Contemporary Clinical Trials 97 (2020) 106174

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Contemporary Clinical Trials

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Semaglutide for the treatment of non-alcoholic steatohepatitis: Trial design T

and comparison of non-invasive biomarkers
Stephen A. Harrisona, , Salvatore Calannab, Kenneth Cusic,d, Martin Linderb, Takeshi Okanouee,
⁎

Vlad Ratziuf, Arun Sanyalg, Anne-Sophie Sejlingb, Philip N. Newsomeh,i

a
Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DU, UK
b
Novo Nordisk A/S, 2860 Søborg, Denmark
c
Division of Endocrinology, Diabetes & Metabolism, University of Florida, Gainesville, FL 32608, USA
d
Endocrinology, Diabetes and Metabolism, Malcom Randall VA Medical Center, Gainesville, FL 32608, USA
e
Department of Gastroenterology & Hepatology, Saiseikai Suita Hospital, Suita, Japan
f
Sorbonne University, ICAN – Institute for Cardiometabolism and Nutrition, Hôpital Pitié Salpêtrière, 75013 Paris, France
g
Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23284, USA
h
National Institute for Health Research Birmingham Biomedical Research Centre and Liver Unit at University Hospitals Birmingham NHS Foundation Trust, Birmingham,
UK
i
Centre for Liver & Gastrointestinal Research, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK

ARTICLE INFO ABSTRACT

Keywords: Non-alcoholic steatohepatitis (NASH) is a chronic liver disease. There is a clear need to develop pharmacological
Biomarkers treatment for patients with NASH as well as biomarkers that can diagnose the disease. We describe a trial of
Clinical trial protocol semaglutide treatment for NASH, identify key patient characteristics and compare the relationship of patient
General diabetes characteristics and non-invasive biomarkers/scores.
Hepatology
NCT02970942 is a randomised, double-blind, placebo-controlled, multi-national Phase 2 trial of daily sub­
Non-alcoholic fatty liver disease
cutaneous semaglutide (0.1 mg, 0.2 mg, 0.4 mg) in patients with biopsy-confirmed NASH, F1–F3 fibrosis, NAFLD
Semaglutide
Activity Score ≥ 4, and body mass index (BMI) > 25 kg/m2. Exploratory analyses were performed to evaluate
correlations between baseline parameters and biomarkers in NASH.
Mean (standard deviation [SD]) age of 320 randomised patients was 55 (11) years, mean BMI was
36 (6) kg/m2, and 199 (62%) had type 2 diabetes. Of the total patients, 28% had F1 fibrosis, 23% had F2 fibrosis
and 49% had F3 fibrosis. The highest area under the receiver operating characteristic curve (0.69) for accuracy
in classifying fibrosis stage, F2–3 versus F1, was observed for Fib-4 and Enhanced Liver Fibrosis (ELF). No
substantial correlation between BMI or other clinical or biochemical parameters and fibrosis stage was observed.
In this large Phase 2 trial of semaglutide treatment for NASH, the clinical profile of enrolled patients was
typical for patients with NASH. Of the investigated biomarkers/scores, ELF and Fib-4 showed the most apparent
correlation in classifying fibrosis stage, but had only moderate predictive value.

1. Introduction mortality [3]. However, up to 30% of patients with NAFLD progress

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alco­
holic fatty liver disease (NAFLD) [1,2]. NAFLD encompasses a number
of pathological conditions characterised by excessive hepatic fat de­
position and is a leading cause of liver disease in Western countries [1].
The majority of patients with NAFLD have isolated steatosis, which
carries a relatively benign prognosis, with no overall increase in
from steatosis to NASH [3]. Hence, the estimated global prevalence of
NASH is 1.5–6.5% and an increasing number of patients are developing
NASH-related end-stage liver disease [2,4]. NASH is defined as the
presence of hepatic steatosis and inflammation with hepatocyte injury
(ballooning) with or without fibrosis [2]. While NASH is often asymp­
tomatic, it is a complex progressive condition that can develop over
time into end-stage liver diseases and hepatocellular carcinoma [1,2];

Corresponding author.
⁎

E-mail addresses: stephenharrison87@gmail.com (S.A. Harrison), sca@novonordisk.com (S. Calanna), Kenneth.Cusi@medicine.ufl.edu (K. Cusi),
MLIX@novonordisk.com (M. Linder), okanoue@suita.saiseikai.or.jp (T. Okanoue), vlad.ratziu@inserm.fr (V. Ratziu), arun.sanyal@vcuhealth.org (A. Sanyal),
asji@novonordisk.com (A.-S. Sejling), P.N.Newsome@bham.ac.uk (P.N. Newsome).

https://doi.org/10.1016/j.cct.2020.106174
Received 29 May 2020; Received in revised form 6 October 2020; Accepted 6 October 2020
Available online 08 October 2020
1551-7144/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
S.A. Harrison, et al.
therefore, resolution of NASH is a major endpoint in current pharma­
cological studies.
NASH has consistently been identified as one of the leading causes
of liver transplantation across a range of patients globally [5–7]. Type 2
diabetes mellitus (T2D) and metabolic syndrome are well-known risk
factors for advanced liver disease and have been shown to exacerbate
NASH, and, in turn, the presence of NASH has been shown to have a
greater cardiovascular risk than in patients with isolated steatosis
[1,2,8]. The development of cirrhosis due to NASH is also associated
with poor long-term prognosis [3]. Approximately 20–40% of patients
with NASH will develop significant liver fibrosis, resulting in cirrhosis
in 10–30% of those patients [3,9].
Subjects with NASH are often asymptomatic and there is low
awareness among both subjects and care providers [3,10–12]. Liver
biopsy is currently the only accepted and definitive method of NASH
diagnosis [1,2]. The presence of hepatocellular injury, inflammation
and fibrosis are key histological features that distinguish NASH from
isolated steatosis [3]. However, a liver biopsy can be impractical, prone
to sampling error and complications, and costly [2]. The enrolment of
study subjects into NASH clinical trials is challenging as subjects can be
expected to undertake multiple liver biopsies to confirm the presence of
NASH [12]. Moreover, high screen failure rates have been reported in
NASH studies with up to 50% of screened subjects not meeting the
required eligibility criteria [13,14]. Thus, there is a pressing need to
identify a non-invasive test or NASH biomarkers with predictive cap­
ability for the diagnosis of NASH and for selection of patients for
treatment and monitoring [4,15].
Non-invasive laboratory biomarker models, to indicate less ad­
vanced liver fibrosis, have previously been investigated and include
serum aspartate aminotransferase (AST)-to-platelet ratio index (APRI),
fibrosis-4 (Fib-4), NAFLD Fibrosis Score (NFS) and Enhanced Liver
Fibrosis (ELF) score [16]. Of these markers, Fib-4 and ELF scores have
shown diagnostic accuracy (area under the receiver operating char­
acteristic curve [AUROC] > 0.8) for the assessment of fibrosis [16,17].
NFS, Fib-4 and ELF score predict overall mortality, cardiovascular
mortality and liver-related mortality; additionally, NFS predicts in­
cident diabetes, and changes in NFS are associated with mortality [1].
The tests perform best at distinguishing advanced fibrosis from other
fibrosis stages with negative predictive values higher than positive
predictive values, thus allowing first-line risk stratification to exclude
severe disease [1,18,19]. Additionally, cytokeratin-18 (CK-18) frag­
ments have also been investigated extensively as biomarkers for stea­
tohepatitis [2].
There are currently no FDA-approved pharmacological treatments
for patients with NASH, and therefore there is a substantial unmet
medical need. A growing cohort of clinical development programmes
evaluating novel pharmacotherapies for NASH is emerging, primarily
focused on demonstrating improvement in histological characteristics
of NASH, including steatosis, steatohepatitis and fibrosis [20,21]. In
particular, the use of glucagon-like peptide-1 (GLP-1) analogues is
currently being studied as a possible treatment option in NASH. GLP-1
receptor agonists have pleiotropic effects on several aspects of meta­
bolic syndrome relevant to treatment of NASH, including body weight,
inflammation, and glucose and lipid metabolism [22–24]. Liraglutide, a
GLP-1 analogue indicated for treatment of T2D and weight manage­
ment, has been shown to promote NASH resolution without worsening
of fibrosis [25]. Semaglutide, also a GLP-1 analogue indicated for T2D,
is under evaluation for both weight management and NASH treatment
[26]. Semaglutide has been shown to significantly reduce alanine
aminotransferase (ALT) and high sensitivity C-reactive protein (hsCRP)
in a dose-dependent manner in patients with obesity or T2D [26].
With an increasing number of patients developing NASH-related
end-stage liver disease, there is a need for effective pharmacological
treatments, and a need for non-invasive biomarkers for those at high
risk of liver-related complications. Here we describe the study design of
a Phase 2 trial investigating semaglutide for the treatment of NASH
2
Contemporary Clinical Trials 97 (2020) 106174
(ClinicalTrials.gov identifier NCT02970942) and identify key char­
acteristics of patients with NASH from analysis of the enrolled popu­
lation. Moreover, we analysed how to potentially diagnose F2–F3 fi­
brosis by assessing correlations between the key patient characteristics
and specific biomarkers, including FibroScan®, Fib-4 and ELF, with fi­
brosis stage.
2. Methods
2.1. Trial design
The NCT02970942 trial was a randomised, double-blind, placebo-
controlled, six-armed, parallel group, multi-national dose-finding trial
comparing semaglutide with placebo in patients with NASH. A planned
total of 823 patients were to be screened and, on the basis of an as­
sumed 65% screening failure rate, 288 patients were to be randomised.
2.2. Randomisation and treatment
Randomisation for all patients was controlled by the interactive web
response system. Patients were randomised 3:3:3:1:1:1 to receive either
once-daily subcutaneous semaglutide (0.1 mg, 0.2 mg, 0.4 mg) or cor­
responding injection volumes of placebo for 72 weeks of treatment
(Fig. 1). Dose escalation occurred until the target dose was reached; the
starting dose for all treatment arms was 0.05 mg per day with doses
increasing step-wise every fourth week, first to 0.1, then 0.2, 0.3 and
0.4 mg. Patients did not change the dose once the randomised target
dose was reached. The trial was double-blinded within dose levels;
however, the dose levels were not blinded between each other due to
the different dose escalations, target doses, and volumes required.
Randomisation was stratified in five strata based on region
(Japanese or non-Japanese) and, within the non-Japanese group, based
on diabetes status at screening (with or without T2D) and fibrosis stage
for baseline liver biopsy (F1/F2 or F3). Patients in all treatment groups,
including placebo, received nutritional and physical activity counsel­
ling by a member of the trial team according to local practice.
2.3. Assessments
Assessments of randomised patients were carried out throughout the
trial and included body weight, waist circumference, vital signs, liver
enzymes (ALT, AST, gamma-glutamyl transferase [GGT]), hsCRP, lipids
(total cholesterol, free fatty acids, high-density lipoprotein cholesterol,
low-density lipoprotein cholesterol, triglycerides, very low-density li­
poprotein cholesterol) and glucose metabolism (fasting plasma glucose,
HbA1c, fasting insulin, fasting glucagon, and homeostatic model as­
sessment of insulin resistance). FibroScan® measurements of liver
stiffness and liver steatosis (with controlled attenuation parameter)
were taken if FibroScan® equipment was available.
2.4. Outcome measures
The primary endpoint was NASH resolution without worsening of
fibrosis (defined by an increase of ≥ 1 stage of the Kleiner fibrosis
classification) after 72 weeks. The resolution of NASH was defined per
the NASH Clinical Research Network (CRN) criteria as “no more than
mild residual inflammatory cells (0–1) and no ballooning (0)” based on
comprehensive interpretation by two independent pathologists (central
reading) blinded to treatment allocation. The secondary endpoints in­
cluded safety endpoints as well as changes in: liver-related histological
parameters (including at least one stage of liver fibrosis improvement
with no worsening of NASH), biomarkers of NASH disease, weight-re­
lated and glucose-metabolism-related parameters, cardiovascular risk
factors and patient-reported outcomes.
S.A. Harrison, et al. Contemporary Clinical Trials 97 (2020) 106174

Placebo*

• Biopsy-confirmed
NASH
• F1–F3 Placebo*
• NAS ≥4
• With or without
T2D

Placebo*
7-week FU

4 8 12 18 72
Liver biopsy Time (weeks) Liver biopsy
(recent or new)

from 0.05 mg
every 4 weeks

Fig. 1. Trial design.

a
Matching the active arm with corresponding injection volumes once daily.
F1–F3 fibrosis stage 1 to 3, FU follow-up, NAFLD non-alcoholic fatty liver disease, NAS NAFLD Activity Score, NASH non-alcoholic steatohepatitis, s.c. subcutaneous,
T2D type 2 diabetes

2.5. Statistical analysis
The target sample size of 288 patients was based on an aim to detect
a difference in the primary endpoint between the highest semaglutide
dose and placebo with 90% power. After accounting for trial with­
drawals who would be imputed as non-responders, the proportion of
primary endpoint responders was assumed to be 38% with the highest
semaglutide dose versus 14% with placebo.
The primary analysis was based on the Cochran–Mantel–Haenszel
test [27], which was performed separately for the comparisons between
each of the semaglutide and placebo arms. The three different placebo
arms were pooled into one placebo arm in the statistical analyses. The
test adjusted for baseline diabetes status and baseline fibrosis stage. To
account for multiple testing, the comparisons were evaluated hier­
archically according to descending semaglutide dose using a global
significance level of 5%.
2.6. Trial population
Patients were eligible if they were 18–75 years of age (20–75 years
of age in Japan), and had histological evidence of NASH based on
central pathologist evaluation of a liver biopsy obtained up to 21 weeks
before screening. Other inclusion criteria included an NAFLD Activity
Score (NAS) of ≥ 4 with a score of 1 or more in each sub-component
(steatosis, hepatocyte ballooning, and lobular inflammation) of the
score; a NASH fibrosis stage of 1, 2 or 3 according to the NASH CRN
fibrosis staging system; HbA1c ≤ 10%; and a stable weight with a body
mass index (BMI) > 25 kg/m2. Patients were excluded if they had
causes of chronic liver disease other than NASH; positive hepatitis B
surface antigen, anti-HIV or hepatitis C virus-RNA; ALT or AST le­
vels > 5 × the upper limit of normal; significant alcohol consumption;
type 1 diabetes or current or history of pancreatitis (chronic or acute);
or treatment with GLP-1 receptor agonists or sodium glucose trans­
porter-2 inhibitors within 90 days prior to screening or the liver biopsy.
In the original protocol, only patients with a NASH fibrosis stage of
2 or 3 were eligible. However, a protocol amendment was made on
1 March 2018 to include patients with F1 to reflect the view that some
patients may be “fast progressors” who could benefit from pharmaco­
logical treatment. This amendment was also in line with other Phase 2
NASH trials, for which most have included patients with fibrosis stage
0–3 [28], or 1–3 [13]. The trial protocol is available as part of the
electronic supplementary material.
The trial was conducted in accordance with Good Clinical Practice
guidelines and principles of the Declaration of Helsinki. An independent
ethics committee or institutional review board at each site approved the
trial protocol and any subsequent amendments. Informed consent was
obtained from every participant before any trial-related activities took
place, including activities to determine suitability for the trial.
2.7. Biomarker assessments
One liver biopsy was performed during the screening period (on the
condition that no biopsy within 21 weeks before screening was avail­
able) and one biopsy is scheduled at 72 weeks after randomisation.
Exploratory biomarkers (including CK-18 fragments, and monocyte
chemoattractant protein 1 [MCP-1]) and algorithms (such as Fib-4
score and NFS) were assessed. The ELF panel includes measures of
hyaluronic acid, tissue inhibitor of matrix metalloproteinase-1 and
amino terminal propeptide of procollagen type III and was calculated
using the original algorithm by Guha et al. 2008 [18,29]. The Fib-4
score algorithm incorporates measures of platelets, ALT, AST and age
[29]. The calculation of the NFS requires values for age, hypergly­
caemia, BMI, platelet count, albumin, and AST/ALT ratio [29]. In­
formation on adverse events and hypoglycaemic episodes was collected
throughout the trial period.
2.8. Exploratory correlative and diagnostic analyses
The full analysis set was used in the exploratory correlative and
diagnostic analyses and included all randomised participants.
Exploratory analyses were performed to evaluate the diagnostic accu­
racy of baseline parameters and biomarkers to classify the fibrosis at
baseline. The classes of fibrosis stage were defined by a dichotomous
split (e.g. F3 vs F1–2). A positive correlation between biomarker and
fibrosis stage was assumed, i.e. a high value of the biomarker was as­
sumed to indicate a high fibrosis stage and vice versa. Corresponding
analyses were also performed to evaluate the accuracy in classifying
NAS and its components. Geometric mean and coefficient of variation

3
S.A. Harrison, et al.
were presented for baseline characteristics and biomarker parameters,
unless otherwise specified in the results.
The receiver operating characteristic (ROC) curve was calculated to
describe the relationship of the true positive rate (sensitivity) versus the
false positive rate (1 - specificity) as the cut-point on the biomarker was
moved. The AUROC with the associated approximative 95% confidence
interval (CI) was estimated using the Mann–Whitney statistic. If the
AUROC was greater than 0.5 (supporting a positive correlation), an
optimal cut-point was determined by finding the point which max­
imised Youden's index (sensitivity + specificity - 1). The results were
summarised by presenting the AUROC with 95% CI and, where ap­
plicable, sensitivity, specificity, positive predictive value (PPV) and
negative predictive value (NPV) at the optimal cut-point.
2.9. Patient and public involvement
No patients or the public were involved in the study trial design or
dissemination of results.
3. Results
3.1. Patient characteristics
The study was completed in March 2020 and met its primary end
point; these results will be reported separately. A total of 320 patients
with NASH were randomised in the trial; 28% had F1 fibrosis, 23% had
F2 fibrosis and 49% had F3 fibrosis (Table 1). At baseline, the mean
(standard deviation [SD]) NAS for the total study population was 4.9
(0.89). The mean age was 55 years and the mean BMI was 36 kg/m2;
62% of the patients had T2D (Table 2). Serum levels of ALT and AST
were 54 U/L and 43 U/L, respectively. Patients had serum levels of free
fatty acids of 0.5 mmol/L and triglycerides of 1.9 mmol/L. FibroScan®
score for the 212 patients with available data was 11 kPa (SD 57.4).
Serum levels of free fatty acids, triglycerides and fasting plasma glucose
did not differ substantially between the fibrosis stages. There appeared
to be a slight trend for increased ALT and AST levels with F3 fibrosis,
Table 1
Histopathological characteristics.
Characteristic Total FAS, n (%)
N = 320
Fibrosis stage
F1 90 (28%)
F2 72 (23%)
F3 158 (49%)
Overall NAFLD Activity Score, 0–8
Mean, SD 5 (0.9)
NAFLD Activity Score
4 133 (42%)
5 114 (36%)
6 59 (18%)
7 12 (4%)
8 2 (0.6%)
Steatosis score
1 90 (28%)
2 162 (51%)
3 68 (21%)
Ballooning score
1 218 (68%)
2 102 (32%)
Lobular inflammation score
1 135 (42%)
2 174 (54%)
3 11 (3%)
FAS full analysis set, N number of patients, NAFLD non-al­
coholic fatty liver disease, SD standard deviation.
4
Contemporary Clinical Trials 97 (2020) 106174
compared with F1 or F2. Patients aged ≥ 65 years were more likely to
have F3 fibrosis (n = 36, 62% of the 58 patients aged ≥ 65 years) ra­
ther than F1 (n = 6, 10%) or F2 (n = 16, 28%), and a slightly greater
proportion of patients with F3 fibrosis had T2D compared with those
with F1 or F2. No obvious relationship between BMI and fibrosis stage
was observed.
The demographics and baseline characteristics of the enrolled pa­
tients were also assessed in relation to the NAS (Table S1 in the elec­
tronic supplementary material). In patients with a NAS of 7–8, serum
levels of ALT and AST appeared to be higher and T2D was less prevalent
compared with those with a NAS of 4–6. Serum levels of free fatty acids,
triglycerides and fasting plasma glucose did not differ substantially as a
function of NAS.
In the total population, plasma CK-18 fragment M30 (cleaved CK-
18) levels were 537 U/L and CK-18 fragment M65 (intact CK-18) levels
were 1030 U/L (Table 2). Plasma concentrations of CK-18 fragment
M30 and M65 were higher in patients with F3 fibrosis compared with
F1 and F2. Circulating MCP-1 levels were similar in patients with dif­
ferent fibrosis stages. Mean NFS for the enrolled population was −0.6
(Table 2) and did not greatly differ between the different fibrosis stages.
FibroScan® values were 9.2 kPa for patients with F1 fibrosis, 10.3 kPa
for patients with F2 fibrosis and 11.8 kPa for patients with F3 fibrosis
(Table 2). ELF score values ranged from −0.4 in patients with F1 fi­
brosis to 0.2 in patients with F3 fibrosis. The Fib-4 score appeared to
increase with the severity of fibrosis stage (Table 2).
3.2. Accuracy of baseline characteristics and biomarkers in classifying
fibrosis stage
The accuracy of classifying fibrosis stage (F2–3 vs F1 and F3 vs
F1–2) based on baseline characteristics and biomarkers was assessed
(Table 3). The cut-points were determined by maximising Youden
index, and the sensitivity and specificity were evaluated at the specified
cut-point. The highest AUROC of 0.69 was observed for Fib-4 for ac­
curacy in classifying fibrosis stage, F2–3 versus F1, with a sensitivity of
64% and specificity of 70% at a cut-point of 1.3. An AUROC of 0.69 was
also observed for ELF for accuracy in classifying F2–3 versus F1 fibrosis
with a sensitivity of 62% and specificity of 68% at a cut-point of −0.2.
The next highest AUROC of 0.63 was reported for liver stiffness by
FibroScan®, F2–3 versus F1, with a sensitivity of 89% and specificity of
33% at a cut-point of 7.3. For AST alone, the AUROC was 0.63 for F2–3
versus F1 (sensitivity 57%; specificity 68% at a cut-point of 42 U/L).
The third highest AUROC was reported as 0.60 for NFS F2–3 versus F1
with a sensitivity of 66% and specificity of 52% at a cut-point of −0.9.
Accuracy of Fib-4 and ELF for classifying fibrosis stage (F2–3 vs F1)
was slightly higher for patients with T2D (Fib-4 AUROC 0.73, ELF
AUROC 0.71) than those without (Fib-4 AUROC 0.63, ELF AUROC
0.67). Accuracy of both ALT and AST for fibrosis stage (F2–3 vs F1 and
F3 vs F1–2) was also slightly higher for patients with T2D when com­
pared with patients without T2D (data not shown). Limited accuracy for
classifying fibrosis stage (AUROC < 0.5) or any other histopathological
characteristic (NAS, steatosis score, hepatocyte ballooning score, lob­
ular inflammation score) was observed for BMI (data not shown).
3.3. Boxplots of patient baseline characteristics and biomarkers
Boxplots of biomarkers are provided by fibrosis stage in Fig. 2 and
additional boxplots of baseline characteristics by fibrosis stage are
available in Figs. S1–S4 in the electronic supplementary material. The
box plots show the median, and 1st and 3rd quartiles. The whiskers
extend to minimum and maximum values (including possible outliers).
In agreement with the ROC analyses, the most apparent correlation
with fibrosis stage was seen for ELF and Fib-4.
S.A. Harrison, et al. Contemporary Clinical Trials 97 (2020) 106174

Table 2
Demographics and baseline characteristics by fibrosis stage.
Characteristic Fibrosis stage, n (%) Total FAS, n (%)

1 2 3 N = 320

n = 90 n = 72 n = 158

Female 51 (57%) 47 (65%) 96 (61%) 194 (61%)

Age group, years
18–64 84 (93%) 56 (78%) 122 (77%) 262 (82%)
≥ 65–74 6 (7%) 16 (22%) 33 (21%) 55 (17%)
≥ 75 0 (0%) 0 (0%) 3 (2%) 3 (1%)
Mean (SD) 52 (9) 56 (11) 56 (11) 55 (11)
Ethnicity
Hispanic or Latino 10 (11%) 13 (18%) 17 (11%) 40 (13%)
NA 2 (2%) 4 (6%) 11 (7%) 17 (5%)
Not Hispanic or Latino 78 (87%) 55 (76%) 130 (82%) 263 (82%)
BMI, kg/m2
< 25 0 (0%) 1 (1%) 3 (2%) 4 (1%)
≥ 25 to < 30 14 (16%) 12 (17%) 38 (24%) 64 (20%)
≥ 30 to < 35 19 (21%) 22 (31%) 46 (29%) 87 (27%)
≥ 35 to < 40 32 (36%) 19 (26%) 35 (22%) 86 (27%)
≥ 40 25 (28%) 18 (25%) 36 (23%) 79 (25%)
Mean (SD) 37 (6) 36 (7) 35 (6) 36 (6)
ALT, U/L 51 (64) 49 (58) 59 (58) 54 (60)
AST, U/L 37 (50) 39 (50) 49 (49) 43 (51)
GGT, U/L 57 (72) 54 (78) 72 (79) 63 (79)
HbA1c, mmol/mol
Mean (SD) 51 (14) 50 (14) 50 (13) 50 (13)
Total bilirubin, umol/L 11 (50) 10 (49) 11 (47) 11 (48)
Alkaline phosphatase, U/L 81 (27) 82 (32) 83 (33) 82 (31)
Total chol., mmol/La 5 (23) 5 (29) 5 (23) 5 (25)
High-density lipoprotein chol., mmol/Lb 1 (23) 1 (25) 1 (24) 1 (24)
Very low-density lipoprotein chol., mmol/Lb 0.9 (48) 0.9 (45) 0.8 (45) 0.8 (47)
Triglycerides, mmol/Lb 2.1 (55.7) 2.0 (48.4) 1.7 (47.3) 1.9 (50.9)
Free fatty acids, mmol/Lc 0.5 (46.8) 0.5 (56.6) 0.6 (50.3) 0.5 (50.6)
Type 2 diabetes mellitus, n (%) 49 (54%) 40 (56%) 110 (70%) 199 (62%)
Fasting plasma glucose, mmol/L
Mean (SD)d 7.4 (2.5) 7.6 (3.0) 7.4 (2.2) 7.4 (2.5)
CK-18 fragments M30, U/Lf 482 (86) 463 (87) 609 (73) 537 (81)
CK-18 fragments M65, U/Lf 906 (79) 934 (70) 1158 (60) 1030 (69)
MCP-1, pg/mLf 361 (35) 374 (39) 375 (36) 371 (36)
High sensitivity C-reactive protein, mg/L 4 (165) 4 (183) 4 (157) 4 (164)
Fibrosis-4 scoreg 1.1 (52.2) 1.3 (58.1) 1.6 (58.1) 1.4 (59.3)
NAFLD Fibrosis Scoreg
Mean (SD) −0.9 (2) −0.6 (2) −0.4 (1) −0.6 (2)
ELFa
Mean (SD) −0.4 (0.7) −0.1 (0.9) 0.2 (0.8) −0.0 (0.8)
Liver stiffness by FibroScan®, kPae 9.2 (51) 10.3 (45) 11.8 (64) 10.6 (57)

N (%) or geometric mean (CV) unless otherwise indicated.

ALT alanine aminotransferase, AST aspartate aminotransferase, BMI body mass index, CK-18 cytokeratin-18, CV coefficient of variation, ELF enhanced
liver fibrosis, FAS full analysis set, GGT gamma-glutamyl transferase, MCP-1, monocyte chemotactic protein 1, N number of patients, NA not available,
NAFLD non-alcoholic fatty liver disease, SD standard deviation.
a
n = 316.
b
n = 315.
c
n = 308.
d
n = 319.
e
n = 212.
f
n = 318.
g
n = 307.

3.4. Strengths and limitations
The strengths of the study are its large trial population and a diverse
clinical profile of enrolled patients, typical for patients with NASH.
The presented ROC analyses are subject to limitations due to the
trial being primarily designed to evaluate the effect of semaglutide on
NASH rather than to qualify diagnostic biomarkers. Most importantly,
there is a spectrum bias present since the analyses were conducted on a
population that excluded patients with NAFLD or NASH at the least
severe end of the spectrum as well as those at the most severe end. The
consequences are that both the sensitivity and the specificity would
likely be lower than would be the case in a broader population in which
the biomarker would be used in practice. For example, the accuracy for
classifying F3 versus F1–F2 fibrosis will be lower compared with clas­
sifying F3–F4 versus F0–F2. There may also be other less obvious
sources of spectrum bias such as a non-representative prevalence of
comorbidities, such as T2D. The measures of accuracy such as AUROC
should not therefore be compared with studies using different inclusion
criteria. However, the spectrum bias does not necessarily impact the
internal validity, which means the comparisons of accuracy between
different biomarkers within the same trial would be valid.
Another limitation is the potential presence of a partial verification
bias due to the investigator selecting patients for screening based on
some of the parameters that were evaluated as biomarkers in this paper.

5
S.A. Harrison, et al. Contemporary Clinical Trials 97 (2020) 106174

Table 3
Accuracy of baseline characteristics and biomarkers in classifying fibrosis stage.
Biomarker Fibrosis stage Cut-point Sensitivity Specificity PPV NPV AUROC (95% CI)

Fibrosis-4 score 2–3 vs 1 1.3 64% (139/218) 70% (62/89) 84% (139/166) 44% (62/141) 0.69 (0.62;0.75)
3 vs 1–2 1.3 69% (103/150) 64% (101/157) 65% (103/159) 68% (101/148) 0.67 (0.61;0.73)
NAFLD Fibrosis Score 2–3 vs 1 −0.9 66% (144/218) 52% (46/89) 77% (144/187) 38% (46/120) 0.60 (0.53;0.67)
3 vs 1–2 −0.9 71% (106/150) 48% (76/157) 57% (106/187) 63% (76/120) 0.58 (0.51;0.64)
ALT, U/L 2–3 vs 1 54.0 53% (121/230) 60% (54/90) 77% (121/157) 33% (54/163) 0.55 (0.48;0.62)
3 vs 1–2 68.0 41% (65/158) 74% (120/162) 61% (65/107) 56% (120/213) 0.58 (0.52;0.64)
AST, U/L 2–3 vs 1 42.0 57% (132/230) 68% (61/90) 82% (132/161) 38% (61/159) 0.63 (0.56;0.70)
3 vs 1–2 37.0 73% (116/158) 52% (84/162) 60% (116/194) 67% (84/126) 0.66 (0.60;0.72)
GGT, U/L 2–3 vs 1 70.0 40% (93/230) 72% (65/90) 79% (93/118) 32% (65/202) 0.56 (0.49;0.63)
3 vs 1–2 68.0 49% (77/158) 72% (117/162) 63% (77/122) 59% (117/198) 0.62 (0.56;0.68)
ELF score 2–3 vs 1 −0.2 62% (141/226) 68% (61/90) 83% (141/170) 42% (61/146) 0.69 (0.63;0.76)
3 vs 1–2 −0.1 67% (104/155) 63% (101/161) 63% (104/164) 66% (101/152) 0.68 (0.62;0.74)
CK-18 fragments M30, U/L 2–3 vs 1 260.0 90% (206/229) 26% (23/89) 76% (206/272) 50% (23/46) 0.56 (0.48;0.63)
3 vs 1–2 260.0 94% (149/158) 23% (37/160) 55% (149/272) 80% (37/46) 0.59 (0.53;0.65)
CK-18 fragments M65, U/L 2–3 vs 1 545.0 90% (207/229) 29% (26/89) 77% (207/270) 54% (26/48) 0.58 (0.50;0.65)
3 vs 1–2 545.0 95% (150/158) 25% (40/160) 56% (150/270) 83% (40/48) 0.60 (0.54;0.66)
MCP-1, pg/mL 2–3 vs 1 497.2 21% (48/229) 87% (77/89) 80% (48/60) 30% (77/258) 0.52 (0.45;0.59)
3 vs 1–2 245.1 93% (147/158) 14% (22/160) 52% (147/285) 67% (22/33) 0.51 (0.44;0.57)
Liver stiffness by FibroScan®, kPa 2–3 vs 1 7.3 89% (135/151) 33% (20/61) 77% (135/176) 56% (20/36) 0.63 (0.54;0.71)
3 vs 1–2 11.5 56% (58/104) 71% (77/108) 65% (58/89) 63% (77/123) 0.65 (0.58;0.73)

The cut-points were determined by maximising Youden index. Sensitivity, specificity, PPV and NPV were evaluated at the specified cut-point.
ALT alanine aminotransferase, AST aspartate aminotransferase, AUROC area under the receiver operating characteristic curve, CI confidence interval, CK-18 cy­
tokeratin-18, ELF enhanced liver fibrosis, GGT gamma-glutamyl transferase, MCP-1 monocyte chemotactic protein 1, NAFLD non-alcoholic fatty liver disease, NPV
negative predictive value, PPV positive predictive value.

Potentially affected biomarkers include standard assessments such as
the liver enzymes (ALT, AST and GGT), indices that can be derived from
such assessments (Fib-4 and NAS) as well as FibroScan® where avail­
able. If patients with low values on these biomarkers tended to be ex­
cluded from screening (and consequently from randomisation), it
would lead to an artificial increase in sensitivity but primarily a de­
crease in specificity. There may also be operator-related variation in
liver stiffness measurements made by transient elastometry that were
conducted locally by a number of different operators. A general lim­
itation common for most studies of biomarkers for NASH is that the
reference standard (i.e. the histological score) is itself just a proxy of the
underlying severity of the disease and is known to be imperfect. The
true accuracy of a biomarker that is as good as, or better than, the
reference standard may consequently be underestimated.
4. Conclusions
In this large Phase 2 trial of semaglutide treatment for NASH, the
baseline demographics of the enrolled participants appeared to be
generally consistent with patients with NASH described in other trials

8
2
NAFLD Fibrosis score
Fibrosis-4 score

6
0
4
-2
2
-4
0
1 2 3 1 2 3
Fibrosis stage Fibrosis stage

50
2
Serum ELF-derived

40
1
30
0
20
-1
10
-2 0
1 2 3 1 2 3
Fibrosis stage Fibrosis stage

Fig. 2. Biomarkers by fibrosis stage. Boxplots of Fibrosis-4 score, NAFLD Fibrosis Score, enhanced liver fibrosis score and liver stiffness by FibroScan® by fibrosis
stage.
NAFLD non-alcoholic fatty liver disease.

6
S.A. Harrison, et al.
[13,28,30]. Patients with F3 fibrosis tended to be older and have higher
ALT and AST levels, compared with patients with F1 or F2 fibrosis.
However, serum levels of free fatty acids, triglycerides, and fasting
plasma glucose did not differ substantially between the fibrosis stages.
Similarly, BMI did not appear to be related to fibrosis stage. More pa­
tients with NAS 4–5 had a BMI ≥ 40 kg/m2, were aged ≥ 65–74 years
and were of Hispanic/Latino ethnicity than patients with NAS 6–8.
Overall, there was a higher percentage of patients with T2D with NAS
4–6 than NAS 7–8. AST and ALT levels appeared to be higher in patients
with NAS 7–8. Similar to the fibrosis stages, serum levels of free fatty
acids, triglycerides, and fasting plasma glucose did not differ sub­
stantially between NAS.
Additionally, we performed correlation analyses to determine if
there were any clinical or biochemical data that could identify histo­
logical disease severity among patients with NASH. Both invasive and
non-invasive methods are currently used to detect and monitor NAFLD
and NASH. Non-invasive imaging techniques have improved over time
and may be used to determine the progression of steatosis, fibrosis and
liver stiffness; however, liver biopsies are still considered to be the gold
standard for confirmatory diagnosis of NASH [1,2]. There is a need for
less invasive methodologies that can provide an accurate and reliable
diagnosis of NASH.
FibroScan® is a validated non-invasive method for the assessment of
liver fibrosis and has been used for screening and detection of com­
plications in patients with NAFLD [3,31]. In this trial, FibroScan® did
not appear to have substantial predictive value for screening for ad­
vanced fibrosis stage. However, liver fibrosis assessments using Fi­
broScan® have been shown in some literature to significantly correlate
with Fib-4 values, APRI scores, and AST/ALT ratios for patients with
NAFLD [19,31].
In the present study, Fib-4 and ELF appeared to be better at clas­
sifying fibrosis stage in patients with NASH than other biomarkers.
However, the performance/results were modest and further investiga­
tion of these biomarkers is warranted. The findings are in line with
previous studies in which Fib-4 has been found to have more promising
clinical utility for prediction of fibrosis against some other non-invasive
scoring systems, including NFS, in studies of patients with NAFLD
[19,32–34]. In one study, the AUROC for Fib-4 for identifying patients
with F3–F4 fibrosis was 0.8, which was similar to the NFS (0.77) but
higher than the other scoring systems it was compared against (all <
0.75) in a cohort of 541 patients with NAFLD from the NASH CRN
network [34]. A Fib-4 score of ≥ 2.67 has been shown to have an 80%
PPV and a Fib-4 score of ≤ 1.30 had a 90% NPV for advanced fibrosis
[34]. For ELF, Guha et al. 2008 reported an AUROC of 0.90 for dis­
tinguishing advanced fibrosis (≥ F3; sensitivity 80%, specificity 90%
with a threshold of 0.4) in a study of 196 patients with NAFLD [18].
The ELF score has been endorsed by the National Institute of Health and
Care Excellence (United Kingdom) in their guidelines for management
of NAFLD as a promising predictor of advanced fibrosis stage [35]. It
should be noted that the results from the present study cannot be di­
rectly compared with those obtained from studies due to the different
eligibility criteria for the different studies.
In this study, AST and ALT showed some correlation with fibrosis
stage. Results from the literature are mixed, with some studies finding
typically higher ALT levels in the presence of NASH or advanced fi­
brosis, but others finding no such relationship [36–39]. Moreover, up to
80% of patients with NAFLD [40] and 30–60% of patients with biopsy-
confirmed NASH have normal ALT levels [41]. Overall, ALT and/or AST
may not have high diagnostic capability on their own, but could con­
tribute to the accurate prediction of NASH or fibrosis stage with other
biomarkers. Recently, combination of type IV collagen 7S and AST have
shown a promising ability to predict both NASH and NASH-related fi­
brosis in patients with NAFLD [42]. ALT and/or AST are currently used
in different diagnostic panels, such as the FibroTest [43], NashTest
[44], NFS [45], FibroMeter™ [46], Fib-4 index [47] and other different
models [48,49].
7
Contemporary Clinical Trials 97 (2020) 106174
As part of this trial, patients were stratified by presence of T2D.
Overall, accuracy of Fib-4, ELF, AST and ALT for classifying fibrosis
stage appeared to be fractionally higher for patients with T2D than
those without T2D. Circulating MCP-1 levels, CK-18 fragments M30 and
M65, BMI, HbA1c and fasting plasma glucose showed poor accuracy in
classifying fibrosis stage in patients with or without T2D. Overall, the
diagnostic accuracy of baseline characteristics and biomarkers was not
conclusive for the full population, patients with T2D or patients without
T2D.
In conclusion, the clinical profile of enrolled patients in the
NCT02970942 study was typical for patients with NASH. Fib-4 and ELF
appeared to be better at classifying fibrosis stage than other biomarkers
in the trial. To our knowledge, these data represent one of the largest
analyses of baseline characteristics and biomarkers in patients with
NASH and highlight that there is a need for further investigation of non-
invasive biomarkers for the diagnosis and monitoring of NASH.
Funding
This trial was supported by Novo Nordisk.
Authorship
SAH, SC, KC, ML, TO, VR, AS and PNN were involved in the study
design. SAH, KC, TO, VR, AS and PNN collected the data. ML performed
the statistical data analysis. All authors participated in interpretation of
the data and drafting and revision of the manuscript. All authors re­
viewed and approved the final submitted version.
PNN was supported by the National Institute of Health Research
(NIHR) Birmingham Biomedical Research Centre (BRC). The views
expressed are those of the authors and not necessarily those of the NHS,
the NIHR or the Department of Health.
Medical writing, editorial, and other assistance
Editorial assistance in the preparation of this article was provided
by Anna Bacon of Articulate Science. Support for this assistance was
funded by Novo Nordisk.
Disclosures
SAH reports research grants from Gilead, Intercept, Genfit, Cirius,
NGM Biopharmaceuticals, Novo Nordisk, Novartis, Galmed, Immuron,
Galectin, Madrigal, Conatus, Pfizer, Tobira/Allergan and CymaBay.
SAH has served as an advisor or consultant for Echosens, Allergan,
Metacrine, Perspectum, Prometheus, Galmed, CiVi Biopharma, Corcept,
Madrigal, Pfizer, NGM Biopharmaceuticals, Bristol-Myers Squibb,
Gilead, Intercept, Histoindex, Cirius, Axcella, Genfit, Novo Nordisk,
Novartis, Pharmaceutical Product Development, Medpace, IQVIA,
CymaBay, Chronic Liver Disease Foundation, Innovate, Albireo,
Hightide, Terns, ConSynance, Galectin, Second Genome and Akero, and
he has served as a speaker or a member of a speaker's bureau for AbbVie
and Alexion. KC reports grants and non-financial support for research
support as a Principal Investigator (PI) from Cirius, Inventiva, Janssen,
Novartis, and Zydus; grants, personal fees and non-financial support for
research support as a PI/consultant from Novo Nordisk; and personal
fees for consulting from Bristol-Myers Squibb, Deuterex, Eli Lilly and
Company, Janssen Research and Development LLC, Poxel, and Amgen.
VR reports consultancy from Allergan, Genfit, Intercept, Novartis,
Boehringer Ingelheim, Novo Nordisk, Galmed and Pfizer; he reports
grants from Gilead and Intercept. AS reports grants and consultancy
from Bristol-Myers Squibb, Gilead, Intercept, and Novartis; grants from
Echosens, Merck, and Tobira; consultancy from Galectin, Nitto Denko,
Nimbus, Ardelyx, Vivelix, Teva Pharmaceutical Industries Ltd., Can-
Fite, Boehringer Ingelheim, Pfizer, Salix, and Enyo; royalties from
UpToDate; stocks in Akarna, Durect, Exalenz, HemoShear and Natural
S.A. Harrison, et al.
Shield; board membership from Sanyal Bio; and board membership and
stocks from Genfit and Tiziana. PN reports grants, consultancy and non-
financial support from Novo Nordisk; grants and consultancy from
Boehringer Ingelheim; consultancy from Afimmune, Gilead Sciences,
Intercept Pharmaceuticals, Pfizer and Shire; and speaker fees from
Norgine. SC, ML and A-SS are employees of Novo Nordisk A/S. TO
declares no competing interests.
Compliance with ethics guidelines
An independent ethics committee or institutional review board at
each site approved the trial protocol and any subsequent amendments.
Informed consent was obtained from every participant before any trial-
related activities took place, including activities to determine suitability
for the trial.
Data availability
Deidentified participant data will be made available for this Article
on a specialised SAS data platform. Datasets from Novo Nordisk will be
available permanently after completion of the study and completion of
data analysis. Access to data can be made through a request proposal
form and the access criteria can be found online (novonordisk-trials.
com). Data will be shared with bona fide researchers submitting a re­
search proposal requesting access to data. Data use is subject to ap­
proval by the Independent Review Board.
Thanking patient participants
We gratefully acknowledge the trial participants and all personnel
involved in the trial.
Patient involvement
No patients were involved in the study trial design or dissemination
of results.
Prior presentation
No part of the manuscript has been previously shared nor is based
on work that has been previously presented.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.cct.2020.106174.
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