Research Article

Cirrhosis and Liver Failure

Granulocyte-colony stimulating factor (G-CSF) to treat

acute-on-chronic liver failure: A multicenter
randomized trial (GRAFT study)

Graphical abstract Authors

Cornelius Engelmann, Adam Herber,

Annegret Franke, ., Peter R. Galle,
Acute-on-chronic liver failure Granulocyte colony stimulating factor
Anett Schmiedeknecht, Thomas Berg

Coagulation failure
Correspondence
thomas.berg@medizin.uni-leipzig.
Brain failure de (T. Berg).

Lay summary
Liver failure Inefficacy Granulocyte-colony stimulating
factor was considered as a novel
treatment for acute-on-chronic
Cardiocirculatory
Respiratory failure
liver failure (ACLF). We performed
failure
the first randomized, multicenter,
Kidney failure controlled phase II trial, which
showed that G-CSF did not improve
survival or other clinical endpoints
in patients with ACLF. Therefore, G-
Highlights CSF should not be used to treat liver
 G-CSF was expected to be a novel therapy for acute-on-chronic disease outside clinical studies.
liver failure.
 In this first multicenter, randomized phase II trial, G-CSF did not
improve patient survival.

 G-CSF was unable to reduce the rate of complications and did not
improve organ function.

https://doi.org/10.1016/j.jhep.2021.07.033
© 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. J. Hepatol. 2021, 75, 1346–1354
Research Article
Cirrhosis and Liver Failure

Granulocyte-colony stimulating factor (G-CSF) to treat acute-on-

chronic liver failure: A multicenter randomized trial (GRAFT study)
Cornelius Engelmann1,2,3, Adam Herber1, Annegret Franke4, Tony Bruns5,6, Philipp Reuken6,
Ingolf Schiefke7, Alexander Zipprich8, Stefan Zeuzem9, Tobias Goeser10, Ali Canbay11,
Christoph Berg12, Jonel Trebicka9,13,14, Frank E. Uschner9,14, Johannes Chang14, Tobias Mueller2,
Niklas Aehling1, Moritz Schmelzle15, Katrin Splith15, Frank Lammert16, Christian M. Lange9,17,
Christoph Sarrazin9,18, Christian Trautwein5, Michael Manns19, Dieter Häussinger20,
Jan Pfeiffenberger21, Peter R. Galle22, Anett Schmiedeknecht4, Thomas Berg1,*
1
Division of Hepatology, Department of Medicine II, Leipzig University Medical Center, Leipzig, Germany; 2Department of Hepatology and
Gastroenterology, Charité Universitätsmedizin Berlin, Berlin, Germany; 3Berlin Institute of Health (BIH), Berlin, Germany; 4Clinical Trial
Centre (ZKS) Leipzig, Faculty of Medicine, University Leipzig, Leipzig, Germany; 5Department of Medicine III, Aachen University Hospital,
Aachen, Germany; 6Clinic for Internal Medicine IV, University Hospital Jena, Jena, Germany; 7Clinic for Gastroenterology, Hepatology and
Endocrinology, St. Georg Hospital, Leipzig, Germany; 8University Hospital for Internal Medicine 1, Martin-Luther-University Halle-Wittenberg,
Halle, Germany; 9Department of Internal Medicine I, University Hospital Frankfurt, Frankfurt, Germany; 10Clinic for Gastroenterology and
Hepatology, University Hospital Cologne, Cologne, Germany; 11Medical Department, University Hospital Ruhr-University Bochum, Bochum,
Germany; 12Department of Internal Medicine I, University Hospital Tuebingen, Tuebingen, Germany; 13European Foundation for the Study of
Chronic Liver Failure, Barcelona, Spain; 14Department of Internal Medicine I, University of Bonn, Bonn, Germany; 15Department of Surgery,
Campus Charité Mitte | Campus Virchow-Klinik, Charité - Universitätsmedizin Berlin, Berlin, Germany; 16Department of Medicine II, Saarland
University Medical Center, Homburg, Germany; 17Clinic for Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany;
18
Medical Clinic 2, St. Josefs Hospital Wiesbaden, Wiesbaden, Germany; 19Department of Gastroenterology, Hepatology and Endocrinology,
Hannover Medical School, Hannover, Germany; 20Clinic and Policlinic of Gastroenterology, Hepatology and Infectious Disease, Heinrich Heine
University Duesseldorf, Düsseldorf, Germany; 21Department of Gastroenterology and Hepatology, University Hospital Heidelberg, Heidelberg,
Germany; 22Department of Internal Medicine, University Medical Center Mainz, Mainz, Germany

Background & Aims: Based on positive results from small single
center studies, granulocyte-colony stimulating factor (G-CSF) is
being widely used for the treatment of patients with acute-on-
chronic liver failure (ACLF). Herein, we aimed to evaluate the
safety and efficacy of G-CSF in patients with ACLF.
Methods: In this multicenter, prospective, controlled, open-label
phase II study, 176 patients with ACLF (EASL-CLIF criteria) were
randomized to receive G-CSF (5 lg/kg daily for the first 5 days
and every third day thereafter until day 26) plus standard
medical therapy (SMT) (n = 88) or SMT alone. The primary effi-
cacy endpoint was 90-day transplant-free survival analyzed by
Cox regression modeling. The key secondary endpoints were
overall and transplant-free survival after 360 days, the devel-
opment of ACLF-related complications, and the course of liver
function scores during the entire observation period.
Results: Patients treated with G-CSF had a 90-day transplant-
free survival rate of 34.1% compared to 37.5% in the SMT group
(hazard ratio [HR] 1.05; 95% CI 0.711–1.551; p = 0.805).
Transplant-free and overall survival at 360 days did not differ
between the 2 arms (HR 0.998; 95% CI 0.697–1.430; p = 0.992
Keywords: cirrhosis; inflammation; regeneration; stem cell mobilization; ACLF;
transplantation; organ failure.
Received 16 April 2021; received in revised form 8 July 2021; accepted 20 July 2021;
available online 5 August 2021
* Corresponding author. Address: Division of Hepatology, Department of Medicine II,
Leipzig University Medical Center, Liebigstrasse 21, 04103 Leipzig, Germany;
Tel.: 00493419712330.
E-mail address: thomas.berg@medizin.uni-leipzig.de (T. Berg).
https://doi.org/10.1016/j.jhep.2021.07.033
and HR 1.058; 95% CI 0.727–1.548; p = 0.768, respectively). G-CSF
did not improve liver function scores, the occurrence of in-
fections, or survival in subgroups of patients without infections,
with alcohol-related ACLF, or with ACLF defined by the APASL
criteria. Sixty-one serious adverse events were reported in the G-
CSF+SMT group and 57 were reported in the SMT group. In total,
7 drug-related serious adverse reactions occurred in the G-CSF
group. The study was prematurely terminated due to futility
after conditional power calculation.
Conclusions: In contrast to previous findings, G-CSF had no
significant beneficial effect on patients with ACLF in this multi-
center controlled trial, which suggests that it should not be used
as a standard treatment for ACLF.
ClinicalTrials.gov number: NCT02669680
Lay summary: Granulocyte-colony stimulating factor was
considered as a novel treatment for acute-on-chronic liver failure
(ACLF). We performed the first randomized, multicenter,
controlled phase II trial, which showed that G-CSF did not
improve survival or other clinical endpoints in patients with
ACLF. Therefore, G-CSF should not be used to treat liver disease
outside clinical studies.
© 2022 The Authors. Published by Elsevier B.V. on behalf of European
Association for the Study of the Liver. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Acute-on-chronic liver failure (ACLF) develops in more than
30% of patients with acutely decompensated cirrhosis and is

Journal of Hepatology 2021 vol. 75 j 1346–1354

characterized by development of organ failures.1 Cirrhosis ac-
counts for about 1 million deaths worldwide and most of these
patients die during an ACLF episode.2 Its overall 90-day mortality
is approximately 50%, which is substantially higher than the 14%
seen in patients with acute decompensation without organ fail-
ures.3 Liver transplantation is the only established treatment
approach,4,5 but due to scarcity of donor organs, costs and con-
traindications it is only accessible to a minority of patients
(approximately 5%).4
Novel treatment approaches aim to target ACLF-specific
pathomechanisms, dominated by the co-existence of exagger-
ated inflammatory responses on the one hand, and immune
paralysis on the other, leading to mitochondrial dysfunction, cell
death and regenerative incapacity.6–9 Bone marrow-derived
stem cells have been shown to be potent in modulating im-
mune functions and promoting regenerative capacities, thus
mitigating various types of liver injury in experimental and
clinical studies.10–12 However, costs, high technical requirements
and peri-interventional complications limit the general applica-
bility of this approach.
Granulocyte-colony stimulating factor (G-CSF) mobilizes he-
matopoietic stem and immune cells and represents an alterna-
tive for exogenous stem cell infusions.13,14 Several randomized
controlled trials in India and China using G-CSF in patients with
ACLF reported improved survival and decreased rates of disease-
related complications, such as bacterial infections.15,16 Conse-
quently, G-CSF is being used as standard of care in many parts of
the world. However, its role in treating ACLF is still under debate
as data supporting its efficacy have been tested in relatively
small clinical trials outside of Europe and the US, and in a limited
number of centers. Therefore, the aim of our study was to
explore the efficacy of G-CSF in patients with ACLF in a large
multicenter, randomized and controlled trial.
Patients and methods
Trial design
The GRAFT study was an investigator-initiated, multicenter,
randomized, controlled trial, which recruited patients with ACLF
in 18 German tertiary centers funded by the German research
foundation (DFG). The trial protocol (supplementary trial pro-
tocol) was approved by the leading ethics committee (University
Leipzig) and by the German competent authority (BfArM). The
trial design followed the CONSORT guidelines and complied with
the principals of the declaration of Helsinki from 1975 (Version
Sommerset West 1996),17 as well as all pertinent national laws
and the ICH guidelines for Good Clinical Practice (GCP) issued in
June 1996 and CPMP/ICH/135/95 from September 1997.
Study population
Patients with ACLF according to the criteria of the European
Foundation for the Study of Chronic Liver Failure (EASL-CLIF)
consortium18 with underlying liver cirrhosis and aged >
−18 years
were eligible for the trial. ACLF was defined as acutely
decompensated cirrhosis (acute development or worsening of
one of the following complications: ascites, hepatic encepha-
lopathy, gastrointestinal hemorrhage, bacterial infection)
combined with one or more organ failure(s) (definitions in
supplementary information).3
Patients with prior non-curatively treated or active malig-
nancies, sickle cell disease, septic shock (defined by the
presence of bacteriaemia, systemic inflammatory response
Journal of Hepatology 2021 vol. 75 j 1346–1354
syndrome, and vasopressor support) or white blood cell (WBC)
count of >50 x 109/L were excluded (complete list of inclu-
sion and exclusion criteria is included in the supplementary
information). All patients gave their written consent before
study inclusion.
Intervention
Patients were randomized in a 1:1 ratio to receive either stan-
dard medical therapy (SMT) (SMT group) or recombinant G-CSF
(G-CSF, Ratiograstim®) plus SMT (G-CSF+SMT group). Patients
were centrally randomized with a Web-based system by using a
minimization algorithm with a factor for center and a probability
of 0.2 for random allocation, to avoid potential loss of allocation
concealment within centers. Patients randomized to the G-
CSF+SMT group were scheduled to receive in total 12 doses of G-
CSF subcutaneously injected on a daily basis for the first 5 days
after randomization and every third day thereafter until day 26.
The dosing scheme used corresponded to that published by Garg
et al.15 which was chosen based on its superior efficacy over a
shorter treatment duration applied by Duan et al.16 Further
randomized efficacy trials were not available at the time of trial
preparation. Injections were performed at 12 am (± 1 hour) on
every treatment day. Using pre-filled syringes provided by the
supplier (30 Mio IU or 48 Mio IU), as part of the clinical routine,
G-CSF doses were guided by body weight using a cut-off value of
70 kg (< −70kg 30 Mio IU G-CSF, >70 kg 48 Mio IU G-CSF) (Fig. 1)
(Supplementary CTAT Table).
A placebo controlled and blinded design was not considered
feasible in the study population for 2 main reasons. First, pa-
tients with ACLF can have pre-existing systemic inflammatory
responses and a high risk of infectious complications1; thus,
blinding could lead to misinterpretation of WBC count, as it will
be unknown if an infection or G-CSF treatment causes the
response in WBC count. Alternative diagnostic approaches are
not reliable to detect infectious complications in cases of G-CSF-
related WBC increases.19 Second, according to the drug manu-
facturer’s instruction, G-CSF treatment was transiently stopped
in cases where WBC rose to >70 x109/L and re-started at a WBC
<50 x109/L, mainly because WBC exceeding 100 x109/L can be
accompanied by a high risk of symptomatic hyperviscosity syn-
drome.20 Any subsequent new ACLF episode, which developed
after the qualifying ACLF episode for inclusion into the trial, was
treated with SMT in both treatment arms.
G-CSF + SMT Prim. endpoint Long-term follow-up
Enrolment
ACLF eligibility Rando Intervention: G-CSF 5 μg/kg s.c. day 1-5 once daily,
EF-CLIF criteria informed 1:1 day 6-26 every third day
(according to the body weight using a cut off value of 70 kg
consent
(≤70 kg 30 Mio IU G-CSF, >70 kg 48 Mio IU G-CSF))
SMT Prim. endpoint Long-term follow-up
Screening + randomisation Intervention Prim. endpoint Follow-up
48 hours 26 days 90 days 12 month
Baseline V1-V4 V5 V6 V7 V8
[D1, D4, D7, D14] [D28] [D90] [D180] [D360]
Fig. 1. Study design. After enrolment patients with G-CSF were randomized to
receive either SMT or SMT+G-CSF for 26 days. The primary endpoint was
assessed after 90 days. Patients were followed up for in total 360 days. There
were 9 study visits (baseline, V1-V8) at which clinical data, adverse events and
blood samples were collected. ACLF, acute-on-chronic liver failure; D, day of
study visit; G-CSF, granulocyte-colony stimulating factor; SMT, standard
medical therapy; V, study visit.
1347
Research Article
Follow-up
After randomization patients were followed-up for 360 days or
until premature study termination defined as:
 withdrawal of informed consent (drop-out)
 complete loss of contact (drop-out)
 death
During follow up visits V1-V8 clinical data and samples were
collected as outlined in Fig. 1.
Endpoints
The primary endpoint was defined as transplant-free survival 90
days after inclusion into the study, with death and orthotopic
liver transplantation (OLT) counting as events. Patients were
censored at the time point of last information still alive and
without OLT. The key secondary endpoints were the overall and
transplant-free survival after 360 days, the development of ACLF-
related complications, bacterial infections and the course of liver
function scores, such as the model of end-stage liver disease
(MELD) score and Chronic Liver Failure-consortium (CLIF-C) or-
gan failure (OF) score, during the entire observation period. A
complete list of endpoints and definitions is presented in the
supplementary information. We abstained from using the CLIF-C
ACLF score18 for further analysis, because it includes WBC count
as part of the scoring system, making it susceptible to artificial
WBC alteration by G-CSF-related cell mobilization. Safety was
evaluated at each visit based on clinical data and physical ex-
amination. Severe adverse events (SAEs) were reported within
24 hours after awareness and monitored until resolution. The
SAE reporting period started on day 0 and ended at day 28
except for newly developed malignancies. Annual safety analyses
were performed. Safety reports and data analyses were reviewed
by the data monitoring and safety board (DMSB) as requested or
at least annually.
Statistical analysis and sample size calculation
Based on the CANONIC Study,3 we expected a transplant-free
survival rate of 42% at day 90 with SMT. Two small single-
center, randomized G-CSF trials in patients with ACLF reported
significant effects at improving survival from 26% to 66% after 60
days15 and from 21% to 48% after 90 days.16 We aimed to detect
an absolute difference of 20% (from 42% to 62%, HR 1.815) in
transplant-survival at day 90. Assuming a 10% loss to follow-up,
we originally determined that 292 patients would be required to
have 90% power, at a 2-sided significance level of 5%. One interim
analysis was scheduled once the primary endpoint was available
for 50% of patients, allowing for detection of marked superiority
of G-CSF (of >
−33%) on the primary endpoint, with a power of at
least 80%. Multiplicity adjustment followed O’Brien,21 with
nominal significance levels of p = 0.005 at interim analysis and
p = 0.048 at final analysis. All patients enrolled up to this point
were followed up until regular end of observation or an
endpoint. Cox regression was used to estimate the treatment
effect on hazard ratio (HR) scale with 2-sided 95% CIs provided in
time-to-event endpoints. Two-sided chi-square or Fisher’s exact
tests were used to compare qualitative variables, as appropriate.
Courses of prognostic scores (MELD; CLIF-C OF score) were
analyzed by repeated-measures ANCOVA, imputing single in-
between values by linear interpolation and adding the last
value observed in cases of death, OLT or dropout. We considered
OLT competing with death and death/OLT competing with
1348 Journal of Hepatology 2021 vol. 75 j 1346–1354
Cirrhosis and Liver Failure
occurrence of disease-related complications in competing risk
analyses (Fine and Gray proportional subdistribution hazard
regression model, R version 3.6.3). Additional analyses were
performed in the per protocol (PP) population. These patients
fulfilled all inclusion and exclusion criteria and received at least
75% of the intended G-CSF total dose, if medical reasons did not
result in premature end of treatment. Ninety-day transplant-free
survival analysis was also calculated post hoc in various sub-
populations. Detailed definitions are provided in the supple-
mentary information. Analyses were performed with SSPS
software, version 25 (IBM inc.) and R (Supplementary CTAT
Table). Safety issues were carefully monitored by standardized
SAE reporting and annual safety reports (according to legal re-
quirements), and presented to the DMSB. All results shown here
were calculated from the intention-to-treat (ITT) population
unless otherwise specified.
Results
Patient and treatment characteristics
After registering 180 patients in total, 176 were randomized be-
tween March 2016 and April 2019 to receive either G-CSF with
SMT or SMT alone (n = 88 G-CSF+SMT group, n = 88 SMT group)
(Fig. 2). Table 1 summarizes the baseline characteristics for both
groups, which did not differ significantly, indicating good
randomization. All patients had underlying cirrhosis. The mean ±
SD age was 54.4 ± 10.2 years in the G-CSF+SMT group and 57.1 ±
9.6 years in the SMT group. The majority of patients enrolled
were male (56.8% G-CSF+SMT group, 69.3% SMT group) and had
an increased mean BMI >28 kg/m2. Most patients had ACLF grade
1 or 2, with a mean number of organ failures of 1.7 ± 0.7 in the G-
CSF+SMT group vs. 1.4 ± 0.6 in the SMT group. The mean baseline
CLIF-C OF score and MELD score were 10.4 ± 1.9 and 24.4 ± 6.3 in
the G-CSF+SMT group and 10.3 ± 2 and 24.5 ± 6.1 in the SMT
group, respectively. Liver and kidney failures were the predom-
inant types of organ failure accounting for more than 60% of
cases in both groups. Alcohol abuse, bacterial infections and
gastrointestinal bleeding were the most frequent precipitating
events (Table 1). At baseline, 96.6% of patients in both groups had
ascites, and bacterial infections were present in 56.8% in the G-
CSF+SMT group and 51.1% in the SMT group.
In the G-CSF+SMT group, 83 patients (94.3%) received at least
1 G-CSF injection whereas 5 patients remained untreated due to
early death (n = 4) and other medical reasons (n = 1). The mean
number of G-CSF injections was 8.6 ± 3.9 and the mean duration
of G-CSF treatment was 17.5 days ± 9.7. In total, 29 patients (33%)
received all 12 injections and 51 patients >
−9 injections. Therefore,
80 patients in the G-CSF+SMT arm and 84 patients in the SMT
arm fulfilled criteria for the PP cohort. Patients treated with G-
CSF showed increasing WBC count during the treatment course,
whereas it remained unchanged in the SMT group (p
<0.001) (Fig. S1).
Patient outcomes
The primary endpoint, defined by death or OLT within 90 days
after study enrolment, was reached in 54 patients (61.4%) in the
G-CSF+SMT group vs. 51 patients (58.0%) in the SMT group from
the ITT cohort. Five patients from the G-CSF+SMT group and 9
from the SMT group received an OLT. The HR for the risk of death
or transplantation when treated with G-CSF was 1.05 (95% CI
0.711–1.551; p = 0.805) (Fig. 3 and 4). The median transplant-free
survival was 35 days (95% CI 19.8–50.2) in the G-CSF+SMT group
 Registered
n = 180

Screening failure n = 4
- n = 3 not fulfilling the inclusion criteria
[no ACLF acc. to EF-CLIF criteria]
- n = 1 no reason provided

Randomized
n = 176

SMT + G-CSF SMT

ITT n = 88 ITT n = 88

Drop-outs n = 10 Drop-outs n = 18
n = 7 until day 90 n = 11 until day 90
n = 3 after day 90 until day 360 n = 7 after day 90 until day 360
Types of drop-out Types of drop-out
n = 5 withdrawal of consent n = 14 withdrawal of consent
n = 3 lost to follow-up n = 4 lost to follow-up
n = 2 other reasons*

Outcome Day 90 Outcome Day 90

n = 27 alive in follow-up n = 26 alive in follow-up
n = 50 deaths without OLT n = 45 deaths without OLT
n = 4 OLT (n = 1 death after OLT) n = 6 OLT (n = 2 death after OLT)

Outcome Day 360 Outcome Day 360

n = 16 alive at end of observation n = 9 alive at end of observation
n = 57 deaths without OLT n = 52 deaths without OLT
n = 5 OLT (n = 1 death after OLT) n = 9 OLT (n = 3 death after OLT)

Fig. 2. CONSORT flow chart of patients. After registering 180 patients, 176 patients fulfilled the inclusion and exclusion criteria, were randomized (n = 88
patients in each arm) and analyzed according to the ITT analysis. Patients were followed up for 360 days. *Other reasons for dropouts were: treatment of bronchial
carcinoma n = 1 and medical decision n = 1; ACLF, acute-on-chronic liver failure; EF-CLIF, European Foundation for the Study of Chronic Liver Failure; ITT,
intention-to-treat (all patients within the full analysis set were treated as randomized); OLT, orthotopic liver transplantation; SMT, standard medical care.

and 34 days (95% CI 9.9–58.1) in the SMT group. Neither the
overall survival (HR 1.058; 95% CI 0.727–1.548; p = 0.768), nor
360-day transplant-free survival (HR 0.998; 95% CI 0.697–1.430;
p = 0.992) were statistically different between arms in the ITT
cohort. Similar results were observed in the PP analysis. The HR
for the primary endpoint was calculated as 1.034 (95% CI
0.698–1.531; p = 0.869] (Fig. 4). For overall survival, the HR was
1.059 (95% CI 0.724–1.548; p = 0.769) and for transplant-free
survival 0.998 (95% CI 0.694–1.436; p = 0.992) when treated
with G-CSF (Fig. 4).
During the study period most fatalities were caused by ACLF
or other liver-related complications in both arms (G-CSF+SMT:
27/88 [30.7%]; SMT: 21/88 [22.7%]). Gastrointestinal bleeding
leading to death was more frequent in the SMT arm (G-CSF+SMT:
2/88 [2.3%]; SMT 8/88 [9.1%]). In total 8 deaths, 3 in the G-
CSF+SMT arm and 5 in the SMT arm, were related to bacterial
infections (Table S2)
As liver transplant and dropouts were competing with death,
we performed a competing risk analysis by calculating the cu-
mulative incidence of death, which confirmed that G-CSF treat-
ment failed to show a significant benefit on survival in patients
with ACLF (HR 1.11; 95% CI 1.61–0.77) (Fig. S2). For futility
analysis, we calculated a conditional power of 1-b=0.0266 for
transplant-free survival after 90-days and 0.0046 for overall
survival, which led to the premature termination of patient
recruitment after consultation with the study’s DMSB.
Secondary endpoints
Fatalities occured predominently in patients with increasing
MELD score during the treatment period until day 28 in both
groups (Fig. S5). The distribution of patients with increasing or
decreasing MELD or CLIF-C OF score, did not differ between the 2
groups (ANCOVA analysis: significance between groups p = 0.884
for MELD and p = 0.757 for CLIF-C OF score; Fig. 5 and S5).
Patients who recovered from the first ACLF episode were
followed up for development of subsequent ACLF episodes (ACLF
recurrence). The risk of developing further ACLF episodes was
not different between the 2 groups (HR 1.94; 95% CI 0.83–4.53;
p = 0.13) (Fig. S4) although the number of patients with ACLF
recurrence randomized into the G-CSF+SMT group was slightly
higher (65.2% vs. 34.8 %, p = 0.19) (Table S3). Groups of patients
with or without ACLF recurrence were further characterized at
baseline and at the time point of remission from the first ACLF
episode. Patients with ACLF recurrence more often had ACLF
grade 1 at baseline (87% vs. 55%, p = 0.019). At the time point of
remission the same patients had higher values for MELD score

Journal of Hepatology 2021 vol. 75 j 1346–1354 1349

Research Article Cirrhosis and Liver Failure

Table 1. Baseline characteristics for the 2 study arms.

1.0
Parameter G-CSF+SMT SMT Treatment arm
group (n = 88) group (n = 88) G-CSF+SMT
SMT
0.8
General

Cumulative survival
Age (years) 54.4 ± 10.2 57.1 ± 9.6
Gender (w/m) 38 (43.2%)/50 27 (30.7%)/61 0.6
(56.8%) (69.3%)
BMI (kg/m2) 28.9 ± 4.8 28.8 ± 5.6 0.4
Mean arterial 79.9 ± 12 82.7 ± 13.2
pressure (mmHg) 0.2 HR 1.05 [95% CI 0.711; 1.551 (p = 0.805)]

Disease severity
ACLF grade* 0.0
Grade 1 39 (44.3%) 45 (51.1%) 0 15 30 45 60 75 90 105
Grade 2 37 (42.0%) 28 (31.8%) Time from baseline in days
Grade 3 12 (13.6%) 15 (17%) Patients at risk
Number of organ failures 1.7 ± 0.7 1.4 ± 0.6 G-CSF + SMT 88 61 42 35 29 28 27
CLIF-C OF score 10.4 ± 1.9 10.3 ± 2 SMT 88 54 43 35 31 28 26
MELD score 24.4 ± 6.3 24.5 ± 6.1
CLIF-C ACLF score 51.9 ± 8.7 51.2 ± 7.4 Fig. 3. 90-day transplant-free survival in ITT cohort. Out of 176 patients with
Organ failures ACLF, 88 were randomized to receive G-CSF+SMT (G-CSF+SMT group) or SMT
Liver failure 58 (65.9%) 56 (63.6%) only (SMT group). Death or liver transplantation were considered as events in a
Kidney failure 58 (65.9%) 57 (64.8%) Cox Regression analysis calculating HR for G-CSF therapy. ACLF, acute-on-
Brain failure 5 (5.7%) 5 (5.7%) chronic liver failure; G-CSF, granulocyte-colony stimulating factor; HR, haz-
Circulatory failure 8 (9.1%) 6 (6.8%) ard ratio; ITT, intention-to-treat; SMT, standard medical therapy.
Coagulation failure 22 (25%) 15 (17%)
Respiratory failure 2 (2.3%) 6 (6.8%)
Precipitating events# Bacterial infections occurred in 71 (80.8%) patients in the G-
Alcohol related 49 (55.7%) 46 (52.3%)
CSF+SMT group and 69 (78.4%) in the SMT group (p = 0.709). Of
Bacterial infection 36 (40.9%) 33 (37.5%)
Bleeding 13 (14.8%) 7 (8%)
those, 50 out of 88 (56.6%) in the G-CSF+SMT arm and 45 out of
Others 7 (8%) 8 (9.1%) 88 (51.1%) had an infection at baseline and 31 out of 88 (35.2%)
Unknown 16 (18.2%) 18 (20.5%) and 32 out of 88 (36.4%) in the respective treatment arms
Complications of cirrhosis developed an infection during the observational period. Urinary
Ascites 85 (96.6%) 85 (96.6%) tract infections, spontaneous bacterial peritonitis, pneumonia
Hepatorenal syndrome 57 (64.8%) 58 (65.9%) and blood stream infections were the most common sites of
Hepatic encephalopathy 55 (62.5%) 61 (69.3%)
infection during the study. There was a predominance of spon-
Bacterial infection at baseline 50 (56.8%) 45 (51.1%)
taneous bacterial peritonitis in the G-CSF+SMT arm and blood
Laboratory values
stream infections in the SMT arm. There were no major differ-
Bilirubin (mg/dl) 18.0 ± 12.0 18.9 ± 14.5
ALT (U/L) 76.0 ± 144.7 81.7 ± 167.0 ences in the rate of infections across the different ACLF grades in
AST (U/L) 139.0 ± 217.0 124.1 ± 136.5 comparison between both arms (Table S4). There were 6 fungal
GGT (U/L) 195.2 ± 287.5 165.0 ± 224.0 infections in each cohort with Candida being the most frequent
ALP (U/L) 150.7 ± 80.6 148.8 ± 116.4 causative pathogen (6 out of 12). Two patients (1 in each group)
Creatinine (mg/dl) 2.4 ± 1.6 2.4 ± 1.5
had a fungal peritonitis (Table S5).
Urea (mg/dl) 101.5 ± 60.9 115.3 ± 61.3
INR 2.2 ± 0.8 2.1 ± 1.0
There was no difference between both groups with respect to
Albumin (g/dl) 3.0 ± 0.7 3.0 ± 0.7 the development of other complications, such as gastrointestinal
C-reactive protein (mg/dl) 39.9 ± 29.2 41.3 ± 39.6 bleeding, hepatic encephalopathy, and hepatorenal syndrome
Procalcitonin (mg/L) 1.7 ± 2.1 1.4 ± 1.4 during the observation period (Table 2).
WBC count (109/L) 14.8 ± 12.4 11.2 ± 7.1
ACLF, acute-on-chronic liver failure; ALP, alkaline phosphatase; ALT, alanine amino-
transferase; AST, aspartate aminotransferase; CLIF-C, Chronic Liver Failure-con- Post hoc sensitivity analyses
sortium; G-CSF, granulocyte-colony stimulating factor; GGT, gamma Sensitivity analyses focused on subgroups of patients with
glutamyltransferase; INR, international normalized ratio; MELD, model for end-stage
liver disease; OF, organ failure; SMT, standard medical therapy.
different disease severities (Fig. 4A) (ACLF grades and types of
#
Precipitating events counted individually in patients with more than one precipi- organ failures), on subgroups of patients who were included in
tating event. previous G-CSF trials (Fig. 4B)15,22 (ACLF defined according to the
*These values represent the number of patients in every respective ACLF grade.
APASL criteria, ACLF due to an alcohol-related precipitating
Values were calculated based on individual parameters. However, patients were
included based on ACLF grade allocations provided by the study centers. Based on event) and on subgroups with a higher likelihood of treatment
back calculations, 2 patients in the SMT group and 1 patient in the G-CSF+SMT group response (such as patients without infections, 7-day survivors
did not fulfil the criteria for ACLF. Percentages shown here were calculated against and patients fulfilling criteria for best cases). In none of these
the overall number of patients included in the trial.
cohorts did G-CSF show a significant effect on transplant-free
survival after 90 days (Table S1).
(17.8 vs. 13.1, p = 0.002), bilirubin (13.5 vs. 7, p = 0.024) and C- Multivariate Cox regression analysis identified risk factors for
reactive protein (38.9 vs. 27.2; p = 0.004) and more often had mortality such as age (HR 1.043; 95% CI 1.019–1.066; p <0.001),
high-grade ascites (60.9% vs. 22.5%, p = 0.002) (Table S3). the ACLF severity grade at baseline (HR 2.249; 95% CI

1350 Journal of Hepatology 2021 vol. 75 j 1346–1354

A Kidney failure 0.724 (0.453; 1.156), p = 0.176

Liver failure 1.013 (0.634; 1.617), p = 0.958

ACLF grade 3 1.162 (0.511; 2.640), p = 0.720

ACLF grade 2 1.037 (0.544; 1.979), p = 0.911

ACLF grade 1 1.005 (0.547; 1.846), p = 0.911

Overall cohort (per protocol) 1.034 (0.698; 1.531), p = 0.869

Overall cohort (intention to treat) 1.050 (0.711; 1.551), p = 0.805

0.1 1 10

B
Best case 1.138 (0.417; 3.1), p = 0.801

ACLF-APASL criteria 1.233 (0.762; 1.963), p = 0.404

Alcohol related ACLF 1.047 (0.594; 1.843), p = 0.875

7-day survivors 0.901 (0.583; 1.39), p = 0.64

No infections 1.055 (0.521; 2.137), p = 0.883

Infection 1.415 (0.777; 2.575), p = 0.254

Overall cohort (per protocol) 1.034 (0.698; 1.531), p = 0.869

Overall cohort (intention to treat) 1.050 (0.711; 1.551), p = 0.805

0.1 1 10

Fig. 4. Forrest plot for risk of all sub-populations analyzed. (A) Cox regression analyses calculating the hazard ratio (HR) for G-CSF therapy in subgroups with
different ACLF severity grades (ACLF grade 1 n = 83, ACLF grade 2 n = 64, ACLF grade 3 n = 27) and organ failures (liver failure n = 114, kidney failure n = 115) were
performed to evaluate the impact of disease severity on treatment efficacy. G-CSF was unable to improve the 90-day transplant-free survival in any of the severity
subgroups. (B) Cox regression analyses adjusted for ACLF grades were performed to calculate the hazard ratio of death or transplant until 90 days of observation
in additional subgroups. Along with the ITT cohort and per protocol sets of patients, analyses were performed in a variety of subgroups of patients with pre-
defined clinical conditions of potentially different prognosis. Best case (n = 65) – patients who survived regular end of treatment without transplant for at least 14
days and received at least 75% of intended dose if applicable; ACLF-APASL criteria (n = 114) – patients who fulfilled the APASL criteria for ACLF; alcohol-related
ACLF (n = 95) – patients who had an alcohol-related precipitating event; 7-day survivors (n = 155) – patients who survived randomization for a minimum of 7
days; no infection (n = 73) – patients without detectable infection at inclusion; infection (n = 69) – patients with a bacterial infection as a precipitating event.
ACLF, acute-on-chronic liver failure; APASL, Asian Pacific Association for the Study of the Liver; G-CSF, granulocyte-colony stimulating factor; ITT, intention-
to-treat.

1.703–2.970; p <0.001) and the WBC count at baseline (HR 1.044;
95% CI 1.02–1.066; p <0.001) whereas treatment with G-CSF, the
presence of an alcohol-related precipitating event or bacterial
infection at baseline (precipitating events) did not impact on
transplant-free survival (Table 3).
Safety characteristics
Overall, 403 adverse events were reported from 80 patients in
the G-CSF+SMT group and 354 from 78 patients in the SMT
group. Sixty-one SAEs were reported in 54 patients from the G-
CSF+SMT group, as well as 57 SAEs in 47 patients from the SMT
group, including 37 and 36 deaths during the SAE reporting
period, respectively. ACLF was the most frequent cause of death
in both groups (n = 17/37 [46%] and 16/36 [44%], respectively, p =
1.0), followed by hemorrhages (n = 6 [16%] and 8 [22%], respec-
tively, p = 0.564), and infection-related deaths (n = 4 [11%] and
n = 5 [14%], respectively, p = 0.736). In the G-CSF+SMT group, G-
CSF-related adverse reactions were reported in 18 patients and 7
serious adverse reactions were reported in 4 patients (Table S6).
The study treatment was paused in 3 out of 4 patients with
serious adverse reactions, one of which was related to a WBC
count above the threshold of 70 x109/L. In total, G-CSF therapy
had to be transiently stopped due to a WBC increase in 8 pa-
tients (9.1%).
Discussion
This is the first large multicenter study evaluating G-CSF as a
potential novel therapy for patients with bona fide ACLF. Despite
G-CSF having immune-modulatory and pro-regenerative capac-
ities,23 our study confirmed the overall dismal prognosis of ACLF
and failed to demonstrate superiority of G-CSF over SMT. Hence,
the data provided here are in strong disagreement with 2 single-
center trials that showed a marked improvement in survival and
reduced infectious complications after G-CSF therapy13,14; thus,

Journal of Hepatology 2021 vol. 75 j 1346–1354 1351

Research Article Cirrhosis and Liver Failure

trials, G-CSF reduced the number of infectious complications,

A DeltaOF-Score - Overall cohort
improved immune dysfunction by increasing the number of
10 G-CSF+SMT
dendritic cells whilst decreasing interferon-producing T-cells
DeltaOF-Score between baseline

SMT

p = 0.757 and enhanced the homing of CD34+ hematopoietic cells, which

5 are believed to promote regenerative capacities.14,15,24 However,
in our trial, G-CSF was neither able to reduce the number of
and day 28

infections or any other complication nor improve severity scores,

0
such as the MELD score and CLIF-C score significantly. This is in
line with recently published data in children with ACLF,25 and
-5 supports other studies that did not find any beneficial effect of G-
CSF.24,25 Notably, some pre-clinical studies have even shown a
worsening of liver injury upon G-CSF use in ACLF.26
-10 There are several reasons that may explain the discrepancies
B DeltaMELD - Overall cohort
in G-CSF treatment outcome observed in our and previous
20 single-center trials.15,16 A recent meta-analysis summarized all
G-CSF+SMT
SMT G-CSF studies which enrolled patients with severe alcoholic
DeltaMELD between baseline

10 p = 0.884 hepatitis and underlined the huge heterogeneity among studies

and pointed towards a conflict between Asian and European
results,26 suggesting that a selection bias might play a role.
and day 28

0
-10
-20
-30
Fig. 5. Development of CLIF-C OF score during the treatment course. The
distribution of patients with increasing and decreasing (A) CLIF-C OF score and
(B) MELD score (delta between baseline and day 28) was not different when
comparing the G-CSF (G-CSF+SMT; blue bars) arm and the control arm (SMT;
grey bars) (between groups effect: DeltaOF-score p = 0.757, DeltaMELD score
p = 0.884). Difference between groups over time was calculated by ANCOVA.
Missing values due to death or transplant were imputed by individuals’ last
value. ACLF, acute-on-chronic liver failure; CLIF-C, Chronic Liver Failure-
consortium; G-CSF, granulocyte-colony stimulating factor; MELD, model for
end-stage liver disease; OF, organ failure; SMT, standard medical therapy.
our data do not support the use of G-CSF as a potential standard
therapy for patients with ACLF.23 We showed that 90-day
transplant-free survival (HR 1.05, p = 0.805) and overall sur-
vival (HR 1.058, p = 0.768) were unaffected by G-CSF adminis-
tration, and this observation was also confirmed in the subgroup
of patients treated strictly according to the protocol (PP) with an
HR of 1.034 (p = 0.869) for 90-day transplant-free survival and of
1.059 (p = 0.769) for overall survival. In smaller single-center
However, when we stratified our patients according to criteria
that were used in the Asian studies, most importantly patients
with an alcohol-related precipitating event and ACLF according
to the APASL criteria, we were not able to demonstrate superi-
ority of the G-CSF approach compared to SMT. In this context, it
has to be mentioned that the definition of an alcohol-related
precipitating event was at investigators discretion and not part
of the study protocol. It has to be assumed that this cohort
comprised predominantly patients with alcoholic hepatitis.
A recent review has indicated already that disease severities
and patient selections were comparable among Asian and Eu-
ropean studies.12 Furthermore, G-CSF’s inefficacy was confirmed
across the different ACLF severity grades and types of organ
failures. Overall, these results indicate that selection criteria and
the study design do not fully explain differences in efficacy data
among previous studies and ours.
It might be tempting to say that the high number of infections
in our study has driven an overwhelming inflammatory
response, which was aggravated by G-CSF thereby inhibiting its
beneficial effect. And indeed, patients with ACLF recurrence after
recovery from the first ACLF episode had higher C-reactive pro-
tein values and tend to be treated more often with G-CSF. The
discrepancy between a high number of overall infections
developing during the study and low number of infection-related
deaths is intriguing but might be related to the fact that only

Table 2. Development of cirrhosis-related complications during the observational period.*

Complication Treatment group (G-CSF+SMT) Control group (SMT) p value
ACLF remission observed1 33/872 (37.9%) 30/862 (34.9%) 0.685
ACLF re-episode3 15/87 (17.2%) 8/862 (9.3%) 0.134
Ascites 87 (98.9%) 87 (98.9%) 1.0004
Hepatic encephalopathy 64 (72.7%) 67 (76.1%) 0.6044
Hepatorenal syndrome 71 (80.7%) 69 (78.4%) 0.7094
Bacterial infections 71 (80.8%) 69 (78.4%) 0.7094
Variceal bleeding 7 (8%) 7 (8%) 1.0004
ACLF, acute-on-chronic liver failure; G-CSF, recombinant granulocyte colony stimulating factor; SMT, standard medical therapy.
*Groups were balanced regarding history of complications.
1
Remission before an endpoint-related/competing event or end of individual observational period.
2
Only patients with ACLF grade of > −1 at baseline were included.
3
ACLF re-episodes were considered only in case of a previous ACLF remission.
4
Group comparisons were performed by Chi-Square test.
5
Competing risk analysis by cumulative incidence function and hazard ratio.

1352 Journal of Hepatology 2021 vol. 75 j 1346–1354

Table 3. Multivariate Cox regression analysis for 90-day transplant-free survival.
Baseline factor
Treatment arm (risk for G-CSF+SMT)
Age (years)
ACLF grade
Precipitating event bacterial infection
Precipitating event alcohol related
WBC count (109/L)
ACLF, acute-on-chronic liver failure; G-CSF, recombinant granulocyte colony stimulating factor; SMT, standard medical therapy; WBC, white blood cell.
primary causes of death were documented by investigators and
data on a potential link between cause of death and underlying
infection was not systematically collected.
Stimulating the immune response may be an effect also
related to G-CSF per se independent of infections as shown by
several pre-clinical studies. The rational for treating ACLF with
GCSF comes from animal models showing pro-regenerative and
anti-inflammatory effects,27,28 and the earlier Asian studies
seemed to confirm these findings in humans.15,16 It has to be
emphasized that G-CSF may act like a double-edged sword. Toll-
like receptor 4 (TLR4)-activation was observed to be a major
driver of disease progression in ACLF.29,30 As shown in septic
liver injury models, G-CSF might enhance sensitivity to endo-
toxins and increase hepatocyte uptake of lipopolysaccharide
through the lipopolysaccharide-binding protein/TLR4 axis,
thereby aggravating tissue injury, as shown by HMGB1 trans-
location and mortality in rat models of septic liver injury.31,32 G-
CSF also activates neutrophils that carry TLR433 and migrate into
injured tissues, potentially causing additional cellular damage.34
Therefore, the effect of G-CSF, either beneficial or detrimental,
might depend on the disease stage. In our trial, the number of
possibly drug-related SAEs and the trend that patients with ACLF
recurrence were more often treated with G-CSF points towards a
potential clinical risk when using G-CSF, although these data
were purely descriptive. The comparison with previous trials is
difficult as WBC-related or drug-related severe complications
were not reported.15,16
The study presented here has several limitations. Firstly, pa-
tient recruitment had to be prematurely terminated due to fu-
tility after performance of conditional power analyses (in a
variety of sub-populations) in parallel to the planned interim
analysis. A calculated conditional power of 0.0266 for the pri-
mary endpoint and <0.005 for overall survival made it unethical
to continue the publicly funded trial although there was no
safety concern. We have also not provided any data about the
mode of action of G-CSF in ACLF. We did not specifically address
potential ethnic differences which could be a potential issue.
However, so far there is no evidence to suggest that G-CSF itself
acts differently or that the ethnic background of patients in-
fluences ACLF’s phenotype beyond the different diagnostic
criteria applied in Asia and Europe. Open labeled trials are al-
ways at risk of bias. However, given the high number of positive
previous study results one would expect an overestimation of a
therapeutic effect rather than failing to proof the benefit of G-
CSF. We therefore consider a bias of the effects associated with
the study design to be very unlikely. Unfortunately, we were not
collecting data on the etiology of the underlying chronic liver
disease. That was related to the fact that our focus was on a clear
diagnosis of ACLF according to the EASL-CLIF criteria and
Journal of Hepatology 2021 vol. 75 j 1346–1354
Multivariate analysis
Hazard ratio 95% CI p value
0.946 0.635–1.408 0.785
1.043 1.019–1.066 <0.001
2.249 1.703–2.970 <0.001
1.023 0.938–1.117 0.604
1.041 0.943–1.149 0.426
1.044 1.022–1.066 <0.001
collection of acute precipitating events, which we considered as
important determinants of the disease course.
This multicenter randomized study confirms the dismal
prognosis of patients with ACLF and failed to demonstrate any
beneficial effect of treating this condition with G-CSF mono-
therapy. G-CSF was associated with drug-related serious adverse
reactions and should not be used outside clinical trials for the
treatment of ACLF.
Abbreviations
ACLF, acute-on-chronic liver failure; AE, adverse events; APASL,
Asian Pacific Association for the Study of the Liver; CLIF-C,
Chronic Liver Failure-consortium; DMSB, data monitoring and
safety board; EASL-CLIF, European Foundation for the Study of
Chronic Liver Failure; G-CSF, granulocyte-colony stimulating
factor; HR, hazard ratio; ITT, intention-to-treat; MELD, model of
end-stage liver disease score; OF, organ failure; OLT, orthotopic
liver transplantation; PP, per protocol; SAEs, severe adverse
events; SMT, standard medical therapy; TLR4, Toll-like receptor
4; WBC, white blood cell.
Financial support
The German Research Foundation (DFG) – EN 1100/1-1, funded
the study. Cornelius Engelmann is participant in the BIH-Charité
Clinician Scientist Program funded by the Charité – Uni-
versitätsmedizin Berlin and the Berlin Institute of Health. Moritz
Schmelzle and Katrin Splith received grants for ancillary studies
funded by the German Research Foundation (DFG) (SCHM2661/
3-1, SCHM2661/3-2). Jonel Trebicka is supported by grants from
the German Research Foundation (DFG) (SFB TRR57 to P18),
European Union’s Horizon 2020 Research and Innovation Pro-
gramme (Galaxy, No. 668031 and MICROB-PREDICT, No. 825694),
and Societal Challenges - Health, Demographic Change and
Wellbeing (No. 731875), and Cellex Foundation (PREDICT).
Conflict of interest
CE: has on-going research collaboration with Merz Pharmaceu-
tical and Novartis. He has received speaker fees from Novartis,
Gilead and Merz Pharmaceuticals. TB: Receipt of honoraria or
consultation fees or participation in a company sponsored
speaker’s bureau: Abbvie, Alexion, Bayer, Gilead, Eisai, Falk
Foundation, Intercept, Ipsen, Janssen, MSD/Merck, Novartis, and
Sequana Medical. Receipt of grants/research supports: Abbvie,
BMS, Gilead, MSD/Merck, Humedics, Intercept, Merz, Sequana
Medical. MS: Receipt of honoraria or consultation fees or
participation in a company sponsored speaker’s bureau: Merck
Serono GmbH, Bayer AG, ERBE Elektromedizin GmbH, Amgen
Inc., Johnson&Johnson Medical GmbH, ERBE Elektromedizin
GmbH, Takeda Pharmaceutical Limited, Olympus K.K., Medtronic
1353
Research Article
GmbH, Intuitive Surgical Inc.. JT: has received speaking and/or
consulting fees from Gore, Bayer, Alexion, MSD, Gilead, Intercept,
Norgine, Grifols, Versantis, and Martin Pharmaceutical. All other
authors declared no conflict of interest.
Please refer to the accompanying ICMJE disclosure forms for
further details.
Authors’ contributions
CE, TB, MS, KS, AF and AS were substantially involved the
conception and design of the project. CE, TB and AF were
involved in data analysis and interpretation. All authors were
involved in data acquisition, revised the manuscript critically,
approved the final version to be published and agreed on the
accuracy and integrity of data presented here.
Data availability statement
Data are available upon request from the corresponding author.
Acknowledgement
We would like to thank Laura A. Kehoe for her excellent English
language editing. We also like to acknowledge Prof. Reiner Wiest,
Prof. Tilmann Sauerbruch and Prof. Walter Lehmacher for their
deliberate and critical contribution as members of the DMSB.
Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jhep.2021.07.033.
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