Research article

Comprehensive assessment of ECM turnover using serum

biomarkers establishes PBC as a high-turnover autoimmune
liver disease
Mette Vesterhus,1,2,3,* Mette Juul Nielsen,4 Johannes Roksund Hov,1,5,6,7 Francesca Saffioti,8,9 Tina Manon-Jensen,4
Diana Julie Leeming,4 Bjørn Moum,5,10 Kirsten Muri Boberg,1,5,6,7 Massimo Pinzani,8 Tom Hemming Karlsen,1,5,6,7
Morten Asser Karsdal,4 Douglas Thorburn8

1
Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo
University Hospital Rikshospitalet, Oslo, Norway; 2Department of Internal Medicine, Haraldsplass Deaconess Hospital, Bergen, Norway; 3Department of
Clinical Science, University of Bergen, Bergen, Norway; 4Fibrosis Biology and Biomarkers, Nordic Bioscience, Herlev, Denmark; 5Institute of Clinical
Medicine, University of Oslo, Oslo, Norway; 6Section of Gastroenterology, Department of Transplantation Medicine, Oslo University Hospital, Oslo,
Norway; 7Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; 8UCL Institute for Liver and Digestive Health,
Division of Medicine, University College London & Royal Free Hospital, London, UK; 9Department of Clinical and Experimental Medicine, Division of
Clinical and Molecular Hepatology, University of Messina, Messina, Italy; 10Division of Medicine, Department of Gastroenterology, Oslo University
Hospital, Oslo, Norway

JHEP Reports 2021. https://doi.org/10.1016/j.jhepr.2020.100178

Background & Aims: Primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH)
are phenotypically distinct autoimmune liver diseases that progress to cirrhosis and liver failure; however, their histological
fibrosis distribution differs. We investigated the extracellular matrix (ECM) profiles of patients with PSC, PBC, and AIH to
establish whether the diseases display differential patterns of ECM turnover.
Methods: Serum samples were retrospectively collected from the UK (test cohort; PSC n = 78; PBC n = 74; AIH n = 58) and
Norway (validation cohort; PSC n = 138; PBC n = 28; AIH n = 27). Patients with ulcerative colitis without liver disease (n = 194)
served as controls. We assessed specific serological biomarkers of ECM turnover: type III and V collagen formation (PRO-C3,
PRO-C5), degradation of type III and IV collagen (C3M, C4M), biglycan (BGM) and citrullinated vimentin (VICM).
Results: Most of the ECM markers showed elevated serum levels in PBC compared with PSC or AIH (p <0.01). PRO-C3
correlated well with liver stiffness and showed the most striking differences between advanced and non-advanced liver
disease; several of the other ECM markers were also associated with stage. PRO-C3 and other ECM markers were inversely
associated with ursodeoxycholic acid treatment response in PBC and remission in AIH. All ECM remodelling markers were
significantly elevated (p <0.05) in patients with PSC, PBC, or AIH compared with ulcerative colitis.
Conclusions: In this first study comparing ECM turnover in autoimmune liver diseases, we found increased ECM turnover in
PBC compared with either PSC or AIH. The study indicates that ECM remodelling is different in PSC, PBC, and AIH, suggesting
differing opportunities for therapeutic intervention.
© 2020 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction 30–40% of ursodeoxycholic acid non-responders exist and

The autoimmune liver diseases primary biliary cholangitis (PBC), progress; AIH patients receive immune modulating therapy
primary sclerosing cholangitis (PSC) and autoimmune hepatitis which induces remission in the majority but maintain a 10 times
(AIH) share scientific and clinical challenges. In PSC, an incom- increased risk of progression to liver transplantation or death
plete comprehension of the pathogenesis and a lack of validated compared with the general population. Thus, a substantial
tools to evaluate effect of novel treatment, have hindered the number of patients with each of the 3 diseases progress to
development of improved therapy. Currently, PBC patients are cirrhosis and liver failure and, although each is rare, collectively,
treated with ursodeoxycholic acid which successfully halts dis- they represent an important indication for liver transplantation.
ease progression in the majority whereas a substantial group of A better comprehension of the pathways driving each individual
disease might facilitate the successful development of highly
warranted effective therapies.1–3
Keywords: Biomarker; Fibrosis; Primary sclerosing cholangitis; Primary biliary
Biomarkers of fibrosis have demonstrated utility in the pre-
cholangitis; PRO-C3.
Received 30 March 2020; received in revised form 21 August 2020; accepted 25 August diction of prognosis in a spectrum of chronic liver diseases,
2020; available online 3 September 2020 including the autoimmune liver diseases, PBC, PSC, and AIH.
* Corresponding author. Address: Department of Internal Medicine, Haraldsplass Histological liver fibrosis stage was demonstrated to predict
Deaconess Hospital, P.O. Box. 6165, N-5892 Bergen, Norway. Tel.: +47-5597-8500.
E-mail address: [email protected] (M. Vesterhus).
clinical outcomes but is currently not recommended in standard
 Research article

diagnostics in PBC and PSC.4,5 Non-invasive markers of fibrosis Norway. Characteristics of the study population are shown in
offer better opportunity for repeated assessments over time and Table 1. Diagnosis of PSC was based on typical cholangiographic
have been reported as effective risk stratifiers for clinical out- findings according to acknowledged criteria; the first patholog-
comes. In PBC and PSC, independently validated biomarkers ical cholangiography defined the time of diagnosis of PSC.20 PBC
include the enhanced liver fibrosis (ELF) test, a serum marker and AIH were diagnosed based on acknowledged criteria.3,20
panel, and liver stiffness measurement (LSM) using transient Duration of disease was defined as the time from the date of
elastography (TE; Fibroscan, Echosens, Paris, France).6–10 How- diagnosis to the date of serum sampling. Cases of PSC or PBC
ever, these tools, developed for chronic liver diseases in general, with features of AIH were included in the PSC and PBC patient
are usually considered as measures of fibrosis load and may not panels, respectively (Table 1). Cases of secondary cholangitis or
capture the diversity in fibrosis distribution and progression rate small duct PSC were excluded. Control sera from 194 patients
between the autoimmune liver diseases reflecting differences in with ulcerative colitis (UC) where PSC had been excluded (all had
pathogenesis, nor the dynamic process of fibrogenesis and normal cholangiograms by magnetic resonance cholangiography
fibrosis degradation and remodelling (reflecting disease activity) and normal alkaline phosphatase [ALP]) were retrieved for
at any given stage of fibrosis. Serological biomarkers specifically comparison from the 20-year follow-up visit of a population-
targeting the extracellular matrix (ECM) remodelling may better based Norwegian cohort.21 All patients provided informed
assess these variations and dynamics. consent in writing. The protocol was in accordance with the
We hypothesised that valuable additional information about Declaration of Helsinki and approved by the regional committee
the dynamics of fibrosis evolution in autoimmune liver disease for research ethics in southeastern Norway (ref. 2011/2572) and
would be provided by estimating the ECM turnover profile using the UK.
serological biomarkers specifically targeting the ECM remodel- For the test panel patients and the validation panel PSC pa-
ling, differentiating between the formation and degradation tients, the respective research databases were revised for infor-
processes related to the various compartments (the interstitial mation on clinical and laboratory data, including ascites,
matrix and the basement membrane). New knowledge regarding encephalopathy, oesophageal varices, variceal bleeding, inflam-
the differences in pathogenesis underlying PSC and the other matory bowel disease (IBD) status, and colorectal or hep-
autoimmune liver diseases could indicate differing opportunities atobiliary malignancy at the time of serum extraction. An IBD
for therapeutic intervention. Furthermore, a dynamic evaluation diagnosis was based on findings at colonoscopy and histology.
of the disease activity using specific markers of ECM turnover Diagnosis of UC and Crohn’s disease were established by
could lead to the identification of sensitive biomarkers for dis- accepted criteria.
ease monitoring and assessment of treatment response in ther- For the test cohort, advanced liver disease was defined based
apeutic trials, responding to an unmet need for surrogates for on LSMs using published cut-off values for TE in PSC and PBC8,9
clinical events particularly in PSC.11 and LSMs or histology in AIH. PBC response was defined ac-
We have previously shown that the marker of type III collagen cording to the Toronto criteria (ALP <1.67 xULN) at 24 months.
formation (PRO-C3) is a strong predictor of prognosis in PSC.12 In AIH remission was defined according to published criteria as
this study, we explored several serological biomarkers specif- normalisation of IgG and alanine aminotransferase (ALT). For the
ically targeting ECM remodelling. Many of these have been PBC and AIH validation and IBD control panels, limited pheno-
demonstrated to be related to various chronic liver diseases as typic information was available. Liver stiffness was not available
either diagnostic,13–15 prognostic,16,17 or surrogate efficacy for the validation panel.
markers.18,19 These markers specifically target the end-product of Biochemical analyses were performed using standard routine
tissue remodelling, that is a neo-epitope resulting from a specific laboratory protocols for tests including platelets, creatinine, in-
protein cleaved by a specific protease, which is released into the ternational normalised ratio (INR), aspartate aminotransferase
circulation and may serve as biomarker for that pathological (AST), ALT, ALP, and gamma-glutamyltransferase (GGT). The AST
process. Combining both the protease and the protein may better to platelet index (APRI) and PSC-specific revised Mayo risk score
assess the dynamic activity of a disease state, compared with were calculated using the published algorithms.22,23
other biomarkers targeting the intact protein. Using this tech- The date of serum sample extraction was identical for the
nique, we investigated the ECM turnover profile of patients with frozen sera used for analyses of biomarkers of ECM turnover and
PSC compared with PBC and AIH in 2 independent cohorts to the ELF test, and routine laboratory biochemical analyses,
evaluate associations with liver stiffness and disease stage for respectively, in all cases of PSC in the validation panel and in all
each disease and to establish whether differential patterns of but 6 cases in the test panel (n = 2 for each of the 3 diseases).
ECM turnover are seen between the diseases.

Biomarkers of ECM turnover

Materials and methods We used validated competitive ELISAs (Nordic Bioscience, Herlev,
Patient panels Denmark) to assess true formation of interstitial matrix collagen
We adopted a 2-step study design (Fig. S1). We explored char- PRO-C3 and type V (PRO-C5), degradation of interstitial matrix
acteristics of ECM turnover in a test panel consisting of PSC pa- collagen type III (C3M) and basement membrane type IV
tients compared with PBC and AIH patients (n = 80, 76, and 57, collagen (C4M), degradation of the proteoglycan biglycan (BGM)
respectively) prospectively recruited at the Royal Free Hospital, and citrullinated type III intermediate filament protein vimentin
UK, then validated main findings in a previously described in- (VICM), in serum samples from all of the patients in each patient
dependent validation panel of Norwegian PSC patients (n = 138) panel. All biomarkers were assessed in a blinded manner ac-
compared with PBC and AIH patients (n = 28 and 27, respec- cording to the manufacturer24–29 and samples were measured
tively), all retrospectively collected from the NoPSC Biobank, within the detection range.

JHEP Reports 2021 vol. 3 j 100178 2

Table 1. Baseline characteristics.

Test panel Validation panel Controls

†
PSC PBC AIH p value* PSC PBC AIH p value UC

N 80 76 57 138 28 27 194
Males, n (%) 54 (67.5) 7 (9.2) 9 (15.8) <0.001 107 (77.5) 4 (13.8) 11 (40.7) <0.001
Age, years, median (range) 46 (20–80) 60 (29–83) 51 (21–76) <0.001 60 (16–72) 60 (31–72) 43 (19–81)
Age at diagnosis, years, 36 (16–80) 49 (29–78) 43 (11–70) <0.001 34 (14–72) n.a. n.a. – n.a.
median (range)
Disease duration, years, 5 (0–28) 7 (0–22) 6 (0–47) n.s. 1.7 (–0.6–29) n.a. n.a. – n.a.
median (range)
Features of AIH, n (%) 7 3 n.a. <0.001 11 1 n.a. <0.001 n.a.
IBD ever, n (%) 56 (70.0) 1 (1.3) 3 (5.2) <0.001 102 (74.4) n.a. n.a. – 194
UC, n (% of all) 49 (61.3) 0 (0) 1 (1.8) <0.001 81 (59.1) n.a. n.a. – 194
(100)
Colorectal malignancy, n (%) 1 0 0 n.s. 5 n.a. n.a. – n.a.
Hepatobiliary malignancy, n (%) 1 1 0 n.s. 1 n.a. n.a. – n.a.
Liver transplantation, n (%) 6 1 0 0.025 31 n.a. n.a. – n.a.
Death, n (%) 2 1 0 n.s. 16 n.a. n.a. – n.a.
Time-follow-up, years, 0.4 (0–1.5) 0.6 (-0.1–1.5) 0.7 (0–1.8) n.s. 0.5 (0–4.2) n.a. n.a. – n.a.
median (range)
Disease stage measures
LSM, kPa, median (range) 10.3 (2.5–75.0) 7.7 (3.0–37.4) 6.9 (2.6–75.0) n.s. n.a. n.a. n.a. –I n.a.
Advanced disease, n (%) 30 (38.0)# 27 (38.0) 18 (33.3) 0.05§ (<0.02)§§ 68 (52.7)#
41 (53.9)‡
ELF score, median (range) 10.1 (7.3–14.3) 10.1 (8.3–12.4) 9.9 (9.4–10.3) n.s. 9.7 (7.1–15.7) n.a. n.a. – n.a.
APRI score, median (range) 0.5 (0.1–10.4) 0.4 (0.09–4.9) 0.3 (0.1–25.3) 0.030 0.5 (0.1–23.7) n.a. n.a. – n.a.
Mayo risk score, -0.3 (-2.3 to 3.7) n.a. n.a. NI 0.1 (–2.4 to 4.1) n.a. n.a. – n.a.
median (range)
Mayo risk score, 49/30 n.a. n.a. NI 61/68 n.a. n.a. – n.a.
low/intermediate-high
groups, n (%)
Laboratory values
ALP, U/L median (range) 198 (21–807) 173 (49–959) 85 (34–218) <0.001 224 (51–1459) n.a. n.a. – n.a.
AST, U/L median (range) 50 (16–919) 43 (17–318) 27 (10–1437) <0.001 68 (16–1219) n.a. n.a. – n.a.
ALT, U/L median (range) 48 (9–796) 48 (11–472) 28 (9–519) <0.001 85 (14–885) n.a. n.a. – n.a.
Albumin, g/L median (range) 44 (21–807) 44 (30–50) 44 (23–53) n.s. 41 (23–50) n.a. n.a. – n.a.
Total bilirubin, lmol/L 13 (3–274) 8 (2–330) 9 (3–124) <0.001 20 (3–532) n.a. n.a. – n.a.
median (range)
INR, median (range) 1 (0.7–3.8) 1 (0.8–1.4) 1 (0.9–2.1) <0.001 1 (0.8–1.8) n.a. n.a. – n.a.
Platelet count, 109/L 244 (53–536) 259 (83–658) 225 (44–480) 0.044 248 (22–903) n.a. n.a. – n.a.
median (range)
Creatinine, median (range) 74 (45–138) 70 (43–166) 68 (43–100) 0.043 65 (37–111) n.a. n.a. – n.a.
GGT, median (range) 186 (13–1594) 116 (27–818) 33 (8–324) <0.001 248 (22–1620) n.a. n.a. – n.a.
* The p-value represents comparison between PSC, PBC, and AIH within the test panel.
†
The p-value represents comparison between PSC, PBC, and AIH within the validation panel.
‡
Defined by LSM using the published cut-off value for F3: n = 41 (53.9%); §PSC (LSM) vs. PBC; §§PSC (LSM) vs. AIH.
#
Defined by Mayo score. The Mann-Whitney U test was used for comparisons between continuous non-normally distributed parameters; Student t test was used when
appropriate. AIH, autoimmune hepatitis; ALP, alkaline phosphatase; ALT, alanine transferase; APRI, AST to platelet ratio index; AST, aspartate transferase; ELF, enhanced liver

ELF test Statistical analyses

We analysed frozen serum samples from the PSC patients using We tested continuous variables for normal distribution and
the commercially available ELF test (Siemens Medical Solution applied the Student t test or the Mann-Whitney U test as
Diagnostics, Inc., Tarrytown, NY, USA). The assays for analysis of appropriate. The collagen III turnover was calculated as PRO-C3/
serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), C3M. We calculated the PSC-specific Mayo risk score for the PSC
hyaluronic acid (HA) and intact N-terminal procollagen type III patients and categorised these patients into low- (<−0), medium-
(PIIINP) were performed using the Siemens ELF test kits con- (>0 to <− 2) and high- (>2) risk groups using published cut-off
taining assays designed specifically for the purpose of generating values. Data are presented as median (range). We explored cor-
the ELF test and an ADVIA Centaur XP analyser (Siemens Medical relations between the novel ECM markers and continuous vari-
Solutions Diagnostics, Inc.). ables using Spearman’s rank test. For calculation of 95%
confidence intervals of rho, the Fisher r-to-z transformation was
used {tanh[arctanh(r) ± 1.96/sqrt(n - 3)]}. Advanced liver disease
Liver stiffness measurements was defined based on published cut-off values; in PSC by LSMs
Prospectively collected LSMs using TE for the assessment of liver (>
−F3; PSC: >9.6 kPa; test panel) or Mayo risk score >0 (both
fibrosis were available for n = 211 patients in the test panel. LSMs panels), in PBC and AIH by LSMs (> −F3; 10.7 and >10.4 kPa,
were performed at median 0 month from the time of biobanking respectively).8,9,30 In addition, we defined advanced disease as
in each diagnostic group (>6 months for n = 5, 4 and 2 patients APRI score >1 for secondary analyses (Supplementary material).
with PSC, PBC, and AIH, respectively). The power of ECM biomarkers and the ELF test to discriminate

JHEP Reports 2021 vol. 3 j 100178 3

 Research article

between patients with and without advanced disease was eval- [7.3–14.3] vs. 10.1 [8.3–12.4], respectively; p = 0.87) (Table 1).
uated by the area under the receiver operating characteristics Information regarding disease stage was not available for PBC or
curve (AUROC) analysis; differences between AUROCs were AIH for the validation panel.
compared with the method of DeLong.31 Optimal cut-off values Comparing PSC test and validation panels, patients showed
to discriminate between patients were obtained from the AUROC similar ELF tests (test panel: n = 45; 10.1 [7.3–14.3] and 9.7
analysis according to the Youden index. We explored associa- [7.2–15.7], respectively, p = 0.66) and APRI scores (p = 0.22)
tions between clinical and laboratory variables and advanced indicating similar levels of fibrosis; whereas the Mayo score was
disease by univariate logistic regression analysis. ECM markers higher and disease duration shorter in the validation panel
were not normally distributed and therefore normalised to ter- compared with the test panel (Mayo score: median [range] -0.3
tiles before analyses; non-normally distributed standard labo- [-2.3–3,7] and 0.1 [-2.4–4.1], p = 0.009; duration: p <0.001).
ratory tests (thrombocytes) were transformed by the natural
logarithm. AUROCs are presented with 95% confidence interval Biomarkers of ECM remodelling in PBC, PSC, and AIH
(CI). Values of p <0.05 were considered significant. Statistical compared with UC controls
analyses were performed using MedCalc (Statistical Software A majority of ECM markers, including PRO-C3, PRO-C5, C3M,
version 16.8.4, MedCalc Software bvba, Ostend, Belgium) and C4M, and BGM, showed an overall difference in serum levels
SPSS (version 24, SPSS Inc., Chicago, IL, USA). Graphs were between PSC, PBC, and AIH in both test and validation panels (p
designed using GraphPad Prism version 8.1.2 (GraphPad Soft- <0.01, Fig. 1). Interestingly, the ECM turnover was overall higher
ware, La Jolla, CA, USA). in PBC compared with the other autoimmune liver diseases.
PRO-C5, C3M, C4M, and BGM were significantly higher (p <0.05
Data availability to p <0.001) in PBC sera compared with PSC as well as AIH in the
Data are available upon request and an appropriate institutional 2 independent panels (Fig. 1). Concerning PRO-C3, findings
collaboration agreement. indicated elevated levels in PBC compared with PSC or AIH but
were inconsistent between the panels (Fig. 1). None of the other
markers showed any consistent significant differences between
Results liver aetiologies. All ECM remodelling markers were significantly
Patients elevated (p <0.05) in patients with PSC compared with UC con-
Patient characteristics are shown in Table 1. We included a test trols in both the test and validation panels (Fig. 1).
panel of 78 PSC patients, 74 PBC patients, and 58 AIH patients as The ratio between formation and degradation of type III
well as an independent validation panel of 138 PSC patients, 28 collagen, that is PRO-C3/C3M is shown in Fig. S2. The ratio was
PBC patients, and 27 AIH patients. Patients with UC with normal elevated in PSC as compared with PBC and AIH in the test panel;
bile duct imaging by magnetic resonance cholangiography (n = however, no differences between aetiologies were found in the
194) served as controls. The test and validation liver disease validation panel, although there was a trend towards higher ratio
panels were similar for each aetiology as regards median age, in PBC as compared with PSC and AIH. In both panels, the ratio
gender distribution (except for a higher male proportion in the was significantly increased as compared with UC controls.
AIH validation panel), the proportion of IBD in PSC patients, and
the proportion of PSC or PBC patients with features of AIH. Associations of the ECM markers with liver disease stage
However, as expected, gender distribution was not equal We explored whether the levels of the ECM markers were
across diagnostic groups, with a male majority amongst PSC different between patients with non-advanced vs. advanced
patients and a strong female predominance for PBC patients disease (see Materials and methods section for definitions).
(males: 67.5, 9.2, and 15.8% in PSC, PBC, and AIH, respectively; Several ECM markers showed elevated levels in advanced dis-
p <0.001). PBC patients were older compared with PSC (median ease, with the most striking differences between advanced and
age 60 vs. 45 years; p <0.001) and median age at diagnosis non-advanced disease for PRO-C3 for all aetiologies (percent
differed between the 3 diagnoses (Table 1). Overall, 70.0% vs. reduction in non-advanced compared with advanced disease:
74.4% of PSC patients had IBD in the test and validation panels, 68%, 56%, and 39% in PSC, PBC, and AIH, respectively) (Table 2),
with UC affecting 61.3% and 59.1%, respectively. Comorbidity underscoring an important association of collagen III formation
with IBD was lower in PBC and AIH patients (Table 1). In the test with advanced disease for all 3 autoimmune liver diseases.
panel, 36 (69.2%) PBC patients were documented ursodeox- In PSC, elevated levels of PRO-C3 and PRO-C3/C3M ratio
ycholic acid responders and 24 AIH patients (46.2%) were in (reflecting the balance between collagen III formation and
remission. degradation) were observed in advanced compared with non-
There was no significant difference in the proportion of pa- advanced disease (Table 2), whereas no significant difference
tients with advanced disease between PSC and PBC patients was demonstrated for the other markers. In multivariate logistic
within the test panel (53.9% vs. 38.0% in PSC and PBC, respec- regression analyses including age, sex, disease duration, ALP,
tively; p = 0.053), whereas in AIH patients there was a proportion Mayo risk score, and a single ECM marker at a time, we
with advanced disease (33.3%) which was similar to PBC demonstrated independent association with advanced PSC for
(p = 0.59) but lower compared with PSC (p <0.02). Disease PRO-C3 as the single variable in the final model (5.57 [2.38,
duration was similar between patients with PSC, PBC, and AIH 13.05], p <0.001). No independent association was found for
(median duration [years]: 5 [0–28], 7 [0–22] and 6 [0–47], other ECM markers. To compare findings in the test and valida-
respectively; p = 0.60). Furthermore, there were no significant tion panels, we further defined advanced compared with
differences between PSC and PBC patients regarding markers non-advanced disease using the Mayo score, confirming the
related to stage including LSMs (10.3 [2.5–75.0] vs. 7.7 [3.0–37.4], associations for PRO-C3 and PRO-C3/C3M ratio with advanced
respectively; p = 0.09), APRI score (0.62 [0.15–12.65] vs. 0.53 PSC. Furthermore, these analyses showed significantly elevated
[0.13–7.07]; p = 0.43) or, in subsets (n = 45), ELF test (10.1 levels in advanced disease for PRO-C5 and C3M in both panels.

JHEP Reports 2021 vol. 3 j 100178 4

 A B Overall p <0.001

***
C Overall p <0.001

Overall p = 0.001 *
*** Overall p <0.001
80 * 3,000 Overall p <0.001 *** 30 *

PRO-C5 (ng/ml)
¤¤ ¤¤ *** ¤
PRO-C3 (ng/ml)

***

C3M (ng/ml)
60 Overall p <0.001 *** ¤¤
¤¤¤ 2,000 20
* *** $
¤¤¤
40
*** ###
$$$ 1,000 $$$
¤ 10
20 ###
###

0 0 0

H
UC

UC

UC

H
AI

AI
PS

PB

AI

PS

PB

AI

PS

PB

PS

PB

AI

PS

PB

PS

PB

AI
Test panel Validation panel Test panel Validation panel Test panel Validation panel

D Overall p <0.001
E Overall p <0.001
Overall p <0.001 F
Overall p <0.001 ** *
***
80 * 30 *** 20
*** ¤¤¤ **
**

VICM (ng/ml)
BGM (ng/ml)

***
C4M (ng/ml)

60 15
¤¤¤ ¤¤ 20 $$$
40 ## ¤ 10
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20 5 ¤¤¤ ¤¤¤
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UC

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AI

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AI
Test panel Validation panel Test panel Validation panel Test panel Validation panel

Fig. 1. ECM markers in patients with PSC, PBC, AIH and UC. Levels of all ECM remodelling markers were higher in all 3 autoimmune liver diseases compared
with UC controls. PBC patients showed higher levels of most ECM markers compared with PSC and AIH. (A) PRO-C3 (marker of type III collagen formation), (B)
PRO-C5 (marker of type V collagen formation), (C) C3M (marker of type III collagen degradation), (D) C4M (marker of type IV collagen degradation), (E) BGM
(marker of biglycan degradation), and (F) VICM (marker of citrullinated vimentin degradation). Comparisons made using the Student t test; asterisks indicate
statistical significances p <0.05. Differences within test and validation panels are indicated by *; differences between test and validation panel are indicated by ¤;
differences between ulcerative colitis (UC) controls and test panel are indicated by $; differences between UC controls and validation panel are indicated by #.
AIH, autoimmune hepatitis; ECM, extracellular matrix; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; UC, ulcerative colitis.

In PBC and AIH, PRO-C3, C3M, and C4M (in both PBC and AIH) of the aetiologies. In PSC, PRO-C3 displayed strong correlation
and PRO-C3/C3M ratio and BGM (in PBC), showed significantly with LSMs (rho >0.5, p <0.001) and excellent correlation with ELF
elevated levels in advanced compared with non-advanced dis- test (validation panel; rho 0.83, p <0.001; Table 4). C3M (both
ease whereas VICM showed lower values in advanced PBC as panels) and C4M (validation panel only) also showed significant
well as AIH (Table 2). Multivariate analysis as above but correlations with LSMs in PSC, linking collagen degradation to
substituting bilirubin and ursodeoxycholic acid response for the liver stiffness in PSC (Table 4). Supporting an association with
Mayo score showed strong and independent association with fibrosis in PSC, in the validation panel, these markers as well as
advanced PBC for PRO-C3 (12.05, 95% CI [2.89, 50.20], p = 0.001) PRO-C5 showed significant correlations with ELF test, a well-
and PRO-C3/C3M ratio (4.33, 95% CI [1.51, 12.40], p = 0.006). In established fibrosis marker panel associated with prognosis in
AIH, multivariate analyses including sex, AIH duration, remission PSC. No correlation with either LSMs or ELF test was seen for
state and 1 ECM marker at a time showed independent associ- BGM and VICM (Table 4). Finally, in PSC, PRO-C3 correlated well
ation only for PRO-C3 (3.49 [1.17, 10.45], p = 0.03). with the Mayo risk score in both PSC panels (rho 0.59 and 0.70,
We performed AUROC analyses of the ability of the ECM respectively; p <0.001). PRO-C5, C3M, and C4M were also
markers to detect advanced disease in PSC, PBC, and AIH, correlated with the Mayo risk score in both panels (Table 4).
respectively (Table 3). Overall, PRO-C3 performed best, showing In PBC and AIH, PRO-C3 showed good correlation with LSMs
excellent (AUC >0.8; in PSC and PBC) or good (AUC = 0.771; AIH) (rho = 0.56 and 0.48, respectively; both p <0.001). Correlations
ability to discriminate advanced from non-advanced disease. with LSMs were also demonstrated for C3M, C4M, and VICM for
Furthermore, in PSC, PRO-C3, and PRO-C3/C3M ratio discrimi- PBC as well as AIH (Table 4).
nated well (AUC >0.7) between mild and advanced disease as
defined by the Mayo score in both panels, whereas discrimina- Associations of the ECM markers with disease activity in PBC
tory ability was also demonstrated for PRO-C5, C3M, and C4M in and AIH
both panels with AUC >0.6, and for BGM (AUC >0.6) in the vali- Several ECM markers were reduced in PBC patients who were
dation panel only (Table 4). In PBC and AIH, PRO-C5, C3M, and ursodeoxycholic acid responders (n = 36; 69.2%) compared with
C4M (both aetiologies) and VICM and BGM (in PBC) discrimi- non-responders (n = 16; 30.8%) (Fig. 2). Responders showed
nated between advanced and non-advanced disease (Table 3). reduced levels for PRO-C3 (median 37.3 vs. 18.6; p = 0.002), PRO-
C5 (1650.3 vs. 1096.3; 0.001), C3M (26.8 vs. 14.1; p <0.001), C4M
Correlations between the ECM markers and LSMs, fibrosis (58.8 vs. 31.6; p <0.001), and BGM (26.7 vs. 18.8; p = 0.04). In
scores and Mayo risk score. addition, increased levels were found in responders for VICM
PRO-C3 showed the strongest correlation with established (4.7 vs. 9.7; p <0.001). There was no difference for LSMs (median
fibrosis markers compared with the other ECM markers for any [range] 7.4 [3.0, 37.4] vs. 10.9 [4.4, 31.2]; p = 0.06).

JHEP Reports 2021 vol. 3 j 100178 5

 Research article

Advanced disease is defined by liver stiffness measurement using published cut-off values by transient elastography for PSC (F3; 9.6 kPa), PBC (F3; 10.7 kPa) and AIH (F3; 10.4 kPa). Median (range) values are given. The Mann-Whitney
In patients with AIH with available data (n = 52), ECM

0.001

0.67
p value

0.25

0.05
0.02
0.02

0.03
markers PRO-C5, C3M, and C4M showed reduced levels in pa-
tients in remission (n = 24; 46.2%) compared with non-remission
(n = 28; 53.8%) with reduced values in remission for PRO-C5,
(0.6–7.9)
(10.6–82.1)

(4.6–26.9)
(0.7–36.3)
(498.2–1,889.5)
(9.5–33.2)
(19.3–70.9)
C3M, and C4M (median 1114.0 vs. 724.2; 14.6 vs. 10.3; 31.9 vs.
24.7; p = 0.001, 0.001 and 0.002, respectively) (Fig. 3) whereas no

U test is used for comparisons. AIH, autoimmune hepatitis; BGM, biglycan marker; ECM, extracellular matrix; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; VICM, citrullinated vimentin.
Advanced

difference across disease activity state was demonstrated for

BGM, VICM, or LSMs.
Test panel

18

35.5

5.1
19.8
1,119.4
15.6

15.4
1.3
AIH

Outcome prediction
We have previously reported that markers of collagen formation
(PRO-C3, PRO-C5) and degradation (C3M, C4M) are associated
(0.2–2.2)
(7.3–35.5)

(14.6–73.2)
(339.6–3,309.6)
(6.1–32.9)

(4.6–39.9)
(0.7–54.4)

with prognosis in PSC.12 Extending analyses to BGM and VICM,

Non-advanced

AUROC analyses showed modest discriminatory ability for BGM

to discriminate between PSC patients who did and did not reach
liver transplantation or death during follow-up (AUC 0.63; p =
0.02), relating biglycan degradation to clinical outcome in PSC for
12.1

27.1
12.3

13.2
36
1.2

11.3
743.2

the first time. Survival times were reduced in high-risk vs. low-
risk groups (defined by optimal cut-off values as decided by
<0.001
<0.001
0.07

0.01

Youden) for BGM (mean survival 1.80 vs. 2.87 [p = 0.009];

p value

0.03

0.05
0.04

Table S1). BGM (hazard ratio 2.34, p = 0.012) was associated with
reaching the clinical endpoint in univariate Cox-regression
analysis (Table S2). Low event-rate and short follow-up in the
(0.5–6.5)

(699.8–3,496.9)
(9.5–44.8)
27

(9.4–107.6)

(25.0–96.9)
(10.0–96.73)
(0.7–59.2)

test panel and missing outcome data for validation panel PBC
and AIH patients precluded analysis for these panels.
Advanced
Test panel

Discussion
19.6
1.7

1,497.9
40.3

50.3
25.1
5.1
PBC
Table 2. Extracellular matrix markers in advanced compared with non-advanced autoimmune liver disease.

In this study, we have dissected the ECM remodelling in 3

autoimmune liver diseases using highly specific, targeted
markers reflecting the dynamic balance between fibrogenesis
44

(8.0–66.9)

(20.1–91.5)
(4.6–47.2)
(0.7–57.6)
(0.4–4.3)

(8.0–80.2)
(211.7–3,464.4)

and fibrosis degradation in the liver. For the first time, we

Non-advanced

demonstrate a striking difference in ECM remodelling indicating

higher turnover in PBC compared with either PSC or AIH. This
difference could not be explained by differences in stage, as the
proportion of patients with advanced liver disease was not
1.1
17.6

16.1
38.7
1,219.6

19.6
9.2

different between PBC and the other autoimmune liver diseases.

Our results highlight the differences between the 3 diseases as
regards fibrosis composition and handling, underscore the
<0.001
<0.001

0.70
p value

0.81
0.05

0.43
0.53

pathogenetic differences between PSC and PBC, and clearly

establish PBC as a high-turnover disease compared with PSC and
AIH.
41

(14.6–65.7)
(0.7–6.4)

(56.1–3,035.2)

(0.7–35.3)
(11.7–104.0)

(7.2–48.9)

(4.6–48.2)

The difference that we observed between PBC and PSC sup-

ports the notion that, despite the fact that PBC and PSC both give
Advanced

rise to biliary-type fibrosis as evidenced by histology, the path-

ogenesis leading to this result may be fundamentally different
Test panel

between the 2 diseases. Whereas the fibrogenesis in PSC is

3.0

12.8

15.8
795.9

7.2
42.2

29.4
PSC

mainly driven by pro-fibrogenic factors derived from ‘reactive’

cholangiocytes, in PBC, the fibrogenesis is driven by a more
defined immune-mediated inflammation and characterised by
35

(5.6–31.4)

(4.6–64.1)
(0.6–5.0)

(15.0–73.4)
(6.8–84.7)
(359.5–2,990.2)

(0.7–36.2)

ECM degradation associated with cholangiocyte damage. Hence,

Non-advanced

portal fibrosis in PBC is more similar to a chronic wound healing

reaction, which could explain the higher ECM turnover
compared with PSC. In AIH, fibrogenesis is initiated in the liver
lobule instead of the portal tract; hence, different pro-fibrogenic
13.3

10.7

15.8
6.8
1.4

808.0

27.2

cells are likely involved. In addition, the ratio between formation

and degradation of type III collagen was significantly increased in
Pro-C3/C3M

all aetiologies as compared with UC controls, further suggesting

not only an increased turnover of type III collagen, but also an
Pro-C3
Pro-C5

VICM

increased net deposition of type III collagen in all aetiologies

BGM
C3M
C4M

compared with UC.

N

JHEP Reports 2021 vol. 3 j 100178 6

Table 3. AUROC analyses of the discriminatory ability of extracellular matrix markers for detecting advanced disease.

Test panel Validation panel

PSC PBC AIH PSC PSC

LSM* LSM* LSM* Mayo Mayo

N advanced/total (%) 37/70 (53) 25/68 (37) 17/52 (33) 30/78 (38) 68/129 (53)
PRO-C3/C3M 0.830 0.767 0.585 0.761 0.772
(0.727, 0.933) (0.641, 0.892) (0.415, 0.755) (0.656, 0.866) (0.692, 0.853)
p <0.001 p <0.001 p = 0.33 p <0.001 p <0.001
PRO-C3 0.855 0.833 0.771 0.820 0.826
(0.766, 0.944) (0.722, 0.943) (0.641, 0.900) (0.727, 0.913) (0.754, 0.898)
p <0.001 p <0.001 p <0.001 p <0.001 p <0.001
PRO-C5 0.498 0.643 0.676 0.655 0.721
(0.360, 0.636) (0.505, 0.781) (0.529, 0.823) (0.524, 0.786) (0.633, 0.809)
p = 0.98 p = 0.04 p = 0.02 p = 0.02 p <0.001
C3M 0.620 0.673 0.708 0.681 0.716
(0.488, 0.752) (0.540, 0.807) (0.566, 0.850) (0.551, 0.810) (0.628, 0.804)
p = 0.09 p = 0.01 p = 0.004 p = 0.01 p <0.001
C4M 0.510 0.668 0.708 0.644 0.761
(0.373, 0.647) (0.533, 0.804) (0.561, 0.855) (0.511, 0.776) (0.679, 0.843)
p = 0.88 p = 0.02 p = 0.01 p = 0.03 p <0.001
BGM 0.419 0.644 0.548 0.535 0.606
(0.284, 0.553) (0.509, 0.780) (0.377, 0.719) (0.398, 0.673) (0.507, 0.704)
p = 0.24 p = 0.04 p = 0.58 p = 0.62 p = 0.04
VICM 0.470 0.280 0.339 0.488 0.504
(0.331, 0.609) (0.149, 0.411) (0.171, 0.506) (0.357, 0.618) (0.404, 0.605)
p = 0.67 p = 0.001 p = 0.06 p = 0.85 p = 0.93
* For analyses involving LSM: panel restricted to patients with LSM available and AST <175. Published cut-off levels for fibrosis (F3) were used (PSC >9.6 kPa, PBC >10.7 kPa;
AIH >10.4 kPa). Mayo risk score cut-off 0 differentiated mild vs. moderate-high risk. Values are given as AUC (95% CI). AIH, autoimmune hepatitis; AUROC, area under the
receiver operator characteristics curve; BGM, biglycan marker; ELF, enhanced liver fibrosis test; LSM, liver stiffness measurement; Mayo, PSC-specific revised Mayo risk score;
PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; VICM, citrullinated vimentin.

We demonstrated the association of several of the novel in osteoporosis.32 This suggests that therapeutic strategies aimed
markers with fibrosis in autoimmune liver diseases, showing at altering the balance between fibrosis formation and degra-
increased levels of markers of collagen formation as well as dation may benefit patients.
degradation, underscoring that the fibrosis in autoimmune liver PRO-C3 showed the strongest association with fibrosis of
diseases represents a relatively high-turnover condition, as an the ECM markers, as underscored by the tight (in PSC and PBC)
analogy to the balance between bone formation and resorption or moderate (in AIH) correlation of PRO-C3 with LSMs and

Table 4. Correlations between extracellular matrix markers and measures of liver fibrosis or prognosis.

Test panel Validation panel

LSM* Mayo ELF Mayo

PSC PBC AIH PSC PSC PSC

N 70 67 52 79 138 129
PRO-C3/C3M Rho 0.533 0.449 0.246 0.473 0.772 0.598
(95% CI) (0.341–0.682) (0.294–0.660) (-0.029–0.486) (0.281–0.628) (0.695–0.832) (0.474–0.699)
p value <0.001 <0.001 0.08 <0.001 <0.001 <0.001
PRO-C3 Rho 0.649 0.555 0.473 0.591 0.830 0.701
(95% CI) (0.489–0.767) (0.363–0.702) (0.230–0.661) (0.425–0.718) (0.770–0.876) (0.601–0.779)
p value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
PRO-C5 Rho 0.090 0.202 0.217 0.233 0.388 0.446
(95% CI) (-0.148–0.318) (-0.040–0.422) (-0.059–0.462) (0.013–0.432) (0.181–0.478) (0.296–0.575)
p value 0.44 0.09 0.12 0.04 <0.001 <0.001
C3M Rho 0.277 0.243 0.263 0.317 0.399 0.449
(95% CI) (0.045–0.481) (0.003–0.457) (-0.011–0.500) (0.103–0.503) (0.248–0.531) (0.299–0.577)
p value 0.02 0.04 0.06 0.004 <0.001 <0.001
C4M Rho 0.160 0.256 0.300 0.279 0.406 0.506
(95% CI) (-0.078–0.381) (0.017–0.467) (0.030–0.530) (0.062–0.471) (0.256–0.537) (0.365–0.624)
p value 0.17 0.03 0.03 0.01 <0.001 <0.001
BGM Rho -0.089 0.121 -0.025 0.084 0.086 0.202
(95% CI) (-0.317–0.149) (-0.123–0.351) (-0.296–0.250) (-0.140–0.230) (-0.082–0.250) (0.030–0.362)
p value 0.45 0.31 0.86 0.46 0.32 0.02
VICM Rho -0.112 -0.367 -0.375 -0.001 -0.098 -0.043
(95% CI) (-0.338–0.126) (-0.558 to -0.139) (-0.588 to -0.114) (-0.222–0.220) (-0.261–0.070) (-0.214–0.131)
p value 0.34 0.002 0.006 0.99 0.25 0.63
* Analysis restricted to patients with AST <175. Correlations were explored using Spearman’s rank test. AIH, autoimmune hepatitis; BGM, matrix metalloproteinase mediated
degradation of biglycan; C3M, degradation of type III collagen; C4M, degradation of type IV collagen; ELF, enhanced liver fibrosis test; LSM, liver stiffness measurement; Mayo,
PSC-specific revised Mayo risk score; PBC, primary biliary cholangitis; PRO-C3 and PRO-C5, formation of type III and V collagen, respectively; PSC, primary sclerosing
cholangitis; VICM, degradation of citrullinated vimentin.

JHEP Reports 2021 vol. 3 j 100178 7

 Research article

A 150 B 4,000 *** C 50 ***

**
PRO-C3 (ng/ml)

PRO-C5 (ng/ml)
40

C3M (ng/ml)
3,000
100
30
2,000
20
50
1,000
10

0 0 0
Non-responders Responders Non-responders Responders Non-responders Responders

D 150
*** E 150
*
F 80
***
60

VICM (ng/ml)
C4M (ng/ml)

BGM (ng/ml)
100 100
40
50 50
20

0 0 0
Non-responders Responders Non-responders Responders Non-responders Responders

Fig. 2. Extracellular matrix markers are different dependent on disease activity in patients with primary biliary cholangitis. The boxplots show significantly
lower levels of extracellular matrix markers in patients with PBC who were ursodeoxycholic acid responders (n = 36) compared with non-responders (n = 16) for
(A) type III collagen formation (PRO-C3; p = 0.002), (B) type V collagen formation (PRO-C5; p = 0.001), (C) C3M (p <0.001), (D) C4M (p <0.001), (E) biglycan (BGM;
< 0.05; ** p <0.005; *** p −
p <0.05), and increased level for (F) citrullinated vimentin (VICM; p <0.001). * p − < 0.001. PBC, primary biliary cholangitis.

serum-based fibrosis scores such as the ELF test and APRI score. fibrosis, including correlations to LSMs and ELF test, presence of
We demonstrated the presence of higher levels in advanced dis- higher levels in advanced compared with non-advanced disease
ease for PRO-C3 for all aetiologies. Overall, PRO-C3 showed the (in PBC and AIH) and good performance for stage discrimination
most consistent difference between stages in autoimmune liver in PBC and AIH but not in PSC. Interestingly, this points to a
disease. In line with this, PRO-C3 performed best for stage possible role for destruction of the basement membrane, which
discrimination, with good to excellent ability to discriminate type IV collagen is the main component of, in the pathogenesis of
advanced from non-advanced disease in PSC as well as PBC the autoimmune liver diseases. Moreover, the associations be-
and AIH. tween these diseases and a BGM are of interest because biglycan
Of the other markers, collagen degradation markers C3M and is an important TGF-beta binding protein that releases TGF-beta
C4M showed most consistent association with other measures of upon degradation, putatively driving fibrogenesis;26 hence our

A 100 B 4,000 *** C 40 ***

80
PRO-C3 (ng/ml)

PRO-C5 (ng/ml)

3,000 30
C3M (ng/ml)

60
2,000 20
40
1,000 10
20

0 0 0
Non-remission Remission Non-remission Remission Non-remission Remission

D 80 *** E 60 F 60

60
VICM (ng/ml)
BGM (ng/ml)
C4M (ng/ml)

40 40
40
20 20
20

0 0 0
Non-remission Remission Non-remission Remission Non-remission Remission

Fig. 3. Extracellular matrix markers are different dependent on disease activity in patients with autoimmune hepatitis. The boxplots show extracellular
matrix markers in patients with autoimmune hepatitis in remission (n = 24) compared with non-remission for (A) type III collagen formation (PRO-C3), (B) type V
collagen formation (PRO-C5), (C) degradation of type III collagen (C3M), (D) degradation of type IV collagen (C4M), (E) biglycan (BGM) and (F) citrullinated
vimentin (VICM). Levels were significantly lower in patients with remission for PRO-C5, C3M and C4M (p = 0.001). *** p − < 0.001.

JHEP Reports 2021 vol. 3 j 100178 8

observations should lead to further exploration of biglycan as a higher compared with that previously found in other liver dis-
therapeutic target. eases such as NAFLD.34,35 We speculate that this might be caused
Thus, our study confirms increased ECM remodelling in by pro-fibrogenic effects of bile acids via activation of portal fi-
advanced compared with non-advanced disease as previously broblasts. In support of this, findings from NGM Bio show that
reported for other liver diseases.16,18,25 Stage definition and the change in bile acid concentration is positively correlated to
evaluation of the level of fibrosis is not straightforward in PSC the change in PRO-C3 in patients with non-alcoholic steatohe-
and PBC as liver biopsy is not routinely performed and because patitis.36,37 Further studies in larger, disease-specific patient
the patchy distribution of fibrosis may affect the reliability of panels are warranted to establish whether PRO-C3 is a good
histology. Hence, we defined advanced disease based on disease- candidate surrogate marker of disease activity in PBC and other
specific cut-off values for moderate fibrosis (F3) for LSMs by TE, autoimmune liver diseases.
for which the association with histological stage and clinical We have previously demonstrated excellent ability for PRO-
outcome has been validated in independent studies in PBC as C3 to detect patients reaching a clinical endpoint during
well as PSC.8,9 Furthermore, to compare test and validation follow-up in PSC, whereas PRO-C5 and C3M showed significant
panels in PSC, we defined disease severity groups using the PSC- but weaker outcome discriminatory abilities.12 The present
specific Mayo risk score, which is the most commonly used extension of the analysis to novel ECM markers relates biglycan
predictive tool in PSC although not validated at the individual degradation to clinical outcome in PSC for the first time and
level.22 We believe that the evaluation of the ECM markers indicates a role for BGM for risk stratification in PSC, although
against several measures of disease stage or severity, including further studies are warranted.
LSMs, strengthens our analysis; however, independent validation The analysis of a broad panel of novel ECM markers in a
of the associations between the ECM markers and LSMs in larger parallel design of patients with 3 different autoimmune liver
disease-specific patient panels is warranted. Conceivably, ECM diseases and the validation in independent panels for each
marker analysis may allow improved stage distinction in PSC as aetiology represent strengths of the study. The number of pa-
well as PBC compared with LSMs, given that serum markers tients differed between aetiologies (PSC > PBC or AIH) with
better reflect the status of the whole organ compared with the relatively small validation groups for PBC and AIH, and this
limited assessment by LSMs which may not be representative in represents a limitation which may have affected power and
diseases with patchy distribution such as PSC and PBC; however, hence results. Stage definition was complicated by the lack of
the present study does not allow any conclusions in this regards. liver biopsy; however, liver biopsy is not routinely indicated in
Our demonstration of excellent correlation of PRO-C3 with either PSC or PBC, furthermore we employed LSMs to define
LSMs and serum scores of fibrosis and the strong ability to stage in the majority of test panel patients. LSMs was not
discriminate between advanced and non-advanced disease, available in Norway at the time of inclusion of patients in the
support the association of PRO-C3 with stage as a marker of validation panel, but we were able to define stage in PSC vali-
prognosis and hence our previous report showing strong pre- dation patients using the Mayo risk score. The levels of several of
dictive ability of PRO-C3 in PSC.12 PRO-C3 is a marker of forma- the ECM markers were different between the test and validation
tion of collagen III, one of the most abundant collagens in liver panels with in general higher levels in the validation panel for
cirrhosis. In contrast, PIIINP which is used as a marker of liver part or all of the aetiologies for a majority of the markers,
fibrosis alone or as part of the ELF test, reflects the total pool of although all of the samples were analysed by the same protocol,
collagen III, including formation, degradation, and stable ver- in the same batch and the same laboratory. We can only spec-
sions of the collagen. Potentially, this might yield improved ulate that this might be related to differences in ethnicity of the
reflection of the dynamics of fibrogenesis to PRO-C3 as compared patients, with a purely Caucasian validation panel and a mixed-
with PIIINP or the ELF test. ethnicity test panel. The short follow-up time and relatively few
We report higher levels of PRO-C3 and other ECM markers in events in the test panel precluded independent validation of
ursodeoxycholic acid non-responders compared with responders analyses regarding the predictive abilities of the ECM markers.
for PBC and in patients with active disease compared with In this first comparison of ECM markers in autoimmune liver
remission for AIH, indicating an association of ECM markers with diseases we found an overall increased ECM turnover in PBC
disease activity in autoimmune liver diseases. Our findings are in compared with either PSC or AIH, indicating that the ECM
line with previous publications demonstrating that PRO-C3 is remodelling is different in PSC, PBC, and AIH. This might suggest
associated with disease activity in HIV patients under treatment differing opportunities for therapeutic intervention for patients
for C3M and C4M;18 moreover, reduction in PRO-C3 levels as well with PSC, PBC, and AIH; hence, further investigation of ECM
as the ELF test was reported following treatment in PSC with remodelling markers is warranted. Furthermore, we have
NGM282, an FGF19 analogue, as a further indication that PRO-C3 demonstrated for the first time the close and consistent associ-
levels may reflect disease activity.33 Interestingly, the serum ation of the ECM remodelling marker PRO-C3 with liver stiffness
levels of the ECM markers observed in this study were much and stage in PSC, PBC, and AIH, respectively.

Abbreviations confidence interval; ECM, extracellular matrix; ELF, enhanced liver

AIH, autoimmune hepatitis; ALP, alkaline phosphatase; ALT, alanine fibrosis; GGT, gamma glutamyltransferase; HYA, hyaluronic acid; IBD,
aminotransferase; AST, aspartate aminotransferase; APRI, AST to platelet inflammatory bowel disease; INR, international normalised ratio; LSM,
ratio index; AUROC, area under the receiver operator characteristics liver stiffness measurement; PBC, primary biliary cholangitis; PIIINP,
curve; BGM, marker of biglycan degradation; C3M, marker of type III N-terminal procollagen type III; PRO-C3, marker of type III collagen for-
collagen degradation; C4M, marker of type IV collagen degradation; CI, mation; PRO-C5, marker of type V collagen formation; PSC, primary

JHEP Reports 2021 vol. 3 j 100178 9

 Research article

sclerosing cholangitis; TE, transient elastography; TIMP-1, tissue inhibitor [9] Corpechot C, Gaouar F, El Naggar A, Kemgang A, Wendum D, Poupon R,
of metalloproteinase; UC, ulcerative colitis; VICM, marker of citrullinated et al. Baseline values and changes in liver stiffness measured by transient
vimentin degradation. elastography are associated with severity of fibrosis and outcomes of
patients with primary sclerosing cholangitis. Gastroenterology
2014;146:970–979. quiz e915-76.
Funding [10] Ehlken H, Wroblewski R, Corpechot C, Arrive L, Rieger T, Hartl J, et al.
The study was sponsored by the Danish Science Foundation. Validation of transient elastography and comparison with spleen length
measurement for staging of fibrosis and clinical prognosis in primary
sclerosing cholangitis. PLoS One 2016;11:e0164224.
Conflicts of interest [11] Ponsioen CY, Chapman RW, Chazouilleres O, Hirschfield GM, Karlsen TH,
MJN, DJL, TM-J, and MAK are full-time employees at Nordic Bioscience. Lohse AW, et al. Surrogate endpoints for clinical trials in primary scle-
MAK and DJL hold stocks in Nordic Bioscience. MJN, DJL and MAK are rosing cholangitis: review and results from an International PSC Study
among the original inventors and patent holders for PRO-C3, PRO-C5, Group consensus process. Hepatology 2016;63:1357–1367.
C3M, and C4M. MV has received fees as an advisory board member for [12] Nielsen MJ, Thorburn D, Leeming DJ, Hov JR, Nygard S, Moum B, et al.
Intercept. DT has received fees for an advisory board with Intercept and Serological markers of extracellular matrix remodeling predict
speakers fees from Intercept and Falk. JRH has served on advisory boards transplant-free survival in primary sclerosing cholangitis. Aliment Phar-
for Orkla Health and Novartis, and received research support from Biogen, macol Ther 2018;48:179–189.
all unrelated to the present study. [13] Leeming DJ, Karsdal MA, Byrjalsen I, Bendtsen F, Trebicka J, Nielsen MJ,
et al. Novel serological neo-epitope markers of extracellular matrix pro-
Please refer to the accompanying ICMJE disclosure forms for further
teins for the detection of portal hypertension. Aliment Pharmacol Ther
details.
2013;38:1086–1096.
[14] Nielsen MJ, Kazankov K, Leeming DJ, Karsdal MA, Krag A, Barrera F, et al.
Authors’ contributions Markers of collagen remodeling detect clinically significant fibrosis in
Guarantor of the article and supervisor of the project: DT. chronic hepatitis C patients. PLoS One 2015;10:e0137302.
[15] Nielsen MJ, Karsdal MA, Kazankov K, Gronbaek H, Krag A, Leeming DJ,
Study conception and design: DT, MV, MAK, THK, MP.
et al. Fibrosis is not just fibrosis – basement membrane modelling and
Collection of biological samples and clinical data: JRH, FS, KMB, BM.
collagen metabolism differs between hepatitis B- and C-induced injury.
Contributed to the designing of the laboratory analyses: DJL, MAK.
Aliment Pharmacol Ther 2016;44:1242–1252.
Performed the laboratory analyses: MJN, TMJ. [16] Nielsen MJ, Veidal SS, Karsdal MA, Orsnes-Leeming DJ, Vainer B,
Contributed to the ELF Test analyses: DT, MP, FS. Gardner SD, et al. Plasma Pro-C3 (N-terminal type III collagen propeptide)
Designed and performed the statistical analyses: MV. predicts fibrosis progression in patients with chronic hepatitis C. Liver Int
Contributed to the interpretation of the data: DT, JRH, KMB, MP, MAK, 2015;35:429–437.
THK, MJN, MV. [17] Nielsen MJ, Lehmann J, Leeming DJ, Schierwagen R, Klein S, Jansen C, et al.
Drafted the manuscript: MV, MJN, DT. Circulating elastin fragments are not affected by hepatic, renal and he-
Reviewed the manuscript for critical content and approved the final modynamic changes, but reflect survival in cirrhosis with TIPS. Dig Dis Sci
version of the manuscript: all authors. 2015;60:3456–3464.
[18] Leeming DJ, Anadol E, Schierwagen R, Karsdal MA, Byrjalsen I, Nielsen MJ,
et al. Combined antiretroviral therapy attenuates hepatic extracellular
Acknowledgements matrix remodeling in HIV patients assessed by novel protein fingerprint
The authors thank Liv Wenche Thorbjørnsen for assistance in the markers. AIDS 2014;28:2081–2090.
collection of serum samples. M. Pinzani and D. Thorburn gratefully [19] Karsdal MA, Hjuler ST, Luo Y, Rasmussen DGK, Nielsen MJ, Holm Nielsen S,
receive funding from UCL NIHR BRC (funding 2017–2022) and PSC part- et al. Assessment of liver fibrosis progression and regression by a sero-
ners (funding 2017–2019). logical collagen turnover profile. Am J Physiol Gastrointest Liver Physiol
2019;316:G25–31.
Supplementary data [20] European Association for the Study of the Liver. EASL Clinical Practice
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