with thickening of the muscularis
propria.85,86 Obliterative muscularization of the submucosa
(OMUS) has been observed in about one-third of small
intestinal resection specimens of Crohn's disease, usually in
stricturing disease.85 OMUS is especially associated with
small bowel strictures, which are themselves closely
associated with submucosal fibrosis.87
To use biopsy histology in order to measure fibrosis is
actually neither simple nor feasible, given the limited depth
of sampling and the possible sampling error. It is however
possible to look at different intestinal histology components
as surrogates for deeper tissue layer fibrosis, such as using
staining for myofibroblasts (α − smooth muscle actin
(αSMA)), tenascin, and for collagen subtypes. This could
also include non-mesenchymal genes, such as epithelial
keratins, as suggested in animal models of colitis.88
6.3. Functional Cell Assays
Intestinal mesenchymal cells, the main producers of ECM in
intestinal fibrosis, can be readily isolated and cultured from
biopsies of primary human tissue. They can then undergo in
vitro evaluations, such as measurement of matrix production
or proliferation89 that could potentially act as markers of the
success or failure of anti-fibrotic therapies. This approach
could be limited if rapid changes in the cell phenotype occur
in culture, and may require immediate analysis of gene
expression (i.e. via single cell real-time PCR).
6.4. Radiology
As mentioned, endoscopic mucosal biopsies are superficial
and are currently not informative about deeper layers of the
bowel. Edema is related to activity and wall fibrosis may be
associated either with active or with inactive disease. New
1173
imaging techniques are being developed and tested in animal
models to detect intestinal fibrosis and assess their potential
translation for human use. The high MRI T2 signal of the
pathologic bowel wall is directly associated with the presence
of edema in the submucosal layer (active disease). In general,
T2-weighted imaging directly expresses the amount of fluids
within the pathological wall, more sensitively and specifically
than other imaging modalities, including ultrasonography and
CT.90 On the other hand, low T2 signal was correlated with
the presence of fibrosis in the intestinal wall.91 The amount of
collagen and fibroblasts is associated with a reduced T2 signal
in the bowel wall, predominantly in the submucosal and
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muscularis propria layers.90 Therefore, T2-signal intensity of
the intestinal wall was directly associated with: (i) the degree
of edema in the submucosal layer, (ii) dilation of sub-mucosal
lymphatic vessels, (iii) and signs of mucosal inflammation and
ulcers. A high T2 signal of the pathologic intestinal wall was
most associated with the degree of submucosal thickening
and edema at histology. It was reported that 97.9% accuracy
for the diagnostic of fibrotic stenosis in CD was achieved by
MRI T2-signal intensity.92 The fibrostenosing phenotype is
related to variable degrees of wall fibrosis and it is
characterized by a low T2-wall signal associated with variable
degrees of wall thickening and bowel dilatation. Following
gadolinium-chelate injection, variable degrees of layered
wall enhancement may be detected.90
Magnetization transfer (MT) is another new imaging
modality. When applied to rats with peptidoglycan-
polysaccharide (PG-PS)-induced fibrosis, the mean MT ratio
in rats with late phase fibrosis was higher than that in
animals with early inflammation and the MT ratio showed a
correlation with the amount of tissue fibrosis.93 Neverthe-
less, MRI data in murine ileitis or colitis are very limited.94–97
T2 relaxometry was also able to discern between the effects
of different cycles of dextran sulfate sodium (DSS) and
correlated with the observed histological changes, allowing
in vivo monitoring of disease status. Tissue edema is
associated with higher water content per pixel. Since
water has a higher T2 than normal tissue, pixels with
substantial tissue edema will be associated with an
increased T2. Thus, the shift of the colon to lower T2 values
with more cycles of DSS can in part be explained by a
decrease of active inflammation over time. The shift in T2 is
likely due to a combination of a lower water content due to a
reduction in inflammation and progressive fibrosis.88 The
sensitivity and specificity of MT in the detection of human
intestinal fibrosis have not yet been determined.
The use of ultrasonography (US) in the assessment of
intestinal fibrosis has been increasing. Contrast-enhanced
US, for evaluation of mural inflammation in CD, with
histopathology as the reference standard, showed signifi-
cantly negative association between the color Doppler grade
and the pathologic fibrostenotic score.98 Contrast enhanced
ultrasound could be useful in distinguishing fibrotic from
inflammatory strictures.98,99 Ultrasound elasticity imaging
(UEI) is a noninvasive method that allows characterization of
intestinal tissue on accurate estimates of tissue motion
(speckle tracking) between two frames before and after
deformation of the tissue. In UEI the tissue is pushing with an
ultrasound transducer, and the tissue deformation with
real-time ultrasound images produce the excitation. Prelim-
inary results in an animal model demonstrate that UEI can
1174
detect intestinal stiffness changes as a result of fibrosis
development, with reasonably high sensitivity and repro-
ducibility.100 When applied to resected bowel segments
from TNBS animals to look for evidence of inflammation and
fibrosis,101 it was able to differentiate acutely inflamed vs.
chronic fibrotic changes, and between unaffected and
fibrotic intestine in a pilot study of Crohn's disease
patients.102 In this small sample of 7 patients with Crohn's
disease, UEI had 100% sensitivity and specificity in differen-
tiating between fibrotic and normal intestine.102 It was
feasible in humans, and the transcutaneous UEI accurately
measures the tissue properties of stenotic segments of the
bowel in patients with CD when compared with the gold
standard of tissue elastometry.102
Computed tomography enterography (CTE) findings of
mesenteric hypervascularity, mucosal hyperenhancement,
and mesenteric fat stranding predict tissue inflammation.
However, small bowel stricture without CTE findings of
inflammation does not necessarily predict the presence of
tissue fibrosis, and inflammation and fibrosis often occur
together in the small intestine in a mixed phenotype in
Crohn's disease.93 Sensitivity and specificity of CTE for
intestinal fibrosis in Crohn's disease have not been reported
using a gold standard of surgical pathology. Therefore,
caution should be used when using CTE criteria to predict
the presence of scar tissue.93
6.5. Defining a Gold Standard for Use in Intestinal
Fibrosis Research
At this point in time there is no gold standard to detect
fibrosis as an endpoint in clinical investigations; however,
using more than one method looking for different pathways
is advisable. We believe that MRI for transmural evaluation,
endoscopy for luminal narrowing, and histopathology for
mucosal and submucosal characterization all offer value,
and should be evaluated in combination in developing and
testing future markers for intestinal fibrosis studies.
Prospective studies for the evaluation of endpoints for
trials are needed to clarify the role of histopathology in
assessing transmural fibrosis and to create and validate a
histologic score for intestinal fibrosis. We need to further
define whether there is truly intestinal fibrosis without
inflammation as the currently available studies might
present a selection bias. No standard exists to define the
amount and type of fibrosis required to classify the specimen
as predominantly fibrotic. Because of selection bias in
surgery, we do not know if there is an association of all
clinically stricturing disease and a histologically predomi-
nant fibrosis phenotype. Further studies are needed to
distinguish between inflammatory and fibrotic strictures.
7. Summary and Outlook
This summary reflects the discussions and ideas raised during
the European Crohn's and Colitis Organization (ECCO)
Scientific Workshop 4 on intestinal fibrosis. Surprisingly,
while being one of the most troubling issues in the care of
IBD, specifically CD patients, this is one of the least
investigated and least therapeutically developed areas in
IBD. In order to advance research in the field of IBD fibrosis,
F. Rieder et al.
we reviewed IBD markers of fibrosis, and markers of fibrosis
in extraintestinal fibrotic diseases. Additionally, fibrosis
endpoints, as may be seen by histology, endoscopy and
imaging studies were discussed. Finally, we investigated the
potential for discovery of markers of fibrosis using rapidly
developing omics technologies. We believe that further
research in this field should pursue the development and
validation of markers, imaging studies, and histology in
order to have a common denominator to assess fibrosis in
future studies and predict clinical outcomes. We also
illustrate key research questions through several clinical
scenarios.
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Conflict of interest statement
None of the authors has a conflict of interest to disclose.
Acknowledgments
No study sponsors had any involvement in study design, data
collection, interpretation or writing of the manuscript.
None of the authors declared any conflict of interest.
This manuscript is a joint scientific workshop activity.
Hence, F.R., J.D.B., B.T.P., K.K., V.A., P.D.R.H., F.M. & I.D.
participated sufficiently, intellectually or practically, in the
work to take public responsibility for the content of the
article, including the conception, design, data interpreta-
tion & writing of the manuscript. The final version of the
manuscript was approved by all authors.
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