Accepted Manuscript

Increased Level of Interleukin 6 Associates With Increased 90-day and 1-yearMortality in Patients With End-stage Liver Disease

Johannes Remmler, Christoph Schneider, Theresa Treuner-Kaueroff, MichaelBartels, Daniel Seehofer, Markus Scholz, Thomas Berg, Thorsten Kaiser

PII: S1542-3565(17)31106-0DOI: 10.1016/j.cgh.2017.09.017Reference: YJCGH 55449

To appear in: Clinical Gastroenterology and HepatologyAccepted Date: 8 September 2017

Please cite this article as: Remmler J, Schneider C, Treuner-Kaueroff T, Bartels M, Seehofer D, ScholzM, Berg T, Kaiser T, Increased Level of Interleukin 6 Associates With Increased 90-day and 1-yearMortality in Patients With End-stage Liver Disease, Clinical Gastroenterology and Hepatology (2017),doi: 10.1016/j.cgh.2017.09.017.

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Title:

Increased Level of Interleukin 6 Associates With Increased 90-day and 1-year

Mortality in Patients With End-stage Liver Disease

Short Title:

Interleukin 6 and prognosis in liver disease

Authors:

Johannes Remmler1*; Christoph Schneider1*; Theresa Treuner-Kaueroff1; Michael

Bartels2; Daniel Seehofer2; Markus Scholz4; Thomas Berg3; Thorsten Kaiser1

* these authors contributed equally

1 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics,

University Hospital Leipzig, Germany

2 Department of Visceral, Transplant, Thoracic and Vascular, Surgery, University

Hospital Leipzig, Germany

3 Section of Hepatology, Department of Gastroenterology and Rheumatology,

University Hospital Leipzig, Germany

4 Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University

Hospital Leipzig, Germany

Grant Support:

The authors received no specific funding for this work.

Abbreviations:

AUC, Area under the curve

AUROC, Area under receiver operating characteristic

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ACLF, Acute-on-chronic liver failure

CRP, C-reactive protein

ECLIA, Electro-chemiluminescence immunoassay

EDTA, Ethylenediaminetetraacetic acid

IL6, Interleukin 6

INR, International normalized ratio

IQR, Interquartile range

MELD, Model for end-stage liver disease

MELD-Na, Model for end-stage liver disease including serum sodium

ROC, Receiver operating characteristic

WBC, White blood cell count

Correspondence:

Dr. med. Thorsten Kaiser

Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics

University Hospital Leipzig

Paul-List-Str. 13-15

04103 Leipzig

Germany

Telephone: +49 341 9722200

Fax: +49 341 22209

E-mail: thorsten.kaiser@medizin.uni-leipzig.de

Disclosures:

The authors declare no conflict of interests.

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Author Contributions:

study concept and design:

J.R.; C.S.; T.T-K.; M.B.; D.S.; M.S.; T.B.; T.K.

acquisition of data:

C.S.; T.T-K.;

analysis and/or interpretation of data:

J.R.; C.S.; T.T-K.; M.B.; D.S.; M.S.; T.B.; T.K.

statistical analysis:

J.R.; M.S.; T.K.

drafting the manuscript:

J.R.; C.S.; T.K.

critical revision of the manuscript for important intellectual content:

J.R.; C.S.; T.T-K.; M.B.; D.S.; M.S.; T.B.; T.K.

final approval of the version to be published:

J.R.; C.S.; T.T-K.; M.B.; D.S.; M.S.; T.B.; T.K.

study supervision:

T.K.

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Abstract

Background & Aims: Organ allocation for liver transplantation is based on

prognosis, using the model for end-stage liver disease (MELD) or MELD-Na score.

These scores do not consider systemic inflammation and septic complications. Blood

level of C-reactive protein (CRP), in addition to the MELD score, associates with

mortality in patients with end-stage liver disease, whereas levels of interleukin 6 (IL6)

have not been systematically studied.

Methods: We performed a retrospective observational cohort study of 474 patients

with end-stage liver disease (63.5% male; median age, 56.9 years), evaluated for

liver transplantation in Germany, with at least 1 year of follow up. Data were collected

on blood levels of CRP, IL6, and white blood cell count (WBC). Findings were

analyzed in relation to mortality and compared with patients’ model for end-stage

liver disease (MELD) scores and MELD-Na scores. For survival analysis, the cohort

was divided into quartiles of IL6, CRP, and WBC levels, as well as MELD scores.

Log-rank test and the Cox proportional hazards regression model were used to

compare the groups, and area under the receiver operating characteristic (AUROC)

values were calculated.

Results: Blood levels of IL6 and MELD scores associated with mortality: none of the

patients with levels of IL6 below the first quartile (below 5.3 pg/ml) died within 1 year.

In contrast, 67.7% of the patients in the highest quartile of IL6 level (37.0 pg/ml or

more) died within 1 year. MELD score also correlated with mortality: among patients

with MELD scores below 8.7, 0.9% died within 1 year, whereas in patients with

MELD scores of 18.0 or more, 67.4% died within 1 year. The predictive value of level

of IL6 (AUROC, 0.940) was higher than level of CRP (AUROC, 0.866) (P=.009) or

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WBC (AUROC, 0.773) (P<.001) for 90-day mortality. MELD scores associated with

90-day mortality (AUROC, 0.933) (P=.756) as did MELD-Na score (AUROC, 0.946)

(P=.771). Level of IL6 associated with 1-year mortality (AUROC, 0.916) to a greater

extent than liver synthesis or detoxification markers international normalized ratio

(AUROC, 0.839) (P=.007) or bilirubin (AUROC 0.846) (P=.007). Level of IL6 was an

independent, significant risk factor for mortality after adjustment for MELD score,

MELD-Na score, level of CRP, or WBC.

Conclusion: In a retrospective analysis, we found high blood levels of IL6 to

associate with 90-day and 1-year mortality in patients with end-stage liver disease; its

predictive value was comparable to that of MELD or MELD-Na score, and was higher

than that of level of CRP or WBC. Further studies should be performed to confirm the

results in different cohorts.

KEY WORDS: acute-on-chronic liver failure; biomarker; cytokine; cirrhosis.

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Introduction

Liver transplantation is the only curative therapy option for patients with end-stage

liver disease. In most countries, organ allocation is prioritized according to the

estimated risk of mortality (medical urgency), which is assessed using the model for

end-stage liver disease (MELD) score 1–3. Introduction of the MELD-based liver

allocation has reduced waiting list registrations, waiting time and, most importantly,

waiting list mortality 4. The MELD score is composed of bilirubin, creatinine and INR

(international normalized ratio). In the USA, the MELD-Na score is used since 2016,

additionally including serum sodium 5–7. The MELD/MELD-Na score does not

account for inflammation in determining mortality risk. However, complications due to

bacterial infections such as spontaneous bacterial peritonitis are associated with a

dramatically worse prognosis. Inflammation has also been considered a possible

prognostic determinant in patients with end-stage liver disease. Furthermore,

systemic inflammatory response syndrome (SIRS) leads to a poor outcome in

patients with cirrhosis 8,9. SIRS is an independent prognostic factor in these patients

10. Cervoni et al. demonstrated the prognostic value of C-reactive protein (CRP) in

cirrhotic patients 11,12. They were able to show that a prognostic model including

MELD, CRP and age predicts three-month mortality of cirrhotic patients better than

the MELD alone 13. The prognostic value of IL6 levels has not been systematically

studied yet in these patients, even though it is a measure of inflammation that is

detectable earlier and more sensitive than CRP 14. IL6 is produced in monocytes,

macrophages, T cells, fibroblasts and endothelial cells, initiates the production of

acute phase proteins and is an important inductor of infection defense 15,16. The aim

of our study was to compare the mortality prediction of IL6 to that of the

MELD/MELD-Na score, CRP and WBC in a large cohort of patients with end-stage

liver disease.

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Materials and methods

Study population

We performed a retrospective observational cohort study. The study population

consisted of 474 different patients that were evaluated for liver transplantation at the

Leipzig University Hospital. A biobank (-80°C) of s era of these patients was

established, allowing further characterization in addition to the routinely determined

biomarkers. Clinical information was collected from electronic patient records. Follow-

up time was at least one year. IL6, CRP, WBC and MELD were available in 474, 471,

461 and 468 of the patients, respectively. Clinical records of all patients deceased in

the university hospital were systematically analyzed for documented complications

and death causes (79 of 111 patients (71.2 %)). Clinical records of patients who

deceased outside our hospital were not available, in these cases the date of death

was received by contacting the hospital or the relatives of the patient. The study was

approved by the Leipzig University Faculty of Medicine ethics committee (reference

number: 039/14ff).

Quantification of WBC, MELD, IL6 and CRP

WBC and laboratory data for the MELD/MELD-Na score were measured routinely as

part of the evaluation process for liver transplantation. WBC was measured in EDTA

whole blood using the XN 9000 system (Sysmex, Kobe, Japan). Bilirubin (Total DPD

Gen. 2 kit, colorimetric assay), creatinine (Creatinine Plus Ver. 2 kit, enzymatic

method) and sodium (ion sensitive electrode Gen. 2) were measured in serum on the

cobas 8000 analyzer (Roche, Mannheim, Germany). INR was determined in citrate

plasma using the ACL TOP 700 System (Instrumentation Laboratory, Lexington,

USA). IL6 (Elecsys IL-6 kit, ECLIA) and CRP (Cobas c pack CRPL3 kit,

immunoturbidimetry) were determined in serum from the biobank using the cobas

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8000 analyzer. These samples were stored at -80°C a fter acquisition and thawed for

batched analysis.

The MELD and MELD-Na scores were calculated according to the UNOS (United

Network of Organ Sharing) guidelines using the formulas given below 6. In this study,

unlike in the routine procedure, MELD score as well as MELD-Na score were not

rounded to whole numbers for statistical analysis.

MELD score = 10 x {0.957 x Ln (creatinine [mg/dL]) + 0.378 x Ln (bilirubin [mg/dL]) +

1.120 x Ln (INR) + 0.643}

creatinine (mg/dL), bilirubin (mg/dL) and INR values that were lower than 1.0 were set to 1.0 for MELD

calculation. Maximum serum creatinine level was set to 4.0 mg/dL. Similarly, the maximum creatinine

level in dialysis patients was set to 4.0 mg/dL.

MELD-Na score = MELD + 1.32 x (137 - Na) - [0.033 x MELD x (137 - Na)]

Meld-Na score was applied only for patients with an MELD greater than 11. Sodium values less than

125 mmol/L were set to 125, and values greater than 137 mmol/L were set to 137.

The following reference values are used: WBC: 3.5-9.8 *10^9/l, bilirubin: <17,1

µmol/l, CRP: <5 mg/l. IL6 <7 pg/ml, creatinine: 45-84/59-104 µmol/l (female/male,

respectively).

Statistics

Statistical analysis was performed using SPSS 23 (SPSS Inc., Chicago, USA),

MedCalc 12 (MedCalc Software bvba, Ostend, Belgium) and R (R Foundation for

Statistical Computing, Vienna, Austria; www.r-project.org).

The characteristics of the cohort are shown as median and interquartile ranges

(IQR). For survival analysis, the cohort was divided into four groups, according to the

quartiles of the IL6, CRP and WBC measures as well as the MELD score. Log-rank

test and Cox Proportional Hazards Regression Model were used to statistically

compare the groups. Survival data of patients receiving liver transplants were

censored at the date of transplantation. Receiver operating characteristics (ROC)

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and area under ROC (AUROC) were computed to determine the mortality prediction

performance of IL6, CRP, WBC MELD and MELD-Na score. DeLong test was used

to compare AUROCs 17, using the pROC R-package 18. Mann-Whitney U test was

used to compare continuous characteristics between two groups, and Pearson’s chi-

squared test was used to compare categorical data. Optimal cut-off values were

obtained calculating the maximum Youden index.

Results

Study population characteristics

The cohort consisted of patients with end-stage liver disease who were evaluated for

liver transplantation. The baseline characteristics of the cohort are summarized in

table 1. We included 474 patients, of which 63.5% were men. The median age was

56.9 years (IQR: 50.6 - 62.9). The most common cause of end-stage liver disease in

this cohort was alcohol abuse (62.9%), followed by cryptogenic liver cirrhosis (10.3%)

and viral hepatitis (8.4%). The median MELD score was 11.9 (IQR: 8.7 - 18.0).

During follow-up (median follow-up time: 549 days (IQR: 257 - 789)), 15.2% of the

patients received a liver transplant, and 27.6% of those who didn’t receive an organ

died. Clinical patient records revealed upper gastrointestinal bleeding in 26.8%,

hepatocellular carcinoma in 18.6% and spontaneous bacterial peritonitis in 14.6% of

the cases.

Survival analysis

Data of deceased and surviving patients were compared (table 2). There were no

significant differences in the ages or sex of surviving and deceased patients. Highly

significant differences (p < 0.001) could be observed for IL6, CRP and MELD score

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as well as bilirubin, creatinine and INR, and a less distinct difference was observed

for WBC (p = 0.003).

For a more detailed survival analysis the patients were divided into four groups

according to the quartiles of the baseline IL6, CRP and WBC measurements as well

as the baseline MELD score. Figure 1 shows the Kaplan-Meier curves for the first

year of follow-up. The detailed descriptive mortality data of the groups can be found

in Supporting documents, table 1. IL6, CRP and MELD score all showed strong

correlations with mortality, since survival of groups divided according to these

measures differed in a highly significant way (p < 0.001 in all cases, log-rank test with

three degrees of freedom). Survival of the groups divided according to WBC differed

less clearly, but still significantly (p = 0.001). In particular, elevated IL6 levels and

MELD scores were highly predictive for mortality. None of the patients from group Q1

(below the first IL6 quartile, < 5.3 pg/ml) died within one year. In contrast, 67.7% of

the patients in group Q4 (on and above the third IL6 quartile, ≥ 37.0 pg/ml) died

within one year. MELD score also correlated significantly with mortality; one-year

mortality for groups Q1 (MELD < 8.7) and Q4 (MELD ≥ 18.0) were 0.9% and 67.4%,

respectively. The differences in survival were less distinct for CRP groups and clearly

less distinct for WBC groups.

To study the possible role of biomarkers for inflammation on short-term mortality and

long-term survival, we separately analyzed patients who died between day 31 and

365 of follow-up. Here, the mortality differences among the groups were most distinct

for IL6 (0% and 38.5% for groups Q1 and Q4, p < 0.001), followed by MELD score

(0.9% and 34.8% for groups Q1 and Q4, p < 0.001). In contrast to this, no significant

differences in mortality between day 31 and 365 were observed in the WBC groups

Q1 (WBC < 4.6 *10^9/l, mortality 12.3%) and Q4 (WBC ≥ 8.2 *10^9/l, mortality

12.8%).

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The correlation of the baseline IL6, CRP and WBC measures as well as the baseline

MELD score with mortality was analyzed further using the Cox regression (table 3).

For IL6 groups, the risk of mortality was significantly higher for groups Q2, Q3 and

Q4 (hazard ratios: 11.1 (p = 0.021), 36.3 (p < 0.001) and 94.8 (p < 0.001),

respectively) than for group Q1. Baseline CRP levels also correlated strongly with the

risk of mortality, but hazard ratios were lower compared to IL6 (1.2 (p = 0.749), 3.0

(p = 0.003) and 8.7 (p < 0.001) for groups Q2, Q3 and Q4 compared to group Q1).

We also analyzed whether the inflammatory markers were still relevant predictors of

mortality after adjustment for MELD score. This was clearly the case for IL6 and

CRP. Even after adjustment for MELD/MELD-Na, CRP and WBC, IL6 levels

remained an independent factor for mortality (hazard ratios: 8.2/8.0

(p = 0.046/0.049), 17.3/17.6 (p = 0.006/0.006) and 22.5/21.4 (p = 0.004/0.004) for

groups Q2, Q3 and Q4 compared to group Q1). CRP, however, was no longer a

significant risk factor after adjustment for MELD/MELD-Na, WBC and IL6 (hazard

ratio 1.7/1.6 (p = 0.236/0.256) for group Q4 compared to Q1, hazard ratios and p-

values obtained adjusting for MELD/MELD-Na score, respectively).

To analyse the predictive value of the parameters of interest, receiver operating

characteristics (ROC) for baseline IL6, CRP and WBC measurements and

MELD/MELD-Na scores were analyzed. The ROC curves and the calculated area

under ROC (AUROC) values are shown in figure 2. Of the investigated markers, IL6

and MELD/MELD-Na score showed the highest mortality prediction for every time

period considered. For 90-day mortality, AUROC was 0.940/0.933 for IL6 and MELD,

respectively. Importantly, there were no significant differences between the AUROCs

of IL6 and the MELD score (p > 0.3 for all time periods). This was also the case for

comparison of IL6 and MELD-Na score. For 90-day mortality, AUROC for MELD-Na

was 0.946 (p = 0.771 compared to IL6, not shown). CRP was also a powerful

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predictor for mortality, yet it was significantly less predictive than IL6

(p = 0.014/0.009/0.013 for 30/90/365-day mortality, respectively). WBC predicted

mortality less precisely than the other inflammation parameters, especially in the long

term.

The analysis of clinical records of all deceased study patients in our hospital showed

with the exception of fulminant and acute on chronic liver failure that IL6 levels were

not associated with certain clinical complications. However, significantly elevated IL6

levels were found in patients with short time mortality (within 30 days). There was no

continued consumption of alcohol documented in the records (supporting documents,

table 2). The optimal cut-off value for the prediction of 90-day mortality by IL6 was

36.6 pg/ml (sensitivity 89.1 %, specificity 85.5 %) in our cohort. (Results for CRP,

WBC and MELD/MELD-Na with corresponding sensitivity and specificity are given in

supporting documents, table 3.)

Discussion

Our study is the first to identify the importance of IL6 as an excellent mortality

predictor in patients with end-stage liver disease. The predictive value of IL6 alone

was comparable to that of the MELD/MELD-Na score (which is composed of bilirubin,

creatinine, INR, and additional sodium for the MELD-Na score) and significantly

better than that of CRP or WBC. IL6 was also an independent risk factor for mortality

after adjustment for MELD. Some patients in this study had low MELD scores but

died within 90 days; the mortality risk for these patients could have been detected by

using elevated IL6 as an additional predictor of mortality (Supporting documents,

figure 1).

IL6 is not only a sensitive indicator; it also serves as a key mediator for inflammation.

IL6-dependent signaling in the liver is critical for the induction of the acute phase

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response 19. In patients with liver cirrhosis, the acute phase reaction is known to play

a role in clinical complications. CRP is an acute phase reactant and it’s elevation has

recently been associated with increased mortality in patients with liver cirrhosis 11–

13,20. Furthermore, inflammation plays a key role in the pathogenesis of ACLF (acute-

on-chronic liver failure), which is a major cause of unfavorable outcomes in patients

with chronic liver disease. Consequently, the recently introduced chronic liver failure

consortium (CLIF-C) ACLF score incorporates WBC as a measure of systemic

inflammation and predicts mortality better than the MELD score in patients with ACLF

21,22. This study confirms the predictive value of WBC and CRP. However, the

predictive value of IL6 elevation is distinctly higher than that of WBC and also clearly

and significantly higher than that of CRP. Unlike IL6, which is produced by T-cells

and macrophages, CRP is produced exclusively in the liver as a response to IL6 and

other cytokines in an acute phase reaction 15,16,23. Consequently, in the case of

severe liver insufficiency, limited synthesis capacity could lead to low CRP levels.

WBC may also not be an suitable parameter because white blood cell count does not

always increase in cases of severe systemic inflammation; it may also decrease due

to the loss of cells in the periphery 24.

Interestingly, the predictive value of IL6 concerning 365-day mortality (AUROC

0.916) was higher even than that of the liver synthesis and detoxification markers

INR (AUROC 0.839, p = 0.007) and bilirubin (AUROC 0.846, p = 0.007) and tended

to be higher for the prediction of 90-day mortality (AUROCs 0.916 for IL6, compared

to 0.887 for INR (p = 0.058) and 0.894 for bilirubin (p = 0.065), respectively) (Not

shown).

The reason for this observation remains to be elucidated. Even after exclusion of

short-time mortality (within 30 days), high IL6 concentrations in the blood were still

highly significantly associated with increased mortality. In our opinion, this indicates

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that the prognostic value cannot be explained by acute infections or infection-related

mortality alone.

IL6 is a cytokine with pleiotropic effects. It indicates infection complications, drives

inflammation and promotes tumorigenesis. A negative correlation between

biomarkers for liver synthesis and IL6 has been demonstrated by Streetz KL et al. 25.

In patients with acute alcoholic hepatitis higher IL6 levels were associated with

increased mortality 26. Recently, an association between high IL6 levels, impairment

of liver function and increased mortality has been observed in small cohorts of

cirrhotic patients with hepatocellular carcinoma and variceal bleeding 27,28.

On the other hand, experimental studies in rodents have repeatedly shown that IL6

may have protective effects in liver failure. This may be caused by a positive effect of

IL6 on liver regeneration and recovery from liver failure 29–33. However, most of these

studies focused on rodent models of liver disease, liver insufficiency and acute liver

failure. Our contrary findings in patients with end-stage liver disease may be

explained by the pathophysiological differences between acute and chronic liver

failure as well as by different IL6 effects over time in our patients suffering from

chronic liver diseases. Acute and only shortly elevated IL6 levels may be

advantageous for liver regeneration whereas chronic IL6 elevations may have

disadvantageous effects for the liver and other organs. A previous study in mice

demonstrated that IL6 can improve hepatic regeneration and repair in acute

situations, but chronic exposure sensitizes the liver to injury and cell death 34. In our

study increased IL6 levels revealed an unexpected high prognostic value for

mortality. Therefore, we assume a direct deleterious effect of long lasting interleukin

6 levels on the progression of liver cirrhosis in patients with chronic liver disease.

This study has some limitations. It is retrospective and designed to quantify and

compare the prognostic power of the inflammatory biomarkers IL6, CRP and WBC.

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Procalcitonin (PCT) levels were not measured in this study, as similar to CRP PCT is

secondarily induced by IL6 35. Due to the study design, the reasons for the strong

association between IL6 and mortality cannot be determined by this study. Since we

aimed to analyze the diagnostic power of standardized biomarkers available in a

clinical routine, we did not quantify the soluble IL6 receptor or gp130, which

complexes with IL6 36. Furthermore, the study population was heterogeneous in

terms of the etiology of end-stage liver disease. However, the majority (62.9%) of the

patients in this study suffered from alcoholic cirrhosis, and subgroup analysis (i.e., for

patients with HCC) provided comparable results.

To the best of our knowledge, this is the first study of patients with liver disease that

has shown IL6 alone to predict mortality with comparable accuracy as the

MELD/MELD-Na score and significantly better than the inflammatory biomarkers

CRP and WBC. IL6 could be a promising candidate to create an improved prognostic

score for predicting mortality in patients with end-stage liver disease. However,

further studies with cohorts of liver disease patients with different etiologies are

necessary to validate and confirm the predictive value of IL6 37.

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Tables Table 1: Baseline characteristics of the cohort.

Asterisks indicate significantly higher values than the other sex (p-value *<0.05, **<0.01, ***<0.001).

Tx - Transplantation, HBV - Hepatitis B virus, HCV - Hepatitis C virus, PBC - Primary biliary cirrhosis,

PSC - Primary sclerosing cholangitis, NASH - Non-alcoholic steatohepatitis, HCC - hepatocellular

carcinoma, SBP - spontaneous bacterial peritonitis, GI - gastrointestinal.

male (n=301 (63.5%)) female (n=173 (36.5%)) all (n=474)

age [years], median (IQR) 57.3 (51.7 - 62.7) 55.3 (49.4 - 62.9) 56.9 (50.6 - 62.9)

MELD, median (IQR) 12.1 (9.0 - 17.6) n=298 11.5 (7.8 - 19.1) n=170 11.9 (8.7 - 18.0) n=468

bilirubin [µmol/l], median (IQR) 26.9 (14.2 - 52.3) 27.0 (14.7 - 75.8) 27.0 (14.2 - 56.6)

creatinine [µmol/l], median (IQR) 84.0 (69.0 - 110.0) *** 72.0 (57.0 - 97.3) 80.0 (65.0 - 105.0)

INR, median (IQR) 1.3 (1.1 - 1.5) 1.3 (1.1 - 1.6) 1.3 (1.1 - 1.5)

IL6 [pg/ml], median (IQR) 12.4 (5.8 - 40.7) 10.4 (4.7 - 34.3) 11.6 (5.3 - 37.0)

CRP [mg/l], median (IQR) 6.3 (3.0 - 15.2) n=299 * 4.3 (2.1 - 12.8) n=172 5.8 (2.5 - 14.2) n=471

WBC [*10^9/l], median (IQR) 5.8 (4.6 - 7.9) n=295 6.3 (4.5 - 8.6) n=166 6.1 (4.6 - 8.2) n=461

follow-up time [days], median (IQR) 536 (281 - 783) 617 (201 - 793) 549 (257 - 789)

Tx received within follow-up-time (%) 47 (15.6) 25 (14.5) 72 (15.2)

mortality (patients that received Tx during the respective period were excluded)

within 7 days (%) 11 (3.7) n=295 8 (4.7) n=170 19 (4.1) n=465

within 30 days (%) 17 (5.9) n=290 14 (8.4) n=167 31 (6.8) n=457

within 90 days (%) 31 (10.7) n=290 23 (14.1) n=163 54 (11.9) n=453

within 365 days (%) 53 (19.7) n=269 39 (24.7) n=158 92 (21.5) n=427

within total follow-up time (%) 68 (26.8) n=254 43 (29.1) n=148 111 (27.6) n=402

etiology (more than 1 per patient possible)

alcoholic (%) 219 (72.8) *** 79 (45.7) 298 (62.9)

viral hepatitis (%) HBV (%) HCV (%)

28 (9.3) 10 (3.3) 19 (6.3)

12 (6.9) 2 (1.2) 11 (6.4)

40 (8.4) 12 (2.5) 30 (6.3)

autoimmune hepatitis (%) 8 (2.7) 16 (9.2) ** 24 (5.1)

PBC (%) 0 (0.0) 14 (8.1) *** 14 (3.0)

PSC (%) 9 (3.0) 5 (2.9) 14 (3.0)

NASH (%) 20 (6.6) 15 (8.7) 35 (7.4)

other (%) 5 (1.7) 22 (12.7) *** 27 (5.7)

cryptogenic (%) 23 (7.6) 26 (15.0) 49 (10.3)

complications/comorbidities (more than 1 per patient possible)

HCC (%) 74 (24.6) *** 14 (8.1) 88 (18.6)

SBP (%) 46 (15.3) 23 (13.3) 69 (14.6)

upper GI bleeding (%) 82 (27.2) 45 (26.0) 127 (26.8)

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Table 2: Comparisons of deceased and surviving patients.

Patients who received liver transplants during follow-up were excluded from this analysis.

deceased patients (n=111) surviving patients (n=291) p-value

age [years], median (IQR) 57.0 (51.7 - 62.4) 56.3 (50.4 - 63.9) 0.930

sex [m=male, f=female] (%) m: 68 (61.3); f: 43 (38.7) m: 186 (63.9), f: 105 (36.1) 0.622

MELD, median (IQR) 19.7 (14.4 - 26.2) 9.9 (7.8 - 13.2) <0.001

bilirubin [µmol/l], median (IQR) 61.4 (32.4 - 172.8) 18.8 (12.0 - 33.7) <0.001

creatinine [µmol/l], median

(IQR) 105 (72.3 - 159.5) 75 (63.3 - 91.0) <0.001

INR, median (IQR) 1.6 (1.3 - 1.9) 1.2 (1.1 - 1.3) <0.001

IL6 [pg/ml], median (IQR) 56.2 (21.9 - 142.3) 7.7 (4.4 - 14.3) <0.001

CRP [mg/l], median (IQR) 16.5 (7.5 - 37.3) 4.0 (2.0 - 8.2) <0.001

WBC [*10^9/l], median (IQR) 6.6 (4.7 - 10.9) 5.8 (4.5 - 7.9) 0.003

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Table 3: Cox proportional-hazard analysis.

In brackets: adjusted for MELD. In brackets and italics: adjusted for MELD and the other two

inflammation parameters (MELD: adjusted for the three inflammation parameters). Q1 was used as

reference category.

n hazard ratio [95%-CI] p-value

IL6 (pg/ml); reference value <7 pg/ml

Q2 (5.3 ≤ x < 11.6) 118 (116) (115)

11.1 [1.5 - 86.2] (8.4 [1.1 - 65.7]) (8.2 [1.0 - 64.5])

0.021 (0.043) (0.046)

Q3 (11.6 ≤ x < 37.0) 118 (117) (113)

36.3 [4.9 - 266] (21.2 [2.8 - 159]) (17.3 [2.2 - 135])

<0.001 (0.003) (0.006)

Q4 (≥ 37.0) 119 (118) (111)

94.8 [13.2 - 683] (37.5 [5.0 - 283]) (22.5 [2.7 - 184])

<0.001 (<0.001) (0.004)

CRP (mg/l); reference value <5 mg/l

Q2 (2.5 ≤ x < 5.8) 117 (115) (116)

1.2 [0.5 - 2.7] (1.0 [0.4 - 2.4]) (0.8 [0.3 - 2.1])

0.749 (0.985) (0.684)

Q3 (5.8 ≤ x < 14.2) 118 (118) (113)

3.0 [1.4 - 6.1] (1.8 [0.9 - 3.7]) (1.0 [0.5 - 2.3])

0.003 (0.123) (0.980)

Q4 (≥ 14.2) 118 (116) (110)

8.7 [4.4 - 16.9] (3.8 [1.9 - 7.6]) (1.7 [0.7 - 3.9])

<0.001 (<0.001) (0.236)

WBC (*10^9/l); reference value 3.5-9.8 *10^9/l

Q2 (4.6 ≤ x < 6.1) 115 (115) (115)

0.9 [0.5 - 1.7] (1.0 [0.5 - 1.8]) (0.9 [0.5 - 1.6])

0.713 (0.924) (0.671)

Q3 (6.1 ≤ x < 8.2) 112 (110) (109)

1.3 [0.7 - 2.3] (1.1 [0.6 - 2.0]) (1.0 [0.6 - 1.8])

0.435 (0.725) (0.981)

Q4 (≥ 8.2) 119 (118) (118)

2.0 [1.1 - 3.3] (1.4 [0.8 - 2.4]) (0.9 [0.5 - 1.6])

0.014 (0.279) (0.758)

MELD

Q2 (8.7 ≤ x < 11.9) 117 (115)

2.9 [0.9 - 9.0] (1.4 [0.4 - 4.5])

0.066 (0.562)

Q3 (11.9 ≤ x < 18.0) 117 (113)

7.5 [2.6 - 21.5] (3.2 [1.1 - 9.3])

<0.001 (0.037)

Q4 (≥ 18.0) 117 (110)

21.9 [8.0 - 60.0] (5.1 [1.7 - 15.0])

<0.001 (0.003)

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Figure legends

Figure 1: Kaplan-Meier curves for the first 365 days of follow-up.

Patients were divided into four groups (Q1-Q4) according to the quartile levels of the respective

biomarker. Censored survival data due to received transplantation are indicated by +.

Figure 2: ROC-curves for prediction of mortality.

Different time periods were considered and AUROC values were calculated. Patients receiving a liver

transplantation in the analyzed time frame were removed. p-values are given, obtained by comparing

with AUROC values for IL6.

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