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CXCL9 is a prognostic marker in patients with liver cirrhosis receiving transjugular intrahepatic portosystemic shunt
Research Article
CXCL9 is a prognostic marker in patients with liver cirrhosis receiving transjugular intrahepatic portosystemic shunt Marie-Luise Berres1, , Sonja Asmacher2, , Jennifer Lehmann2, Christian Jansen2, Jan Görtzen2, Sabine Klein2, Carsten Meyer3, Holger M. Strunk3, Rolf Fimmers4, Frank Tacke1, Christian P. Strassburg2, Christian Trautwein1, Tilman Sauerbruch2, Hermann Elard Wasmuth1, Jonel Trebicka2,⇑ 1
Department of Internal Medicine III, RTWH Aachen, Aachen, Germany; 2Department of Internal Medicine I, University of Bonn, Bonn, Germany; 3 Institute of Radiology, University of Bonn, Bonn, Germany; 4Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany
Background & Aims: Inflammation, collagen deposition and tissue remodelling are involved in the pathogenesis and complications of cirrhosis with portal hypertension. CXCL9 and other chemokines play an important role in these processes and have been associated with liver injury and complications of liver disease in humans. However, their predictive value in patients with cirrhosis and portal hypertension remains to be established. Methods: 103 patients with liver cirrhosis who had received TIPS (transjugular intrahepatic portosystemic shunt) were included into this study. The TIPS indication was either refractory ascites or recurrent bleeding. Before and after the TIPS procedure portal and hepatic venous blood samples were obtained in 78 patients. In 25 patients blood samples were obtained from the portal vein, hepatic vein, right atrium and cubital vein at TIPS insertion. Serum levels of CXCL9 were measured by cytometric bead array and correlated with clinical parameters and overall outcome. Results: Portal venous levels of CXCL9 decreased after TIPS. Child-Pugh score, refractory ascites, renal dysfunction and alcoholic aetiology of cirrhosis were associated with increased CXCL9 levels. Importantly, low levels of CXCL9 in portal and hepatic vein samples were prognostic factors for the survival of patients receiving TIPS during long-time follow-up.
Keywords: Cirrhosis; Portal hypertension; Transjugular intrahepatic portosystemic shunt; Chemokines. Received 17 June 2014; received in revised form 25 September 2014; accepted 30 September 2014; available online 17 October 2014 ⇑ Corresponding author. Address: Department of Internal Medicine I, University of Bonn, Sigmund-Freud Str. 25, D-53105 Bonn, Germany. Tel.: +49 228 287 15507; fax: +49 228 287 19718. E-mail address: [email protected] (J. Trebicka). These authors contributed equally to this work. Abbreviations: ALT, alanine aminotransferase; BUN, blood urea nitrogen; CBA, cytometric bead array; CHE, choline esterase; CXCL9, chemokine (C-X-C motif) ligand 9; CXCR3, chemokine (C-X-C motif) receptor 3; cGT, c-glutamyltransferase; HE, hepatic encephalopathy; HRS, hepatorenal syndrome; INR, international normalized ratio; MELD, model for end-stage liver disease; NASH, non-alcoholic steatohepatitis; PBC, primary biliary cirrhosis; PHPG, portal hepatic pressure gradient; SD, standard deviation; TIPS, transjugular intrahepatic portosystemic shunt.
Conclusions: The CXCR3 ligand CXCL9 affects the liver and/or is released by the liver and thereby might contribute to hepatic and extrahepatic organ dysfunction. Elevated levels of CXCL9 are associated with shorter survival in cirrhotic patients with severe portal hypertension receiving TIPS. This chemokine should be further evaluated as a novel biomarker for the outcome in patients with cirrhosis and portal hypertension and its modulation as a new therapeutic strategy. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Liver cirrhosis is the end stage of chronic liver injury. This is associated with portal hypertension, a major driver of complications limiting patients’ survival [1,2]. Portal hypertension results from different noxal stimuli, causing hepatic inflammation, fibrosis and angiogenesis [2]. These processes themselves are at least partly initiated and modulated by chemokines [3,4]. In particular, CXCR3 ligands, especially CXCL9, are increased during liver diseases [5–12] and are functionally linked to hepatic injury, inflammation, fibrosis, [8–10,12–18], angiogenesis [6] and complications of cirrhosis [19]. Liver resident cells, such as hepatocytes, Kupffer cells and hepatic stellate cells were found to represent a major source of CXCR3 ligands in the liver [20–25]. The increased intrahepatic levels of CXCR3 ligands during liver disease are also paralleled by elevated serum levels, as demonstrated in various animal and human studies [8,11,13,14,16–19,26–28]. Circulating inflammatory cells, in addition to liver resident cells, might contribute to their synthesis and secretion as shown previously [23,29]. Although the development of portal hypertension during chronic liver disease parallels the increase of intrahepatic and circulating levels of these chemokines, their specific impact and prognostic value in the setting of portal hypertension remains poorly investigated. Increased blood levels of these chemokines reflect an inflammatory syndrome, known to be associated with a worse prognosis in patients with liver cirrhosis. Thus, studies
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JOURNAL OF HEPATOLOGY in patients with end-stage liver disease suggested a direct relationship between complications of chronic liver diseases and the levels of CXCR3 ligands especially CXCL9 [19]. The aim of the present study was therefore: (1) to investigate whether there is a gradient of CXCL9 levels between portal and hepatic venous blood as a hint for their site of release and clearance; (2) to determine whether TIPS implantation changes CXCL9 levels in the splanchnic vascular bed and (3) to assess whether CXCL9 levels at the time of TIPS insertion are predictive for long-term survival in patients with portal hypertension undergoing TIPS procedure.
Patients and methods Patients and data collection 103 patients with liver cirrhosis and severe portal hypertension, selected for TIPS insertion, were enrolled in the study. 78 patients between August 1998 and August 2003, and 25 patients between January 2013 and July 2014, in the Department of Internal Medicine I, University of Bonn, Germany. General clinical characteristics are displayed in Table 1. Inclusion criteria were: age >18 years, clinical signs of liver cirrhosis, absence of signs of infection and an indication for the placement of a TIPS (secondary prophylaxis for recurrent bleeding in n = 40, therapy refractory ascites in n = 50 and both in n = 13). Patients were excluded from the study if they had contraindications for TIPS insertion (e.g. hepatic encephalopathy greater than grade I; bilirubin >5 mg/dl in the absence of acute bleeding; arterial pulmonary hypertension). Biochemical blood analyses were performed using standard tests. The local ethics committee of the University of Bonn approved the study protocol, and patients signed a written inform consent for all procedures. TIPS procedure and pressure measurement In the first cohort of patients (n = 78), enrolled between 1998 and 2003, a bare TIPS (8–10 mm Wallstent, Boston Scientific, MA, USA) was inserted as previously described [30–33]. In these patients, after a mean of fourteen days, an invasive procedure was performed to re-evaluate the function and position of the TIPS (previously specified as routine in our centre’s protocol to optimize TIPS function) [30–33]. Patients of the second cohort (n = 25), enrolled between 2013 and 2014, received covered TIPS-stents, but no invasive re-evaluation procedure was performed. Portal and hepatic venous pressures were invasively measured using a pressure transducer system (Combitrans, Braun Melsungen, Germany) and a
Table 1. Clinical parameters of all patients (n = 103) at admission and outcome.
Parameters Gender (female/male) Age (in years)-median (range) Child-score-median (range) Child category (A/B/C) MELD-score-median (range) Etiology (alcoholic/chronic hepatitis/other) Indication for TIPS insertion (variceal bleeding/refractory ascites/both) Ascites (absent/mild/severe) Hepatorenal syndrome (absent/Type 1/Type 2) Esophageal varices (absent/grade I-II/grade III-IV) Variceal bleeding (absent/present) Hepatic encephalopathy (absent/present) Survival time (in days)-median (range) MELD, model for end-stage liver disease. Median (range)
Value 41/62 57 (29-79) 8 (5-12) 22/66/15 8 (6-20) 67/14/22 40/50/13 24/23/56 86/13/4 12/70/21 54/49 93/10 291 (12-3850)
multichannel monitor (Sirecust, Siemens, Germany). The difference between these pressures is defined as the portal hepatic pressure gradient (PHPG). Arterial pressure and heart rate were non-invasively monitored during the procedures. Biochemical parameters, as well as portal and systemic haemodynamics, were recorded at the placement procedure of the TIPS and during the TIPS follow-up procedure (Table 2). Assessment of circulating levels of chemokines During the TIPS procedure, blood from the portal and hepatic veins were collected from all patients included in the study (n = 103) as soon as the right branch of the portal vein was cannulated to get blood specimen for the determination of CXCL9 levels. For the first cohort comprising 78 patients, a second invasive procedure was performed to re-evaluate the TIPS at the follow-up appointment and a catheter was placed into the portal vein, for pressure measurement and blood collection. In the second cohort of patients receiving covered TIPS (n = 25), additional blood samples from the portal vein, hepatic vein, the right atrium and the cubital vein were collected during the TIPS procedure. After collection, blood samples were centrifuged at 3000 rotations/minute for 15 min at 4°C. Serum samples for chemokine analyses were stored at 80°C until they were used. Serum concentrations of CXCL9 (monokine induced by interferon-c) were assessed with a cytometric bead array (Becton Dickinson, Heidelberg, Germany) according to the manufacturer’s instructions. The chemokine levels were quantified in undiluted serum samples in duplicates. Statistical analysis Data are presented as mean ± standard error (SEM) or medians and ranges. The Wilcoxon test was used for comparison of paired data and the Mann-Whitney and Kruskal-Wallis test for unpaired comparisons. Correlations were analysed with the Spearman correlation coefficient. Kaplan-Meier curves were used to analyse the survival rates of patients using the log-rank test. Cox regression multivariate analysis (forward stepwise likelihood-quotient) was performed to predict survival rates using the log-rank test. p values <0.05 were considered statistically significant. Statistical analyses were performed using SPSS 18.0 for Windows (SPSS Inc. Chicago, IL, USA) and GraphPad Prism version 5.00 (GraphPad Software, La Jolla, USA).
Table 2. Biochemical parameters before TIPS insertion and at the early TIPS check after a median of fourteen days (n = 78).
Biochemical parameters (units) Bilirubin (mg/dl) CHE (U/L) Albumin (g/L) ALT (U/L) AST (U/L) γGT (U/L) INR Serum creatinine (mg/dl) BUN (mg/dl) Serum sodium (mmol/L) Platelet count (G/L) PHPG (mmHg) Portal pressure (mmHg) Central venous pressure (mmHg) Portal venous blood flow velocity (cm/s)
Before TIPS
14 days after TIPS
1.1 (0.4-4.7) 1984 (479-4070) 32 (11-56) 18 (7-94) 18 (8-73) 59 (8-527) 1.17 (0.93-2.23) 1.1 (0.5-8.2) 43 (9-225) 136 (119-145) 100 (27-389) 20 (11-35) 32 (18-49) 11 (0-25)
1.3 (0.6-6.8)b 2000 (411-3795) 29 (16-34) 24 (9-357)b 22.5 (13-275)a 112 (12-515)b 1.2 (0.96-1.98) 0.9 (0.5-7.1)a 28 (1-118)b 138 (118-144)a 136 (65-326)b 9 (1-16)b 21 (12-33)b 12 (5-22)
16 (5-40)
38 (17-72)b
Data are shown as median and range and compared by non-parametric testing (Wilcoxon test). ap <0.05 and bp <0.005 vs. value before TIPS. CHE, choline esterase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cGT, c-glutamyltransferase; INR, international normalized ratio; BUN, blood urea nitrogen; PHPG, portal hepatic pressure gradient.
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Research Article Results Characteristics of patients before and after TIPS The characteristics of patients included in the study are depicted in Table 1. Briefly, patients were predominantly male (60%) with a median age of 57 years. The median Child-Pugh score was eight points and the majority of the patients were in Child-Pugh class B. The median MELD score was eight points. 65% of patients had alcoholic cirrhosis. The indications for TIPS insertion were secondary prophylaxis for recurrent bleeding in 40 patients, refractory ascites in 50 patients, or both indications in 13 patients. Hepatic encephalopathy episodes prior to placement of TIPS occurred in only 10 patients. In the first cohort of 78 patients the median survival time was 595 days, within a follow-up period of more than 10 years. The maximal follow-up of the second cohort was 408 days. The differences between the two cohorts are shown in the Supplementary Table 1. Briefly, patients receiving covered stents showed lower Child-Pugh and MELD scores compared to patients receiving bare stents. At the invasive TIPS follow-up, 14 days after the placement procedure, liver synthesis capacity remained unchanged as shown by the preserved choline esterase and INR levels (Table 2). After TIPS insertion levels of cGT, ALT, AST and bilirubin as markers of liver injury slightly increased, but renal function improved, as shown by the levels of creatinine and sodium as well as by the platelet counts (Table 2). Prior to TIPS insertion, patients showed significant portal hypertension with a median portal hepatic pressure gradient of 20 mmHg and a median absolute portal pressure of 32 mmHg, as well as a decreased portal venous blood velocity, measured by Doppler ultrasound (Table 2). 14 days after portal decompression by TIPS insertion, the portal hepatic pressure gradient remained significantly decreased, and portal venous blood velocity, a read out of preserved TIPS function, had more than doubled (Table 2). Serum levels of CXCL9 in portal and hepatic venous blood before and after TIPS placement Assessment of CXCL9 in the portal and hepatic venous blood before TIPS revealed surprising results (Table 3). CXCL9 levels in the portal vein tended to be higher than in the hepatic vein
without reaching significance (p = 0.192). After TIPS insertion, portal venous levels of CXCL9 significantly decreased (p = 0.031) compared to the corresponding levels before TIPS, while hepatic venous levels showed a tendency towards decrease without reaching statistical significance. When comparing the levels of all patients together, patients receiving a covered stent showed lower portal and hepatic CXCL9 serum levels than those receiving a bare stent. However, when comparing CXCL9 levels between patients of these two cohorts, stratified by Child-Pugh classes, no significant differences were observed (Supplementary Table 2). Importantly, we also assessed CXCL9 levels in blood samples collected from the right atrium and cubital vein from all patients of the second cohort in addition to hepatic levels. We did not observe any significant difference between hepatic and right atrial or cubital venous levels, while the levels of all sites showed excellent correlations with each other (Supplementary Fig. 1). Serum levels of CXCL9 in the portal vein and hemodynamic parameters The portal systemic pressure gradient showed a significant correlation with CXCL9 levels in portal venous blood after TIPS (r = 0.514; p = 0.01, data not shown), while no other association to hemodynamic parameters could be assessed.
Serum levels of CXCL9 in the portal and hepatic vein and hepatic function Serum levels of CXCL9 correlated with the Child-Pugh class. This association was highly significant for levels of CXCL9 in the hepatic vein (Fig. 1A). Interestingly, the difference of CXCL9 levels between different Child-Pugh classes was abrogated after TIPS insertions (Fig. 1A). While patients with Child-Pugh class A and B cirrhosis presented with chemokine levels in a similar range as before TIPS insertions, patients with Child-Pugh class C cirrhosis displayed a dramatic reduction of CXCL9 levels in portal as well as in hepatic venous blood (Fig. 1A). When stratified for aetiology, patients with liver cirrhosis due to excessive alcohol consumption presented with higher levels of CXCL9 in the portal vein as compared to those patients with non-alcoholic cirrhosis (Fig. 1B).
Table 3. CXCL9 levels in portal and hepatic venous blood before and after TIPS insertion as well as correlation between CXCL9 levels and parameters of patients with significant portal hypertension receiving TIPS (n = 103).
CXCL9 median (range) MELD Creatinine BUN Sodium excretion in urine before TIPS
Portal venous levels before TIPS 317 pg/ml (9.9-66,995) r = 0.1 p = 0.3 r = 0.620 p = 0.000 r = 0.590 p = 0.000 r = -0.266 p = 0.116
Hepatic venous levels before TIPS 320 pg/ml (7.85-51,702) r = 0.3 p = 0.016 r = 0.570 p = 0.000 r = 0.500 p = 0.000 r = -0.381 p = 0.017
Portal venous levels after TIPS 165 pg/mla (20-44,208) r = 0.286 p = 0.066 r = 0.446 p = 0.002 r = 0.363 p = 0.081 r = -0.374 p = 0.015
Hepatic venous levels after TIPS 218 pg/ml (34-23,500) r = 0.174 p = 0.274 r = 0.484 p = 0.002 r = 0.532 p = 0.006 r = -0.403 p = 0.015
Data are shown as median and range and compared by non-parametric testing (Wilcoxon test). ap <0.05 vs. portal venous levels before TIPS.
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JOURNAL OF HEPATOLOGY Table 4. Parameters correlating with survival time. Parameters correlating with survival time in univariate Cox regression analysis (A) and CXCL-9 levels correlating with survival time in multivariate Cox regression analysis (B).
A
Parameters Levels of CXCL9 in portal venous blood before TIPS Levels of CXCL9 in hepatic venous blood before TIPS MELD Bleeding before TIPS Hepatorenal syndrome Sodium before TIPS Sodium after TIPS Serum creatinine before TIPS Serum creatinine after TIPS BUN after TIPS Bilirubin after TIPS Portal hepatic pressure gradient after TIPS Portal pressure after TIPS
B
p value
Serum levels of CXCL9 0.0001 below 400 pg/ml in portal blood before TIPS insertion
p value 0.0001 0.0001 0.0001 0.026 0.035 0.023 0.057 0.0001 0.0001 0.0001 0.0001 0.002 0.0001
Hazard Confidence ratio interval of hazard ratio 0.264 0.133-0.524
Interestingly, CXCL9 levels before TIPS insertion differed with regard to the indication for TIPS. Patients receiving TIPS for refractory ascites displayed higher levels of CXCL9 in portal and hepatic venous blood than those who received TIPS for recurrent variceal bleeding (Fig. 1C). Of note, this effect was independent of the magnitude of portal pressure. Importantly, the TIPS procedure significantly decreased levels of CXCL9 in the portal vein in both groups (Fig. 1D and E), although median levels remained significantly higher in patients who underwent TIPS procedure for refractory ascites. Moreover, CXCL9 levels positively correlated with the severity of ascites especially before TIPS procedure (Fig. 1F). Serum levels of CXCL9 in portal and hepatic vein and renal function Serum levels of CXCL9 in portal and hepatic vein were positively correlated with the MELD score (Table 3). The biochemical parameters creatinine, blood urea nitrogen (BUN) as well as sodium urinary excretion as marker of renal function were correlated with CXCL9 levels in portal and hepatic vein (Table 3). Additionally, an inverse correlation was observed with serum albumin (data not shown). Serum levels of CXCL9 and survival of patients receiving TIPS The parameters with impact on survival time were identified by univariate Cox regression analysis and are depicted in Table 4A. Higher serum levels of CXCL9 before TIPS in hepatic and portal venous blood were significantly associated with poor survival in the univariate analysis, as was the MELD score, episodes of bleeding prior to the procedure, and presence of a hepatorenal syndrome. Similarly, this analysis revealed an association of serum sodium and creatinine levels before and after TIPS, as well
as BUN and bilirubin after TIPS with patient survival. As expected portal systemic pressure gradient and portal pressure after TIPS placement were associated with survival as well. In the multivariate Cox regression analysis, in which we included all parameters previously identified to be associated with survival in the univariate analysis (Table 4A), the CXCL9 level in portal blood before TIPS insertion was the only parameter independently predictive for survival in our cohort (Table 4B). Kaplan-Meyer analysis for overall survival were performed using the 50th percentile of CXCL9 levels in the portal vein and hepatic vein before TIPS as a cut-off (Fig. 2A and B). For portal venous blood, the 50th percentile was presented by 400 pg/ml in our cohort. Patients with CXCL9 levels higher than 400 pg/ml in the portal vein showed a median survival time of 113 days vs. 941 days for patients with CXCL9 levels lower than 400 pg/ ml. The calculation of the hazard ratio for overall survival for portal CXCL9 levels below 400 pg/ml revealed a highly reduced ratio of 0.2623 (95% confidence interval 0.133–0.524, p = 0.0001; Table 4B). Of note, we were able to validate the cut-off of 400 pg/ml CXCL in the portal vein during TIPS procedure as a highly significant predictive marker for survival in our second, independent cohort, revealing a statistically significant reduced ratio of 0.04 (p = 0.024), albeit, the short follow-up with a low number of events might limit the statement for this second cohort. Additionally, stratification of patients from the second cohort, using the cut-off of 400 pg/ml CXCL9 in the cubital vein, also revealed a significant survival effect (Supplementary Fig. 2). Finally, ROC analysis were performed to assess the predictive value of portal CXCL9 levels for 6-month and 2 year overall survival (Fig. 2C and D). For both time points, CXCL9 levels in the portal vein revealed a highly predictive value for survival with an AUC >0.77, especially within the first 6 months (Fig. 2C). These results were superior to the predictive values of the MELD score (Fig. 2E and F) and the Child-Pugh score (AUC of 0.6552, p = 0.055 for 6 months after TIPS, AUC of 0.5877, p = 0.2344 for 2 years after TIPS, data not shown) for both periods.
Discussion This study showed for the first time that portal venous levels of the CXCR3 ligand CXCL9 predict survival in patients with liver cirrhosis and severe portal hypertension receiving TIPS. In these patients high circulating levels of CXCL9 in the portal and hepatic vein were associated with hepatic and renal dysfunction. CXCL9 levels showed no direct correlation with hepatic portal pressure, implicating that CXCL9 is not simply a surrogate marker for portal hypertension. It rather is correlated to complications with portal hypertension, such as presence and severity of ascites, and consequently the TIPS procedure resulted in a reduction of CXCL9 levels. CXCR3 ligands have been extensively investigated in animal studies [6,10,13,15,23,34], as well as in humans with liver disease [5,9,11,12,16,18,26,27,35]. Different studies have assessed a relationship between CXCR3 ligands and fibrosis and hepatic injury [8–10,12–18], angiogenesis [6], and complications of liver cirrhosis [19]. The majority of the studies focused on hepatitis C and alcoholic cirrhosis. Some studies analysed primary biliary cirrhosis and autoimmune hepatitis [5,11,35]. These studies revealed that intrahepatic levels of these chemokines are increased during liver injury and liver fibrogenesis [5,6–12]. In vitro studies have
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Fig. 1. Serum levels of CXCL9 correlate with the Child-Pugh score, indication for TIPS insertion, and ascites in cirrhotic patients with severe hypertension. Serum levels of CXCL9 are significantly increased in patients with higher Child-Pugh class before TIPS insertion in portal and hepatic venous blood (n = 78) (A). Serum levels of CXCL9 in patients with alcoholic cirrhosis are elevated compared to patients with non-alcoholic cirrhosis (B). Serum levels of CXCL9 in patients receiving TIPS for recurrent bleeding are significantly lower compared to those patients receiving TIPS for refractory ascites or both indications (C). After TIPS, levels of CXCL9 in the portal vein decrease in patients receiving TIPS for variceal bleeding (D) and refractory ascites (E). Serum levels of CXCL9 in patients with severe ascites are elevated compared to patients with less severe or no ascites (F). Data in (A, B, C, and F) are shown as means +/ standard error of the mean, in (D and E) paired data are shown. Wilcoxon and Kruskal-Wallis tests were used when appropriate.
shown that liver resident cells (e.g. hepatocytes, Kupffer cells, hepatic stellate cells) express these chemokines as a response to cellular injury and stress [20–25], suggesting a hepatic release of these chemokines. However, it was also implicated that circulating immune cells might contribute to elevated chemokine levels [23,29]. Our data do not support a distinct place of production or clearance of CXCL9. They rather suggest that levels of this CXCR3 ligand might be a result of intra- and extrahepatic production and clearance. The present study confirms previous findings, showing an association between the complications of end-stage liver disease and the levels of CXCR3 ligands [19]. In addition, we could demonstrate that CXCL9 levels in the portal vein are higher in patients receiving TIPS for refractory ascites, compared to those receiving TIPS for recurrent bleeding. This might be a result of continuous bacterial translocation and subsequent systemic inflammation in patients with refractory ascites. Importantly, we observed higher levels of CXCL9, especially in the portal vein, in patients with alcoholic cirrhosis, which might be explained by the more pronounced gut barrier dysfunction in alcoholic patients. More interestingly, these associations could provide functional insight on why the portal levels of CXCL9 had the strongest predictive value for survival in patients receiving TIPS. 336
In patients with portal hypertension, TIPS insertion increases the effective blood volume and improves renal perfusion [36]. In our patients, we observed a strong association of CXCL9 levels with parameters of renal function. In TIPS-treated patients, ascites and renal function were improved, which is associated with a drop in CXCL9 levels in the portal vein that could partially be explained by better renal clearance of CXCL9. This might also explain why levels in the portal vein and hepatic vein were not significantly different to those in the cubital vein and right atrium and showed excellent correlation to each other. Importantly, this excellent correlation might also implicate that levels of CXCL9 in cubital venous blood accurately reflect hepatic levels of CXCL9 and might facilitate the applicability of CXCL9 measurement for risk stratification of patients with portal hypertension undergoing TIPS procedure in clinical practise. However, it remains to be demonstrated in extended cohorts that the predictive value of cubital venous CXCL9 levels are comparable to those obtained from portal venous blood. Patients with severe portal hypertension and portal venous CXCL9 levels, elevated over 400 pg/ml, showed a higher mortality after TIPS insertion. Indeed, CXCL9 was an independent predictor of mortality in two independent cohorts, in patients receiving covered as well as bare TIPS. Nevertheless, one could speculate that its predictive value might at least partly be due to its
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JOURNAL OF HEPATOLOGY CXCL9 levels: hepatic vein before TIPS
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Fig. 2. CXCL9 levels are associated with the survival of cirrhotic patients with severe hypertension after TIPS insertion. (A) Kaplan-Meier survival analysis, demonstrating reduced survival of patients with portal venous CXCL9 levels >400 pg/ml (50th percentile of entire cohort) at the time of TIPS insertion during the follow-up period (n = 104). (B) Kaplan-Meier survival analysis showing survival of patients with hepatic venous CXCL9 levels >400 pg/ml (50th percentile of entire cohort) at the time of TIPS insertion during the follow-up period (n = 104). Data were tested by log-rank analyses. (C and D) ROC analyses demonstrate the predictive value of portal CXCL9 levels before TIPS insertion on 6-month (C) and 2-year survival (D) in a cohort of cirrhotic patients with severe hypertension. (E and F) ROC analyses for the predictive value of the MELD score before TIPS insertion on 6-month (E) and 2-year survival (F), respectively.
association with renal function, as renal function had been recently identified as one of the major short- and mid-term predictive factors of patients with chronic liver failure [37]. The association of CXCL9 levels with the Child-Pugh score, MELD-score, and severity of ascites was more pronounced in the hepatic vein. This finding suggests that CXCL9 might be a mediator of extrahepatic complications due to liver insufficiency. The relationship between the hepatic release of this chemokine and its exact mechanism, how it takes influence on other organs for example
by inducing an inflammatory answer in tissues, might be further evaluated. Clinical and biochemical parameters of hepatic and renal dysfunction, observed in our cohort, reflected severity of the liver disease and its extrahepatic complications in these patients. In addition, patients with high levels of the CXCR3 ligand CXCL9 showed poor overall survival. Interestingly, this was independent of other clinical parameters of end-stage liver disease. In our study CXCL9 levels in portal, hepatic and cubital vein bared valuable predictive information on the overall
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Research Article mortality risk in cirrhotic patients with severe portal hypertension receiving TIPS. Our study, thus, not only delivers insight into the association of chemokines with portal hypertension, but also offers proof that specific chemokines can predict mid- and longterm survival in patients receiving TIPS. Before TIPS-placement, portal levels of CXCL9 revealed a predictive value for 6-month and 2-year survival in our cohort of patients with severe portal hypertension receiving TIPS, which was superior to the established MELD and Child-Pugh scoring system. This information might be especially useful for the risk assessment in patients receiving TIPS as bridging strategy before liver transplantation. Further investigations are certainly needed, but this first proof of concept finding is essential to demonstrate the relevance of this chemokine system for the progression of liver disease. This study has several limitations. Portal venous levels of control and cirrhotic patients without TIPS would be desirable, however this is unethical in the usual clinical setting. Furthermore, a higher number of patients would be needed to better characterize potential aetiology-dependent differences between the subgroups receiving TIPS. However, our group of patients showed characteristics of severe portal hypertension. Survival in these patients was associated with already important and well investigated clinical and biochemical parameters and scores. This confirms that the data are consistent with findings, published in the literature. In summary, our study shows for the first time that the CXCR3 ligand CXCL9 is correlated with hepatic, renal and hemodynamic parameters in patients with portal hypertension. CXCR3 ligands should be further mechanistically evaluated in patients with liver cirrhosis and severe portal hypertension. Attempts to modulate these chemokines might help to alleviate symptoms and improve treatment of patients with liver disease, high morbidity, and mortality. Financial support The study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB TRR57 P04, P06, P07, P09, and P18 to MLB, FT, HEW, CT, TS, and JT) and from the H.J. & W. Hector Stiftung (M60.2 to JT).
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Authors’ contributions JT, MLB, HEW: study concept & design, acquisition of data analysis and interpretation of data, drafting of the manuscript, statistical analysis; SA, JL, CJ, HMS, JG, SK: technical support, acquisition of data, critical revision of the manuscript; RF, FT, CPS, CT, TS: technical support, critical revision of the manuscript.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2014.09. 032. 338
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CXCL9 is a prognostic marker in patients with liver cirrhosis receiving transjugular intrahepatic portosystemic shunt Marie-Luise Berres1, , Sonja Asmacher2, , Jennifer Lehmann2, Christian Jansen2, Jan Görtzen2, Sabine Klein2, Carsten Meyer3, Holger M. Strunk3, Rolf Fimmers4, Frank Tacke1, Christian P. Strassburg2, Christian Trautwein1, Tilman Sauerbruch2, Hermann Elard Wasmuth1, Jonel Trebicka2,⇑ 1
Department of Internal Medicine III, RTWH Aachen, Aachen, Germany; 2Department of Internal Medicine I, University of Bonn, Bonn, Germany; 3 Institute of Radiology, University of Bonn, Bonn, Germany; 4Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany
Background & Aims: Inflammation, collagen deposition and tissue remodelling are involved in the pathogenesis and complications of cirrhosis with portal hypertension. CXCL9 and other chemokines play an important role in these processes and have been associated with liver injury and complications of liver disease in humans. However, their predictive value in patients with cirrhosis and portal hypertension remains to be established. Methods: 103 patients with liver cirrhosis who had received TIPS (transjugular intrahepatic portosystemic shunt) were included into this study. The TIPS indication was either refractory ascites or recurrent bleeding. Before and after the TIPS procedure portal and hepatic venous blood samples were obtained in 78 patients. In 25 patients blood samples were obtained from the portal vein, hepatic vein, right atrium and cubital vein at TIPS insertion. Serum levels of CXCL9 were measured by cytometric bead array and correlated with clinical parameters and overall outcome. Results: Portal venous levels of CXCL9 decreased after TIPS. Child-Pugh score, refractory ascites, renal dysfunction and alcoholic aetiology of cirrhosis were associated with increased CXCL9 levels. Importantly, low levels of CXCL9 in portal and hepatic vein samples were prognostic factors for the survival of patients receiving TIPS during long-time follow-up.
Keywords: Cirrhosis; Portal hypertension; Transjugular intrahepatic portosystemic shunt; Chemokines. Received 17 June 2014; received in revised form 25 September 2014; accepted 30 September 2014; available online 17 October 2014 ⇑ Corresponding author. Address: Department of Internal Medicine I, University of Bonn, Sigmund-Freud Str. 25, D-53105 Bonn, Germany. Tel.: +49 228 287 15507; fax: +49 228 287 19718. E-mail address: [email protected] (J. Trebicka). These authors contributed equally to this work. Abbreviations: ALT, alanine aminotransferase; BUN, blood urea nitrogen; CBA, cytometric bead array; CHE, choline esterase; CXCL9, chemokine (C-X-C motif) ligand 9; CXCR3, chemokine (C-X-C motif) receptor 3; cGT, c-glutamyltransferase; HE, hepatic encephalopathy; HRS, hepatorenal syndrome; INR, international normalized ratio; MELD, model for end-stage liver disease; NASH, non-alcoholic steatohepatitis; PBC, primary biliary cirrhosis; PHPG, portal hepatic pressure gradient; SD, standard deviation; TIPS, transjugular intrahepatic portosystemic shunt.
Conclusions: The CXCR3 ligand CXCL9 affects the liver and/or is released by the liver and thereby might contribute to hepatic and extrahepatic organ dysfunction. Elevated levels of CXCL9 are associated with shorter survival in cirrhotic patients with severe portal hypertension receiving TIPS. This chemokine should be further evaluated as a novel biomarker for the outcome in patients with cirrhosis and portal hypertension and its modulation as a new therapeutic strategy. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Liver cirrhosis is the end stage of chronic liver injury. This is associated with portal hypertension, a major driver of complications limiting patients’ survival [1,2]. Portal hypertension results from different noxal stimuli, causing hepatic inflammation, fibrosis and angiogenesis [2]. These processes themselves are at least partly initiated and modulated by chemokines [3,4]. In particular, CXCR3 ligands, especially CXCL9, are increased during liver diseases [5–12] and are functionally linked to hepatic injury, inflammation, fibrosis, [8–10,12–18], angiogenesis [6] and complications of cirrhosis [19]. Liver resident cells, such as hepatocytes, Kupffer cells and hepatic stellate cells were found to represent a major source of CXCR3 ligands in the liver [20–25]. The increased intrahepatic levels of CXCR3 ligands during liver disease are also paralleled by elevated serum levels, as demonstrated in various animal and human studies [8,11,13,14,16–19,26–28]. Circulating inflammatory cells, in addition to liver resident cells, might contribute to their synthesis and secretion as shown previously [23,29]. Although the development of portal hypertension during chronic liver disease parallels the increase of intrahepatic and circulating levels of these chemokines, their specific impact and prognostic value in the setting of portal hypertension remains poorly investigated. Increased blood levels of these chemokines reflect an inflammatory syndrome, known to be associated with a worse prognosis in patients with liver cirrhosis. Thus, studies
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JOURNAL OF HEPATOLOGY in patients with end-stage liver disease suggested a direct relationship between complications of chronic liver diseases and the levels of CXCR3 ligands especially CXCL9 [19]. The aim of the present study was therefore: (1) to investigate whether there is a gradient of CXCL9 levels between portal and hepatic venous blood as a hint for their site of release and clearance; (2) to determine whether TIPS implantation changes CXCL9 levels in the splanchnic vascular bed and (3) to assess whether CXCL9 levels at the time of TIPS insertion are predictive for long-term survival in patients with portal hypertension undergoing TIPS procedure.
Patients and methods Patients and data collection 103 patients with liver cirrhosis and severe portal hypertension, selected for TIPS insertion, were enrolled in the study. 78 patients between August 1998 and August 2003, and 25 patients between January 2013 and July 2014, in the Department of Internal Medicine I, University of Bonn, Germany. General clinical characteristics are displayed in Table 1. Inclusion criteria were: age >18 years, clinical signs of liver cirrhosis, absence of signs of infection and an indication for the placement of a TIPS (secondary prophylaxis for recurrent bleeding in n = 40, therapy refractory ascites in n = 50 and both in n = 13). Patients were excluded from the study if they had contraindications for TIPS insertion (e.g. hepatic encephalopathy greater than grade I; bilirubin >5 mg/dl in the absence of acute bleeding; arterial pulmonary hypertension). Biochemical blood analyses were performed using standard tests. The local ethics committee of the University of Bonn approved the study protocol, and patients signed a written inform consent for all procedures. TIPS procedure and pressure measurement In the first cohort of patients (n = 78), enrolled between 1998 and 2003, a bare TIPS (8–10 mm Wallstent, Boston Scientific, MA, USA) was inserted as previously described [30–33]. In these patients, after a mean of fourteen days, an invasive procedure was performed to re-evaluate the function and position of the TIPS (previously specified as routine in our centre’s protocol to optimize TIPS function) [30–33]. Patients of the second cohort (n = 25), enrolled between 2013 and 2014, received covered TIPS-stents, but no invasive re-evaluation procedure was performed. Portal and hepatic venous pressures were invasively measured using a pressure transducer system (Combitrans, Braun Melsungen, Germany) and a
Table 1. Clinical parameters of all patients (n = 103) at admission and outcome.
Parameters Gender (female/male) Age (in years)-median (range) Child-score-median (range) Child category (A/B/C) MELD-score-median (range) Etiology (alcoholic/chronic hepatitis/other) Indication for TIPS insertion (variceal bleeding/refractory ascites/both) Ascites (absent/mild/severe) Hepatorenal syndrome (absent/Type 1/Type 2) Esophageal varices (absent/grade I-II/grade III-IV) Variceal bleeding (absent/present) Hepatic encephalopathy (absent/present) Survival time (in days)-median (range) MELD, model for end-stage liver disease. Median (range)
Value 41/62 57 (29-79) 8 (5-12) 22/66/15 8 (6-20) 67/14/22 40/50/13 24/23/56 86/13/4 12/70/21 54/49 93/10 291 (12-3850)
multichannel monitor (Sirecust, Siemens, Germany). The difference between these pressures is defined as the portal hepatic pressure gradient (PHPG). Arterial pressure and heart rate were non-invasively monitored during the procedures. Biochemical parameters, as well as portal and systemic haemodynamics, were recorded at the placement procedure of the TIPS and during the TIPS follow-up procedure (Table 2). Assessment of circulating levels of chemokines During the TIPS procedure, blood from the portal and hepatic veins were collected from all patients included in the study (n = 103) as soon as the right branch of the portal vein was cannulated to get blood specimen for the determination of CXCL9 levels. For the first cohort comprising 78 patients, a second invasive procedure was performed to re-evaluate the TIPS at the follow-up appointment and a catheter was placed into the portal vein, for pressure measurement and blood collection. In the second cohort of patients receiving covered TIPS (n = 25), additional blood samples from the portal vein, hepatic vein, the right atrium and the cubital vein were collected during the TIPS procedure. After collection, blood samples were centrifuged at 3000 rotations/minute for 15 min at 4°C. Serum samples for chemokine analyses were stored at 80°C until they were used. Serum concentrations of CXCL9 (monokine induced by interferon-c) were assessed with a cytometric bead array (Becton Dickinson, Heidelberg, Germany) according to the manufacturer’s instructions. The chemokine levels were quantified in undiluted serum samples in duplicates. Statistical analysis Data are presented as mean ± standard error (SEM) or medians and ranges. The Wilcoxon test was used for comparison of paired data and the Mann-Whitney and Kruskal-Wallis test for unpaired comparisons. Correlations were analysed with the Spearman correlation coefficient. Kaplan-Meier curves were used to analyse the survival rates of patients using the log-rank test. Cox regression multivariate analysis (forward stepwise likelihood-quotient) was performed to predict survival rates using the log-rank test. p values <0.05 were considered statistically significant. Statistical analyses were performed using SPSS 18.0 for Windows (SPSS Inc. Chicago, IL, USA) and GraphPad Prism version 5.00 (GraphPad Software, La Jolla, USA).
Table 2. Biochemical parameters before TIPS insertion and at the early TIPS check after a median of fourteen days (n = 78).
Biochemical parameters (units) Bilirubin (mg/dl) CHE (U/L) Albumin (g/L) ALT (U/L) AST (U/L) γGT (U/L) INR Serum creatinine (mg/dl) BUN (mg/dl) Serum sodium (mmol/L) Platelet count (G/L) PHPG (mmHg) Portal pressure (mmHg) Central venous pressure (mmHg) Portal venous blood flow velocity (cm/s)
Before TIPS
14 days after TIPS
1.1 (0.4-4.7) 1984 (479-4070) 32 (11-56) 18 (7-94) 18 (8-73) 59 (8-527) 1.17 (0.93-2.23) 1.1 (0.5-8.2) 43 (9-225) 136 (119-145) 100 (27-389) 20 (11-35) 32 (18-49) 11 (0-25)
1.3 (0.6-6.8)b 2000 (411-3795) 29 (16-34) 24 (9-357)b 22.5 (13-275)a 112 (12-515)b 1.2 (0.96-1.98) 0.9 (0.5-7.1)a 28 (1-118)b 138 (118-144)a 136 (65-326)b 9 (1-16)b 21 (12-33)b 12 (5-22)
16 (5-40)
38 (17-72)b
Data are shown as median and range and compared by non-parametric testing (Wilcoxon test). ap <0.05 and bp <0.005 vs. value before TIPS. CHE, choline esterase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cGT, c-glutamyltransferase; INR, international normalized ratio; BUN, blood urea nitrogen; PHPG, portal hepatic pressure gradient.
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Research Article Results Characteristics of patients before and after TIPS The characteristics of patients included in the study are depicted in Table 1. Briefly, patients were predominantly male (60%) with a median age of 57 years. The median Child-Pugh score was eight points and the majority of the patients were in Child-Pugh class B. The median MELD score was eight points. 65% of patients had alcoholic cirrhosis. The indications for TIPS insertion were secondary prophylaxis for recurrent bleeding in 40 patients, refractory ascites in 50 patients, or both indications in 13 patients. Hepatic encephalopathy episodes prior to placement of TIPS occurred in only 10 patients. In the first cohort of 78 patients the median survival time was 595 days, within a follow-up period of more than 10 years. The maximal follow-up of the second cohort was 408 days. The differences between the two cohorts are shown in the Supplementary Table 1. Briefly, patients receiving covered stents showed lower Child-Pugh and MELD scores compared to patients receiving bare stents. At the invasive TIPS follow-up, 14 days after the placement procedure, liver synthesis capacity remained unchanged as shown by the preserved choline esterase and INR levels (Table 2). After TIPS insertion levels of cGT, ALT, AST and bilirubin as markers of liver injury slightly increased, but renal function improved, as shown by the levels of creatinine and sodium as well as by the platelet counts (Table 2). Prior to TIPS insertion, patients showed significant portal hypertension with a median portal hepatic pressure gradient of 20 mmHg and a median absolute portal pressure of 32 mmHg, as well as a decreased portal venous blood velocity, measured by Doppler ultrasound (Table 2). 14 days after portal decompression by TIPS insertion, the portal hepatic pressure gradient remained significantly decreased, and portal venous blood velocity, a read out of preserved TIPS function, had more than doubled (Table 2). Serum levels of CXCL9 in portal and hepatic venous blood before and after TIPS placement Assessment of CXCL9 in the portal and hepatic venous blood before TIPS revealed surprising results (Table 3). CXCL9 levels in the portal vein tended to be higher than in the hepatic vein
without reaching significance (p = 0.192). After TIPS insertion, portal venous levels of CXCL9 significantly decreased (p = 0.031) compared to the corresponding levels before TIPS, while hepatic venous levels showed a tendency towards decrease without reaching statistical significance. When comparing the levels of all patients together, patients receiving a covered stent showed lower portal and hepatic CXCL9 serum levels than those receiving a bare stent. However, when comparing CXCL9 levels between patients of these two cohorts, stratified by Child-Pugh classes, no significant differences were observed (Supplementary Table 2). Importantly, we also assessed CXCL9 levels in blood samples collected from the right atrium and cubital vein from all patients of the second cohort in addition to hepatic levels. We did not observe any significant difference between hepatic and right atrial or cubital venous levels, while the levels of all sites showed excellent correlations with each other (Supplementary Fig. 1). Serum levels of CXCL9 in the portal vein and hemodynamic parameters The portal systemic pressure gradient showed a significant correlation with CXCL9 levels in portal venous blood after TIPS (r = 0.514; p = 0.01, data not shown), while no other association to hemodynamic parameters could be assessed.
Serum levels of CXCL9 in the portal and hepatic vein and hepatic function Serum levels of CXCL9 correlated with the Child-Pugh class. This association was highly significant for levels of CXCL9 in the hepatic vein (Fig. 1A). Interestingly, the difference of CXCL9 levels between different Child-Pugh classes was abrogated after TIPS insertions (Fig. 1A). While patients with Child-Pugh class A and B cirrhosis presented with chemokine levels in a similar range as before TIPS insertions, patients with Child-Pugh class C cirrhosis displayed a dramatic reduction of CXCL9 levels in portal as well as in hepatic venous blood (Fig. 1A). When stratified for aetiology, patients with liver cirrhosis due to excessive alcohol consumption presented with higher levels of CXCL9 in the portal vein as compared to those patients with non-alcoholic cirrhosis (Fig. 1B).
Table 3. CXCL9 levels in portal and hepatic venous blood before and after TIPS insertion as well as correlation between CXCL9 levels and parameters of patients with significant portal hypertension receiving TIPS (n = 103).
CXCL9 median (range) MELD Creatinine BUN Sodium excretion in urine before TIPS
Portal venous levels before TIPS 317 pg/ml (9.9-66,995) r = 0.1 p = 0.3 r = 0.620 p = 0.000 r = 0.590 p = 0.000 r = -0.266 p = 0.116
Hepatic venous levels before TIPS 320 pg/ml (7.85-51,702) r = 0.3 p = 0.016 r = 0.570 p = 0.000 r = 0.500 p = 0.000 r = -0.381 p = 0.017
Portal venous levels after TIPS 165 pg/mla (20-44,208) r = 0.286 p = 0.066 r = 0.446 p = 0.002 r = 0.363 p = 0.081 r = -0.374 p = 0.015
Hepatic venous levels after TIPS 218 pg/ml (34-23,500) r = 0.174 p = 0.274 r = 0.484 p = 0.002 r = 0.532 p = 0.006 r = -0.403 p = 0.015
Data are shown as median and range and compared by non-parametric testing (Wilcoxon test). ap <0.05 vs. portal venous levels before TIPS.
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JOURNAL OF HEPATOLOGY Table 4. Parameters correlating with survival time. Parameters correlating with survival time in univariate Cox regression analysis (A) and CXCL-9 levels correlating with survival time in multivariate Cox regression analysis (B).
A
Parameters Levels of CXCL9 in portal venous blood before TIPS Levels of CXCL9 in hepatic venous blood before TIPS MELD Bleeding before TIPS Hepatorenal syndrome Sodium before TIPS Sodium after TIPS Serum creatinine before TIPS Serum creatinine after TIPS BUN after TIPS Bilirubin after TIPS Portal hepatic pressure gradient after TIPS Portal pressure after TIPS
B
p value
Serum levels of CXCL9 0.0001 below 400 pg/ml in portal blood before TIPS insertion
p value 0.0001 0.0001 0.0001 0.026 0.035 0.023 0.057 0.0001 0.0001 0.0001 0.0001 0.002 0.0001
Hazard Confidence ratio interval of hazard ratio 0.264 0.133-0.524
Interestingly, CXCL9 levels before TIPS insertion differed with regard to the indication for TIPS. Patients receiving TIPS for refractory ascites displayed higher levels of CXCL9 in portal and hepatic venous blood than those who received TIPS for recurrent variceal bleeding (Fig. 1C). Of note, this effect was independent of the magnitude of portal pressure. Importantly, the TIPS procedure significantly decreased levels of CXCL9 in the portal vein in both groups (Fig. 1D and E), although median levels remained significantly higher in patients who underwent TIPS procedure for refractory ascites. Moreover, CXCL9 levels positively correlated with the severity of ascites especially before TIPS procedure (Fig. 1F). Serum levels of CXCL9 in portal and hepatic vein and renal function Serum levels of CXCL9 in portal and hepatic vein were positively correlated with the MELD score (Table 3). The biochemical parameters creatinine, blood urea nitrogen (BUN) as well as sodium urinary excretion as marker of renal function were correlated with CXCL9 levels in portal and hepatic vein (Table 3). Additionally, an inverse correlation was observed with serum albumin (data not shown). Serum levels of CXCL9 and survival of patients receiving TIPS The parameters with impact on survival time were identified by univariate Cox regression analysis and are depicted in Table 4A. Higher serum levels of CXCL9 before TIPS in hepatic and portal venous blood were significantly associated with poor survival in the univariate analysis, as was the MELD score, episodes of bleeding prior to the procedure, and presence of a hepatorenal syndrome. Similarly, this analysis revealed an association of serum sodium and creatinine levels before and after TIPS, as well
as BUN and bilirubin after TIPS with patient survival. As expected portal systemic pressure gradient and portal pressure after TIPS placement were associated with survival as well. In the multivariate Cox regression analysis, in which we included all parameters previously identified to be associated with survival in the univariate analysis (Table 4A), the CXCL9 level in portal blood before TIPS insertion was the only parameter independently predictive for survival in our cohort (Table 4B). Kaplan-Meyer analysis for overall survival were performed using the 50th percentile of CXCL9 levels in the portal vein and hepatic vein before TIPS as a cut-off (Fig. 2A and B). For portal venous blood, the 50th percentile was presented by 400 pg/ml in our cohort. Patients with CXCL9 levels higher than 400 pg/ml in the portal vein showed a median survival time of 113 days vs. 941 days for patients with CXCL9 levels lower than 400 pg/ ml. The calculation of the hazard ratio for overall survival for portal CXCL9 levels below 400 pg/ml revealed a highly reduced ratio of 0.2623 (95% confidence interval 0.133–0.524, p = 0.0001; Table 4B). Of note, we were able to validate the cut-off of 400 pg/ml CXCL in the portal vein during TIPS procedure as a highly significant predictive marker for survival in our second, independent cohort, revealing a statistically significant reduced ratio of 0.04 (p = 0.024), albeit, the short follow-up with a low number of events might limit the statement for this second cohort. Additionally, stratification of patients from the second cohort, using the cut-off of 400 pg/ml CXCL9 in the cubital vein, also revealed a significant survival effect (Supplementary Fig. 2). Finally, ROC analysis were performed to assess the predictive value of portal CXCL9 levels for 6-month and 2 year overall survival (Fig. 2C and D). For both time points, CXCL9 levels in the portal vein revealed a highly predictive value for survival with an AUC >0.77, especially within the first 6 months (Fig. 2C). These results were superior to the predictive values of the MELD score (Fig. 2E and F) and the Child-Pugh score (AUC of 0.6552, p = 0.055 for 6 months after TIPS, AUC of 0.5877, p = 0.2344 for 2 years after TIPS, data not shown) for both periods.
Discussion This study showed for the first time that portal venous levels of the CXCR3 ligand CXCL9 predict survival in patients with liver cirrhosis and severe portal hypertension receiving TIPS. In these patients high circulating levels of CXCL9 in the portal and hepatic vein were associated with hepatic and renal dysfunction. CXCL9 levels showed no direct correlation with hepatic portal pressure, implicating that CXCL9 is not simply a surrogate marker for portal hypertension. It rather is correlated to complications with portal hypertension, such as presence and severity of ascites, and consequently the TIPS procedure resulted in a reduction of CXCL9 levels. CXCR3 ligands have been extensively investigated in animal studies [6,10,13,15,23,34], as well as in humans with liver disease [5,9,11,12,16,18,26,27,35]. Different studies have assessed a relationship between CXCR3 ligands and fibrosis and hepatic injury [8–10,12–18], angiogenesis [6], and complications of liver cirrhosis [19]. The majority of the studies focused on hepatitis C and alcoholic cirrhosis. Some studies analysed primary biliary cirrhosis and autoimmune hepatitis [5,11,35]. These studies revealed that intrahepatic levels of these chemokines are increased during liver injury and liver fibrogenesis [5,6–12]. In vitro studies have
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Fig. 1. Serum levels of CXCL9 correlate with the Child-Pugh score, indication for TIPS insertion, and ascites in cirrhotic patients with severe hypertension. Serum levels of CXCL9 are significantly increased in patients with higher Child-Pugh class before TIPS insertion in portal and hepatic venous blood (n = 78) (A). Serum levels of CXCL9 in patients with alcoholic cirrhosis are elevated compared to patients with non-alcoholic cirrhosis (B). Serum levels of CXCL9 in patients receiving TIPS for recurrent bleeding are significantly lower compared to those patients receiving TIPS for refractory ascites or both indications (C). After TIPS, levels of CXCL9 in the portal vein decrease in patients receiving TIPS for variceal bleeding (D) and refractory ascites (E). Serum levels of CXCL9 in patients with severe ascites are elevated compared to patients with less severe or no ascites (F). Data in (A, B, C, and F) are shown as means +/ standard error of the mean, in (D and E) paired data are shown. Wilcoxon and Kruskal-Wallis tests were used when appropriate.
shown that liver resident cells (e.g. hepatocytes, Kupffer cells, hepatic stellate cells) express these chemokines as a response to cellular injury and stress [20–25], suggesting a hepatic release of these chemokines. However, it was also implicated that circulating immune cells might contribute to elevated chemokine levels [23,29]. Our data do not support a distinct place of production or clearance of CXCL9. They rather suggest that levels of this CXCR3 ligand might be a result of intra- and extrahepatic production and clearance. The present study confirms previous findings, showing an association between the complications of end-stage liver disease and the levels of CXCR3 ligands [19]. In addition, we could demonstrate that CXCL9 levels in the portal vein are higher in patients receiving TIPS for refractory ascites, compared to those receiving TIPS for recurrent bleeding. This might be a result of continuous bacterial translocation and subsequent systemic inflammation in patients with refractory ascites. Importantly, we observed higher levels of CXCL9, especially in the portal vein, in patients with alcoholic cirrhosis, which might be explained by the more pronounced gut barrier dysfunction in alcoholic patients. More interestingly, these associations could provide functional insight on why the portal levels of CXCL9 had the strongest predictive value for survival in patients receiving TIPS. 336
In patients with portal hypertension, TIPS insertion increases the effective blood volume and improves renal perfusion [36]. In our patients, we observed a strong association of CXCL9 levels with parameters of renal function. In TIPS-treated patients, ascites and renal function were improved, which is associated with a drop in CXCL9 levels in the portal vein that could partially be explained by better renal clearance of CXCL9. This might also explain why levels in the portal vein and hepatic vein were not significantly different to those in the cubital vein and right atrium and showed excellent correlation to each other. Importantly, this excellent correlation might also implicate that levels of CXCL9 in cubital venous blood accurately reflect hepatic levels of CXCL9 and might facilitate the applicability of CXCL9 measurement for risk stratification of patients with portal hypertension undergoing TIPS procedure in clinical practise. However, it remains to be demonstrated in extended cohorts that the predictive value of cubital venous CXCL9 levels are comparable to those obtained from portal venous blood. Patients with severe portal hypertension and portal venous CXCL9 levels, elevated over 400 pg/ml, showed a higher mortality after TIPS insertion. Indeed, CXCL9 was an independent predictor of mortality in two independent cohorts, in patients receiving covered as well as bare TIPS. Nevertheless, one could speculate that its predictive value might at least partly be due to its
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Fig. 2. CXCL9 levels are associated with the survival of cirrhotic patients with severe hypertension after TIPS insertion. (A) Kaplan-Meier survival analysis, demonstrating reduced survival of patients with portal venous CXCL9 levels >400 pg/ml (50th percentile of entire cohort) at the time of TIPS insertion during the follow-up period (n = 104). (B) Kaplan-Meier survival analysis showing survival of patients with hepatic venous CXCL9 levels >400 pg/ml (50th percentile of entire cohort) at the time of TIPS insertion during the follow-up period (n = 104). Data were tested by log-rank analyses. (C and D) ROC analyses demonstrate the predictive value of portal CXCL9 levels before TIPS insertion on 6-month (C) and 2-year survival (D) in a cohort of cirrhotic patients with severe hypertension. (E and F) ROC analyses for the predictive value of the MELD score before TIPS insertion on 6-month (E) and 2-year survival (F), respectively.
association with renal function, as renal function had been recently identified as one of the major short- and mid-term predictive factors of patients with chronic liver failure [37]. The association of CXCL9 levels with the Child-Pugh score, MELD-score, and severity of ascites was more pronounced in the hepatic vein. This finding suggests that CXCL9 might be a mediator of extrahepatic complications due to liver insufficiency. The relationship between the hepatic release of this chemokine and its exact mechanism, how it takes influence on other organs for example
by inducing an inflammatory answer in tissues, might be further evaluated. Clinical and biochemical parameters of hepatic and renal dysfunction, observed in our cohort, reflected severity of the liver disease and its extrahepatic complications in these patients. In addition, patients with high levels of the CXCR3 ligand CXCL9 showed poor overall survival. Interestingly, this was independent of other clinical parameters of end-stage liver disease. In our study CXCL9 levels in portal, hepatic and cubital vein bared valuable predictive information on the overall
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Research Article mortality risk in cirrhotic patients with severe portal hypertension receiving TIPS. Our study, thus, not only delivers insight into the association of chemokines with portal hypertension, but also offers proof that specific chemokines can predict mid- and longterm survival in patients receiving TIPS. Before TIPS-placement, portal levels of CXCL9 revealed a predictive value for 6-month and 2-year survival in our cohort of patients with severe portal hypertension receiving TIPS, which was superior to the established MELD and Child-Pugh scoring system. This information might be especially useful for the risk assessment in patients receiving TIPS as bridging strategy before liver transplantation. Further investigations are certainly needed, but this first proof of concept finding is essential to demonstrate the relevance of this chemokine system for the progression of liver disease. This study has several limitations. Portal venous levels of control and cirrhotic patients without TIPS would be desirable, however this is unethical in the usual clinical setting. Furthermore, a higher number of patients would be needed to better characterize potential aetiology-dependent differences between the subgroups receiving TIPS. However, our group of patients showed characteristics of severe portal hypertension. Survival in these patients was associated with already important and well investigated clinical and biochemical parameters and scores. This confirms that the data are consistent with findings, published in the literature. In summary, our study shows for the first time that the CXCR3 ligand CXCL9 is correlated with hepatic, renal and hemodynamic parameters in patients with portal hypertension. CXCR3 ligands should be further mechanistically evaluated in patients with liver cirrhosis and severe portal hypertension. Attempts to modulate these chemokines might help to alleviate symptoms and improve treatment of patients with liver disease, high morbidity, and mortality. Financial support The study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB TRR57 P04, P06, P07, P09, and P18 to MLB, FT, HEW, CT, TS, and JT) and from the H.J. & W. Hector Stiftung (M60.2 to JT).
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Authors’ contributions JT, MLB, HEW: study concept & design, acquisition of data analysis and interpretation of data, drafting of the manuscript, statistical analysis; SA, JL, CJ, HMS, JG, SK: technical support, acquisition of data, critical revision of the manuscript; RF, FT, CPS, CT, TS: technical support, critical revision of the manuscript.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2014.09. 032. 338
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