The effects of sorafenib on the portal hypertensive syndrome in patients with liver cirrhosis and hepatocellular carcinoma – a pilot study

Authors


Prof M. Peck-Radosavljevic, Division of Gastroenterology/Hepatology, Department of Internal Medicine III, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
E-mail: markus.peck@meduniwien.ac.at

Abstract

Aliment Pharmacol Ther 2012; 35: 83–91

Summary

Background  Increased intrahepatic vascular resistance and hyperperfusion in the splanchnic circulation are the principal mechanisms leading to portal hypertension in cirrhosis. Several preclinical studies have demonstrated a beneficial effect of the multikinase inhibitor sorafenib on the portal hypertensive syndrome.

Aim  To investigate the effect of sorafenib on hepatic venous pressure gradient (HVPG), systemic hemodynamics and intrahepatic mRNA expression of proangiogenic, profibrogenic and proinflammatory genes.

Methods  Patients with liver fibrosis/cirrhosis and hepatocellular carcinoma were treated with sorafenib 400 mg b.d. HVPG measurement and transjugular liver biopsy were performed at baseline and at week 2. Changes in HVPG and intrahepatic mRNA expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), RhoA, tumour necrosis factor-alpha (TNF-α) and placental growth factor (PlGF) were evaluated.

Results  Thirteen patients (m/f = 12/1; Child–Pugh class A/B = 10/3) were included. The most common aetiology of liver disease was alcohol consumption (n = 7). Eleven patients had an elevated portal pressure, including eight patients with clinically significant portal hypertension. A significant decrease of HVPG (≥ 20% from baseline) was observed in four subjects. In HVPG responders, we observed mRNA downregulation of VEGF, PDGF, PlGF, RhoA kinase and TNF-α, while no substantial mRNA decrease was found in nonresponders in any of the five genes. In two of the four HVPG responders we observed a dramatic (43–85%) mRNA decrease of all five investigated genes.

Conclusion  Larger controlled clinical trials are needed to demonstrate any potential beneficial effect of sorafenib on portal hypertension in patients with cirrhosis.

Introduction

Portal hypertension (PHT) is a major complication and the leading cause of mortality in patients with liver cirrhosis.1 Characteristic features of PHT are the development of ascites and the formation of portosystemic collateral vessels including gastric and oesophageal varices, which are prone to rupture and may cause life-threatening bleeding.2

Increased intrahepatic vascular resistance and hyperperfusion in the splanchnic circulation are the principal mechanisms leading to PHT.3 Structural changes during liver fibrogenesis and hyperresponsiveness to and an imbalance of mediators with a shift towards vasoconstrictors lead to increased intrahepatic resistance.4, 5 The hyperdynamic splanchnic circulation is mediated by splanchnic vasodilatation and angiogenesis.6, 7

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a hallmark of PHT as it is crucially involved in the development of increased splanchnic blood flow and the formation of portosystemic collaterals.8–10

Several growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) orchestrate the multiple steps of angiogenesis.11

The oral multikinase inhibitor sorafenib inhibits cell proliferation and angiogensis by targeting several tyrosine kinases such as Raf kinase, VEGF receptor 2 and 3, as well as PDGF receptor β.12 Sorafenib has already been approved as anticancer drug for the treatment of renal cell carcinoma and hepatocellular carcinoma (HCC).13–15 The latter usually develops in patients with liver cirrhosis and PHT but to our knowledge, no clinical data about the effect of sorafenib on liver fibrogenesis and portal pressure in humans have been published until now. Several preclinical animal studies demonstrated that sorafenib may have beneficial effects on both, developing as well as established PHT.16–18

This pilot study investigated the short term effects of sorafenib on (i) hepatic venous pressure gradient (HVPG) and systemic hemodynamics as well as on (ii) intrahepatic mRNA expression of genes involved in liver fibrogenesis, angiogenesis, and inflammation in patients with liver fibrosis/cirrhosis and hepatocellular carcinoma.

Methods

Study design

This study is a sub-study of the SORATACE-1 trial, an open label single arm pilot trial to evaluate the safety of conventional transarterial chemoembolization (cTACE) in combination with continuous sorafenib administration in patients with HCC (ClinicalTrials.gov, registry number: NCT00768937).

Sorafenib was started after baseline measurement of HVPG at a dose of 400 mg orally twice daily. HVPG measurement was repeated 2 weeks thereafter prior to the first scheduled TACE.

The primary endpoint of this sub-study was to evaluate the short-term effect of continuous sorafenib treatment on HVPG. The secondary endpoint was to assess the effect of sorafenib on mRNA expression of genes involved in liver fibrogenesis, inflammation and angiogenesis.

Patient selection

Patients aged ≥ 18 years with cirrhosis of any aetiology and HCC confirmed by histology or European Association for the Study of the Liver (EASL) criteria19 and not suitable for curative treatment like orthotopic liver transplant, resection or local ablation were eligible for inclusion. Additional inclusion criteria were well-to-moderately preserved liver function (Child–Pugh stage A or B),20 Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0–2, Absolute Neutrophil Count > 1 × 109/L, platelet count > 40 × 109/L, prothrombin time ≥ 40%, total bilirubin ≤ 3 mg/dL, serum creatinine < 2.0 mg/dL and a life expectancy of > 3 months.

Patients had to give written informed consent prior to any study-specific procedures.

Patients were excluded if they had complete portal vein thrombosis (PVT) or Child–Pugh stage C. Additional exclusion criteria were prior TACE or TAE, history of acute variceal bleeding within the last 2 weeks, large oesophageal varices without prophylactic band ligation, past or current history of malignancies except for HCC.

This study was approved by the local ethics commitee of the Medical University of Vienna and performed in accordance with good clinical practice and Declaration of Helsinki guidelines. All patients provided written informed consent before enrolment.

Hepatic venous pressure gradient (HVPG) measurement

Hepatic venous pressure gradient measurements were conducted at baseline before the first sorafenib dose and repeated after 2 weeks of continuous sorafenib administration. HVPG measurements were routinely performed with a balloon catheter according to standard methodology as described elsewhere.21–23 According to the hemodynamic response to sorafenib treatment, patients were classified as: (i) responders, when HVPG decreased ≥ 20% from the baseline and (ii) nonresponders, when HVPG increased or decreased < 20% from the baseline.24

The Patients who already received β-blocker therapy (propanolol, carvedilol) for primary or secondary prophyaxis of variceal bleeding before study entry (5/13 patients) continued the therapy during the trial. Beta-blocker-naive patients with elevated HVPG +/− varices at baseline measurement were not put on β-blocker therapy before the second HVPG measurement at week 2. Patients with large oesophageal varices (> 5 mm diameter)25 underwent prophylactic band ligation before sorafenib initiation.

Quantitive real-time RT-PCR

Liver specimens of the nontumoural liver tissue were obtained at baseline and after 2 weeks of sorafenib therapy by transjugular liver biopsy, which was taken right after HVPG measurements.

RNA was isolated according to a standard Trizol-extraction protocol (Invitrogen, Vienna, Austria). On-column RNase digestion was performed using RNase-Free DNase set according to a standard protocol (Qiagen, Hilden, Germany). cDNA was synthesised using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and assessed for gene expression by real-time RT-PCR Taqman System using the following primers: hs00173626_m1 for VEGF, hs00966522_m1 for PDGF, hs00357608_m1 for RhoA, hs00174128_m1 for TNF-α, hs01119261_m1 for PlGF, and 4352935 for β-Actin (Applied Biosystems, Carlsbad, CA, USA).

Statistical analysis

Baseline characteristics were summarised using descriptive statistics. A Chi-squared test was used to compare nominal data. The Wilcoxon matched pairs test was used to compare paired metric data. Overall survival (OS) was defined as the time from start of sorafenib until the date of death or last follow-up. Survival curves were calculated using Kaplan–Meier methods and were compared by means of the log rank test. A P-value < 0.05 was considered significant.

All statistical analyses were performed using spss version 17.0 (SPSS Inc., Chicago, IL, USA).

Results

Patients

Of the 17 patients screened between October 2008 and June 2010, 15 patients met the inclusion criteria and were included into the study. HVPG measurements at both timepoints (baseline and after 2 weeks of sorafenib treatment) were available from 13 patients and only these patients were included into the final analysis (Figure 1).

Figure 1.

 Flow diagram. HVPG, hepatic venous pressure gradient.

The main baseline patient characteristics are shown in Table 1. Most common aetiology of liver disease was alcohol consumption. Child–Pugh class A/B was 10/3. Oesophageal varices were present in eight patients and three patients had a history of variceal bleeding prior to study entry. Five patients received concomitant β-blocker prophylaxis; compliance was checked by measuring heart rate before HVPG measurement. Six subjects had a history of endoscopic variceal band ligation prior to sorafenib initiation.

Table 1.   Patient characteristics at baseline
 N = 13
  1. HCV, hepatitis C virus; NASH, non-alcoholic steatosis hepatis; MELD, model for end-stage liver disease; BCLC, Barcelona clinic liver cancer.

Age (years)
 Mean ± s.d.66 ± 10.4
 Range46–79
Sex
 Male12
 Female1
Aetiology
 Alcohol7
 HCV3
 NASH3
Bilirubin (mg/dL)
 Mean ± s.d.1.44 ± 0.49
 Range0.68–2.34
Albumin (g/L)
 Mean ± s.d.36.5 ± 4.1
 Range28.5–41.2
Child–Pugh stage
 A10
 B3
Child–Pugh score
 Mean ± s.d.6 ± 1.3
 Range5–9
MELD score
 Mean ± s.d.10 ± 2.9
 Range6–14
Ascites
 Absent8
 Present5
Varices
 Absent5
 Present8
Prior variceal bleeding
 Absent10
 Present3
β-Blocker prophylaxis
 No8
 Yes5
Endoscopic band ligation
 Absent7
 Present6
BCLC stage
 B8
 C5

Effect of sorafenib treatment on HVPG

After baseline HVPG evaluation patients received Sorafenib 400 mg twice daily. Patients underwent second HVPG measurement after a median time of 18 days (95% CI, 16–20 days). No dose reduction was performed before the second HVPG measurement. Baseline HVPG measurement revealed that two patients had no PHT (HVPG ≤ 5 mmHg), three patients had PHT (HVPG 6–9 mmHg), and eight subjects had clinically significant PHT (HVPG ≥ 10 mmHg). Four of 11 patients (36%) with PHT at baseline showed a significant decrease of HVPG (≥ 20%) after 2 weeks of sorafenib treatment and were therefore classified as responders (Table 2; Figure 2). Two responders had Child–Pugh A and two had Child–Pugh B cirrhosis. Three subjects showed an increase (6%, 12% and 50% respectively), two patients had a mild decrease (5%, 8% respectively) while two patients experienced no change in HVPG after 2 weeks of sorafenib administration. Of eight subjects with clinically significant PHT three patients (38%) significantly decreased with HVPG at week 2; two of them had concomitant β-blocker prophylaxis at study entry which was continued during the study (Table 2).

Table 2.   HVPG at baseline and after 2 weeks of sorafenib treatment
Patient numberConcomitant β-blockerHVPG (mmHg)Change* (%)Response†
BaselineWeek 2
  1. HVPG, hepatic venous pressure gradient; PHT, portal hypertension; N/A, not applicable.

  2. * Change of HVPG after 2 weeks of sorafenib therapy compared to baseline

  3. † Responder (R), HVPG decrease ≥ 20%; nonresponder (NR), HVPG decrease < 20%

No PHT (HVPG ≤ 5 mmHg)
 1None34+33N/A
 2None45+25N/A
PHT (HVPG 6–9 mmHg)
 3None880NR
 4None84−50R
 5None990NR
Clinically significant PHT (HVPG ≥ 10 mmHg)
 6Carvedilol1211−8NR
 7Propranolol1512−20R
 8None1617+6NR
 9Propranolol1624+50NR
 10None1719+12NR
 11Propranolol2019−5NR
 12None2520−20R
 13Carvedilol2520−20R
Figure 2.

 Individual patient HVPG response to sorafenib (n = 13). HVPG, hepatic venous pressure gradient.

Aetiology of liver disease was associated with response to sorafenib treatment in patients with PHT: of seven patients with alcohol as underlying aetiology four subjects (57%) were responders while no responder was observed in patients with NASH- or HCV-related cirrhosis (n = 4; P = 0.058). All patients with alcohol as underlying aetiology have stopped active drinking at least 3 months before inclusion into the study.

On systemic hemodynamics, sorafenib caused a significant increase in systolic blood pressure (BP; 11 ± 12%; P = 0.012), diastolic BP (6 ± 9%; P = 0.034), and mean BP (7 ± 7%; P = 0.011), but had no substantial effect on heart rate (6 ± 17%; P = 0.168).

Interestingly, median overall survival of HVPG responders (n = 4) was 20.5 months compared with 10.6 months (HR, 0.18; 95% CI, 0.02–1.50; P = 0.079) in all other patients.(n = 9; Figure 3).

Figure 3.

 Kaplan–Meier survival curves for HVPG responders (n = 4) vs. HVPG. nonresponders and patients without portal hypertension (n = 9). HVPG, hepatic venous pressure gradient.

Effect of sorafenib on intrahepatic mRNA expression of genes involved in liver fibrogenesis, angiogenesis and inflammation

We determined the intrahepatic mRNA expression of VEGF, PDGF, PlGF, RhoA, and TNF-α in patients of which adequate liver specimens were available at baseline and at week 2 after sorafenib therapy (n = 8). Six patients had PHT of which four were HVPG responders and two patients were nonresponders; two patients had no PHT.

In HVPG responders, sorafenib treatment effectively decreased mRNA expression of VEGF, PDGF, PlGF, RhoA kinase, and TNF-α (Figure 4a,b). In nonresponders (n = 2) and patients without PHT (n = 2) mRNA levels increased (i.e. VEGF, PDGF and RhoA) or remained stable (i.e. TNF- α, PlGF) after 2 weeks of sorafenib treatment (Figure 4a,c,d). Of note, we observed a dramatic (43–85%) mRNA downregulation of all five investigated genes in two of four HVPG responders.

Figure 4.

 Changes in intrahepatic mRNA expression levels after 2 weeks of sorafenib treatment compared with baseline. (a) HVPG responders (n = 4) vs. HVPG nonresponders/patients without portal hypertension (n = 4), (b) one HVPG responder, (c) one HVPG nonresponder and (d) one patient without portal hypertension. PHT, portal hypertension; HVPG, hepatic venous pressure gradient.

Discussion

The oral multikinase inhibitor, sorafenib, is the new reference standard for the treatment of advanced hepatocellular carcinoma (HCC),14 which develops in cirrhosis in 80–90%26 of cases and is then associated with PHT in the majority of cases.

Several animal studies have already demonstrated a beneficial effect of sorafenib on the PHT syndrome. Sorafenib reduced portal pressure, splanchnic neovascularization, hyperdynamic splanchnic circulation and portosystemic collateral formation and led to a remarkable improvement in intrahepatic fibrosis, angiogenesis and Rho kinase-mediated vascular resistance.16–18

Recently, a French group reported a reversible decrease (54%) of mean portal venous flow by sorafenib as assessed by MRI velocity mapping in a small cohort of seven cirrhotic patients with HCC.27

To our knowledge, no clinical data investigating the effect of sorafenib treatment on HVPG and intrahepatic molecular changes have been published so far.

We investigated the effect of a 2-week sorafenib treatment on HVPG as well as on mRNA expression of selected genes involved in liver fibrogenesis, angiogenesis, and inflammation in patients with liver fibrosis/cirrhosis and HCC in a small pilot study.

We observed a HVPG response rate to sorafenib in 36% of patients with PHT (HVPG ≥ 6 mmHg). Similar response rates (14–38%) have been reported for propanolol,28–31 while higher HVPG response rates were found with carvedilol (54–64%).28, 29 Interestingly, two of four sorafenib responders received concomitant β-blocker prophylaxis suggesting a potential additive effect of sorafenib on the HVPG response rate of β-blockers. However, whether sorafenib indeed could have an additive effect with β-blockers on HVPG has to be subject to further research.

Noteworthy, we observed a trend to better overall survival in responders compared with all other patients (20.5 months vs. 10.6 months; Figure 3). This finding warrants further evaluation of HVPG measurement as biomarker for sorafenib response in patients with hepatocellular carcinoma and PHT.

Several preclinical studies identified key genes for the developement and maintenance of PHT that are regulated by sorafenib.16–18 It is assumed that the effect of sorafenib on hepatic stellate cells (HSC) may contribute to the beneficial effect of sorafenib on HVPG.16 After their activation into fibrogenic myofibroblast-like cells HSC play a key role in the development of liver fibrosis32 and contribute to increased intrahepatic vascular resistance and PHT by expressing high levels of Rho kinase.33–35 PDGF and its receptor PDGF-Rβ are crucial for the viability and activation of HSC.36, 37 Recently, inhibition of PDGF-Rβ by sorafenib reduced number and activity of HSC and significantly improved intrahepatic fibrosis in a rat model.36 Furthermore, the proangiogenic cytokines VEGF and PDGF are overexpressed in partially portal vein ligated (PPVL) rats. Treatment with sorafenib effectively reduced VEGF and PDGF expression levels, which resulted in notable inhibition of splanchnic neovascularization.17, 18 Finally, the developement of intrahepatic inflammation in response to liver damage was suggested to trigger liver fibrogenesis.38 Indeed, Mejias et al.17 observed an increased inflammatory cell infiltration and an elevated expression of proinflammatory cytokines (i.e. TNF-α) in livers of CBDL rats compared with SHAM animals, which could be attenuated by sorafenib therapy.

In this pilot study, we determined the mRNA expression of VEGF, PDGF, PlGF, RhoA and TNF-α in liver specimens obtained at baseline and after a 2-week treatment with sorafenib. We observed a mRNA downregulation of VEGF, PDGF, PlGF, RhoA kinase, and TNF-α predominantly in HVPG responders, while no substantial decrease in any of the five genes was observed in nonresponders (Figure 4a–d). Interestingly, we found an mRNA downregulation of all five investigated genes in two of four HVPG responders.

There are relevant concerns about the use of sorafenib in patients with PHT without HCC. Preclinical39 as well as clinical40, 41 data suggest a sorafenib-induced liver damage, especially in the cirrhotic liver, and side effects of sorafenib may significantly interfere with the patients′ quality of life. It seems to be unlikely that sorafenib at anticancer doses will become a suitable therapeutic option for patients with PHT without HCC. Thus, the effect of low-dose sorafenib treatment in patients with liver cirrhosis and PHT should be investigated in further studies.

Finally, the small sample size and the lack of a control group are the main limitations of this pilot study and obviate definitive conclusions on the effect of sorafenib on PHT in humans. Given the invasive procedures necessary to obtain this information, pilot data are needed to design a larger clinical trial. Exactly these pilot data are provided by our study and could be used to design properly-powered randomised controlled studies.

In conclusion, our pilot study provides some evidence that sorafenib influences HVPG and mRNA expression of genes involved in the pathophysiology of PHT, at least in a proportion of cirrhotic patients with PHT and HCC. Additionally, HVPG response was associated with a trend towards better overall survival. Given that patients with HCC often die from complications of liver cirrhosis and PHT, the beneficial effects of sorafenib on survival may not only be explained by its direct antitumor effect but also by an improvement of PHT.18 Prospective controlled trials are warranted to evaluate the effect of sorafenib treatment on the PHT syndrome in patients with liver cirrhosis.

Acknowledgements

Declaration of personal interests: Markus Peck-Radosavljevic has served as a speaker, a consultant, an advisory board member, and an investigator for and has received research funding from Bayer Schering Pharma. Wolfgang Sieghart has served as a speaker for and has received research funding and travel support from Bayer Schering Pharma. Matthias Pinter and Nataliya Rohr-Udilova have received travel support from Bayer Schering Pharma. The remaining authors disclose no conflicts. We thank Martha Seif and Hubert Hayden for their technical assistance in the molecular biological analyses performed in this study. Declaration of funding interests: This study was partly supported by a grant from Bayer Schering Pharma Austria to Markus Peck-Radosavljevic.

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