The safety of pegylated interferon alpha-2b in the treatment of chronic hepatitis B: predictive factors for dose reduction and treatment discontinuation

Authors


  • 1

    Other members of the HBV 99-01 Study Group are listed in the Appendix.

Dr H. L. A. Janssen, Department of Gastroenterology and Hepatology, Erasmus Medical Center, Dr. Molewaterplein 40, Room Ca 326, 3015 GD Rotterdam, The Netherlands.
E-mail: h.janssen@erasmusmc.nl

Summary

Background : Treatment with interferon-alpha has been shown to be effective in one-third of hepatitis B e antigen-positive chronic hepatitis B patients, but is clinically associated with relevant adverse events.

Aim : To investigate the safety of pegylated interferon alpha-2b in 300 hepatitis B e antigen-positive patients with compensated liver disease.

Methods : Patients were treated with pegylated interferon alpha-2b for 52 weeks combined with either lamivudine 100 mg/day or placebo. Pegylated interferon alpha-2b was administered for 100 μg once a week for 32 weeks; thereafter, the dose was reduced to 50 μg once a week. Adverse events and their effect on study medication were reported at monthly visits in a standardized way.

Results : The most frequently reported side-effects were flu-like syndrome (68%), headache (40%), fatigue (39%), myalgia (29%) and local reaction at the injection site (29%). These symptoms typically occurred within the first month of therapy and subsided during the course of therapy. Neutropenia and thrombocytopenia induced by pegylated interferon alpha-2b increased the risk of infections and bleeding complications, but these complications were rare and mild. The frequency of all side-effects was not different between patients treated with pegylated interferon alpha-2b combined with lamivudine or placebo. In 69 (22%) patients the dose of pegylated interferon alpha-2b was reduced prematurely. Of these dose reductions, 36 (52%) were because of neutropenia. Therapy was discontinued in 28 (8%) patients. The most frequent reasons for early discontinuation were psychiatric side-effects (depression, psychosis) and flu-like symptoms. Multivariate Cox regression analysis showed that low neutrophil count at baseline and cirrhosis were independent predictors of dose reduction or therapy discontinuation.

Conclusion : We conclude that in patients with chronic hepatitis B and compensated liver disease prolonged pegylated interferon alpha-2b therapy is safe, and that pre-existent cirrhosis and neutropenia are the most important predictors of dose reduction or early treatment discontinuation.

Introduction

An estimated 400 million people are chronically infected with hepatitis B virus (HBV). Chronic hepatitis B (CHB) is the single most common cause of liver cirrhosis and hepatocellular carcinoma (HCC) worldwide.1 Interferon-alpha (IFN) is effective in about one-third of the patients.2 Reported HBeAg seroconversion rates range from 15 to 37%,3–5 depending on baseline characteristics such as alanine aminotransferase (ALT) levels and viral load. Response to IFN therapy was shown to result in sustained clearance of hepatitis B e antigen (HBeAg), HBV-DNA and normalization of aminotransferase levels.6–8 Therapy with IFN is associated with considerable side-effects. Most frequently reported side-effects are flu-like syndrome, fatigue, headache and myalgia. Other clinically relevant side-effects, such as depression, anorexia and insomnia, occur less frequently. Cases of suicide attempts during IFN therapy have been reported.9–11 To improve response rates and possibly reduce the number of side-effects, newer forms of IFN have been developed. ‘Pegylated’ forms of IFN (with a polyethylene glycol moiety attached to it) have an improved pharmacokinetic profile with a prolonged half-life time.9 Pegylated interferons have been reported to be safe and more effective than conventional IFN in patients with chronic hepatitis C.12, 13 Until now, the safety data of these pegylated forms of IFN in the treatment of CHB are limited to the study of Cooksley et al.,14 who investigated the safety of peginterferon alpha-2a in CHB patients. In this study, we assessed the safety of peginterferon alpha-2b (PEG-IFN) alone or in combination with lamivudine in HBeAg-positive CHB.

Materials and methods

Patients

In a randomized multicentre trial reported previously,15 307 HBeAg-positive CHB patients with compensated liver disease were treated with PEG-IFN in combination with either lamivudine or placebo. Inclusion criteria were hepatitis B surface antigen (HBsAg)-positivity for at least 6 months, age ≥16 years, ALT at least twice the upper limit of normal (ULN) and HBeAg positive on two occasions within 8 weeks prior to randomization. Patients were excluded if they had been treated with antiviral medication in the previous 6 months or any investigational drug within 30 days of entry in this protocol, were coinfected with hepatitis C, hepatitis D or human immunodeficiency virus (HIV), had alcoholic hepatitis or other causes of liver disease, had pre-existent leucopenia or thrombocytopenia [white blood cell count (WBC) ≤3000/mm3, neutrophils ≤1800/mm3, platelets ≤100 000/mm3], had decompensated liver disease (prothombin time prolonged by ≥3 s, serum albumin <35 g/L, ascites, encephalopathy, history of variceal bleeding) or had hypothroidism or hyperthyroidism. Patients were also excluded in case of pregnancy, inadequate contraception, any significant medical illness potentially interfering with the study or any contraindication specified for IFN. Ethics committees of the participating centres approved of the protocol and all patients provided written informed consent.

Study design

In this double-blinded trial, eligible patients were randomized to one of two treatment regimens (Figure 1). All patients received PEG-IFN for 52 weeks. Patients were treated with a 100 μg dose of PEG-IFN once a week until week 32, whereafter it was reduced to 50 μg once a week. In addition, patients received either placebo or 100 mg lamivudine orally. Patients were examined every 4 weeks during treatment and during the 6-month post-treatment follow-up. At visits, routine physical examination was performed and blood samples were obtained for haematological and biochemical screening [haemoglobin, WBC, neutrophils, platelets, ALT and aspartate aminotransferase (AST)]. All adverse events were reported by the treating physician on standard case-record forms and verified by the trial-coordinating centre. All participating investigators were instructed, received a protocol and were monitored every 3 months in order to ensure uniform scoring of side-effects. Adverse events were graded according to the WHO recommendations for grading of acute and subacute toxicities,16 and reported as mild, moderate, severe or life-threatening. The reported adverse events were also assessed in their relation to therapy by the treating physician and reported as unrelated, possibly related, probably related or related to therapy. Effect on study medication was scored as none, dose reduction or treatment discontinuation. Serious adverse events (SAE) were defined as events resulting in death, events that are life-threatening, require or prolong in-patient hospitalization, as well as events which result in persistent or significant disability or incapacity, pregnancy, any congenital anomaly, cancer, or drug overdose. Hepatitis flares were defined as an increase in serum ALT to at least three times the baseline level. Psychiatric side-effects included mood changes, irritability and depression. Autoimmune phenomena are not reported because they were not systematically investigated with, e.g. longitudinal assessment of autoantibodies. Guidelines on discontinuation of therapy in case of flares were provided to each investigator before the start of the therapy. The decision to stop therapy in patients with flares and signs of diminished liver function was left at the responsibility of the participating physician who treated the patient.

Figure 1.

Treatment schedule.

Statistical analysis

Kaplan–Meier analysis was used to assess time until dose reduction or premature discontinuation of therapy. For univariate analysis, the following factors were considered: age, gender, race, mode of transmission, baseline levels of AST, ALT, bilirubin, neutrophils, platelets and HBV-DNA, previous therapy with IFN or lamivudine and presence of cirrhosis. To assess which variables independently predicted dose reduction or early discontinuation of therapy, variables that were significant or approximated significance in the univariate analysis (P < 0.2) were included in a multivariate analysis. Cox regression was used for multivariate analysis. Chi-square analysis was carried out to compare frequencies of adverse events. All statistical analyses were performed in SPSS version 10 (SPSS Inc, Chicago, IL, USA).

Results

Baseline characteristics

A total of 307 patients were randomized to treatment with PEG-IFN and either lamivudine or placebo (ratio 1:1). The analysis of efficacy, that was reported previously, was performed for 266 patients, and excluded patients who never started treatment (n = 7), were HBeAg-negative at the start of treatment (n = 10), and patients from a single centre where source data could not be verified (n = 24).15 Because safety data were available and could be verified for all patients, the 300 patients who received at least one dose of medication were included in the present safety analysis. The baseline characteristics of these patients are shown in Table 1. A total of 228 males (76%) and 72 females were treated with PEG-IFN in combination with either lamivudine (n = 148) or placebo (n = 152). The mean age of the patients was 35 years (range 16–72). The majority of patients (76%) were Caucasians. For 230 patients, pre-treatment biopsies were available and of sufficient quality to be evaluated. Of these patients, 26 (11%) had cirrhosis at liver biopsy. Among the 300 patients, 71 patients (24%) had been treated with IFN, and 45 (16%) with lamivudine prior to entry in this study.

Table 1.  Patient characteristics at baseline
Characteristicsn = 300
  1. IFN, interferon; ALT, alanine aminotransferase; HBV, hepatitis B virus; ULN, upper limit of normal.

  2. * Mean ± s.d.

Age (years)35 ± 13
Gender, n (%)
 Male228 (76)
 Female 72 (24)
Weight (kg)* 73 ± 14
Race, n (%)
 Caucasian228 (76)
 Asian/other 72 (24)
Mode of transmission, n (%)
 Vertical 66 (22)
 (Homo) sexual 31 (10)
 Parenteral 27 (9)
 Transfusion associated  8 (3)
 Unknown173 (56)
Previous IFN therapy, n (%) 71 (24)
Previous lamivudine therapy, n (%) 45 (16)
Histological diagnosis (%)
 Cirrhosis26 (11)
 No cirrhosis204 (89)
ALT (×ULN)*4.3 ± 3.5
Platelets (109/L)204 ± 60
Neutrophils3.4 ± 1.3
log HBV-DNA*9.0 ± 1.1

Side-effects

All treated patients reported one or more of the known side-effects of IFN. The frequency of all side-effects was not significantly different between the PEG-IFN/placebo group and the PEG-IFN/lamivudine group. Moreover, there was no difference in occurrence of SAE and in the need for dose reduction or premature treatment discontinuation between the treatment groups. Therefore, we combined the data of the two groups for all analyses. The most frequently reported adverse events were flu-like syndrome (68%), headache (40%), fatigue (39%), myalgia (29%) and local reaction at the injection site (29%) (Table 2). These symptoms typically occurred within the first month of therapy and mostly subsided during the course of therapy. Alopecia and psychiatric symptoms (including depression (n =26) and mood changes or irritability without depression (n = 33)) occurred later in the course of therapy (Figure 2). Transient visual impairment was recorded in five patients (2%), but were not a reason for dose reduction or early treatment discontinuation. During therapy, leucopenia (<3.0 × 109 U/L, grade II), neutropenia (<1.5 × 109 U/L, grade II) and thrombocytopenia (<75 × 109 U/L, grade II) occurred in 42, 22 and 12% of the patients, respectively (Table 2). All adverse events were reported less frequently after dose reduction and were completely reversed after the end of therapy (Figure 2).

Table 2.  Frequencies of adverse events
Adverse eventFrequency (%)
  1. * Includes depression, mood changes, irritability.

Flu-like syndrome68
Headache40
Fatigue39
Myalgia29
Local reaction29
Alopecia22
Weight loss (>10 %)19
Psychiatric*20
Abdominal pain15
Haematologic events 
Leucopenia (<3.0 × 109 U/L)42
Neutropenia (<1.5 × 109 U/L)22
Thrombocytopenia (<75 × 109 U/L)12
Figure 2.

(a) Course of frequent adverse events during therapy and follow-up. (b) Adverse events: Frequency of events in relation to peginterferon alpha-2b dose.

Hepatitis flares.  A total of 71 hepatitis flares were reported, occurring during therapy in 31 patients and after the end of therapy in 40 patients. Flares occurred in 25% (37/148) of patients in the PEG-IFN/lamivudine group and in 22% (34/152) of patients in the PEG-IFN/placebo group (P = 0.5). The frequency of on- and post-treatment flares was not different between groups. Two flares led to discontinuation of therapy. One patient had signs of diminished liver function in the course of a flare. This patient had elevated bilirubin levels (62 μmol/L), which resolved after normalization of ALT levels, but no other signs of hepatic decompensation. None of the hepatitis flares resulted in sustained decompensated liver disease or death.

Serious adverse events.  During the study, 33 SAE were reported. Of these 17 were judged as probably related to therapy. Four patients had a serious hepatitis flare, and three developed severe depression. Neutropenia (grade III or IV) was reported in three cases. Other SAE that were probably related to therapy were syncope, seizures, psychosis, pancreatitis, diarrhoea, anxiety and dizziness (resulting in a fall and a head wound). In 14 patients, the serious adverse event led to early discontinuation of therapy [depression (n = 4), flare (n = 2), neutropenia, seizures, pneumonia, psychosis, pancreatitis, severe diarrhoea, allergic reaction with hypotension and pregnancy (all n = 1)].

Bacterial infections and bleeding complications.  Bacterial infections occurred in 12 patients (4%). Nine of these 12 patients had neutropenia of at least grade II (<1.5 × 109/L) (Table 3). The risk of infection increased with an increasing severity of neutropenia. Patients with neutrophil counts below 1.5 × 109/L had a significantly increased risk of infection compared with patients with higher neutrophil counts (13.6% vs. 1.3%; P < 0.001). Infections included urinary tract infection (n = 4), gastroenteritis (n = 2), tonsillitis (n = 2), gingivitis (n = 2), appendicitis (n = 1) and pneumonia (n = 1). Overall, three severe bacterial infections were reported. In two of these cases (pneumonia and appendicitis) the neutrophil count never dropped below 1.5 × 109/L. The third patient had a Salmonella gastroenteritis while his neutrophil count was 0.7 × 109/L. In four cases [pneumonia, appendicitis and gastroenteritis (n = 2)] the infection necessitated hospital admission. All infections resolved without lasting complications.

Table 3.  Bacterial infections in patients with low neutrophil count
Neutrophils (×109 U/L) nBacterial infections (%)
  1. Reported bacterial infections: urinary tract infection (n = 4), gastroenteritis (n = 2), tonsillitis (n = 2), gingivitis (n = 2), pneumonia (n = 1), appendicitis (n = 1).

>1.52343 (1.3)
1.0–1.5427 (16.6)
0.5–1.0151 (6.6)
<0.591 (11.1)

The risk of bleeding complications was increased in patients with thrombocytopenia, especially of grade III (<50 × 109/L) or IV. Reported bleeding complications in these patients were epistaxis (n = 2), gastrointestinal bleeding, subcutaneous bleeding and combined ecchymosis plus epistaxis (Table 4a). Cirrhotic patients (n = 25) had a significantly higher risk of developing thrombocytopenia than non-cirrhotic patients (54% vs. 8%, P < 0.001). Three of the cirrhotic patients (with thrombocytopenia grade II and III) had bleeding complications (Table 4b). The reported bleeding complications were not life-threatening and did not necessitate blood transfusions.

Table 4.  Bleeding complications according to level of thrombopenia (a) and cirrhosis (b) in patients with low platelets
Platelets (×109 U/L)nBleeding complications (%)
(a)
>1002226 (2.7)
75–100412 (4.9)
50–75281 (3.6)
<5092 (22.2)
Platelets (U/L)Cirrhosis (n = 26)No cirrhosis (n = 204)
nBleeding complications n (%)nBleeding complications n (%)
  1. Reported bleeding complications in patients with platelets <100 × 109 U/L: epistaxis (n = 2), gastrointestinal bleeding (n = 1), subcutaneous bleeding (n = 1), ecchymosis and epistaxis (n = 1).

(b)
>75 × 109120 (0)1884 (2)
<75 × 109143 (21)160 (0)

Dose reduction and discontinuation of therapy

In 69 patients (23%), the dose of PEG-IFN was reduced prematurely (Table 5). Of the dose reductions, 37 (54%) occurred in the PEG-IFN/lamivudine group and 32 (47%) in the PEG-IFN/placebo group. The dose of the blinded drug (lamivudine/placebo) was not reduced in any of the patients. The main reasons for dose reduction were neutropenia (52%), thrombocytopenia (10%), flu-like syndrome (10%) and combined haematological abnormalities (8%). In four patients (6%), dose reduction was necessary because of psychiatric symptoms and in seven patients because of flu-like-syndrome. Fifty per cent of the dose reductions occurred within the first 10 weeks. Thereafter, the number of dose reductions decreased. Only two dose reductions were reported after week 32.

Table 5.  Reasons for dose reduction and early discontinuation
 n (%)
Dose reduction
Neutropenia36 (52)
Thrombocytopenia7 (10)
Leucopenia2 (3)
Combined haematological6 (8)
Flu-like syndrome7 (10)
Psychiatric4 (6)
Fatigue2 (3)
Local reaction1 (1)
Anorexia1 (1)
Myalgia1 (1)
Other2 (3)
Total69
Early discontinuation
Psychiatric10 (36)
Flu-like syndrome3 (11)
Patients lost to follow-up4 (14)
Anaemia1 (4)
Neutropenia1 (4)
Thrombocytopenia1 (4)
Flare1 (4)
Seizures1 (4)
Acute pancreatitis1 (4)
Decompensated liver disease1 (4)
Pneumonia1 (4)
Other3 (11)
Total28

PEG IFN therapy was discontinued prematurely in 28 patients (9%). In 24 patients, the blinded drug (lamivudine/placebo) was also discontinued. In 10 patients, therapy discontinuation was due to psychiatric side-effects. The second clinically important reason for discontinuation was flu-like syndrome (n = 3). Other reasons for early discontinuation were acute pancreatitis, flare, decompensated liver disease and seizures (Table 5). Discontinuation of therapy was reported more frequently before the scheduled dose reduction of PEG-IFN at week 32. In univariate analysis, a low neutrophil count (<3 × 109/L) at baseline and presence of liver cirrhosis were associated with an increased risk of dose reduction or discontinuation of therapy (Table 6). Cox regression analysis, including all variables with a P-value <0.2 in the univariate analysis, showed that both low neutrophil count (hazard ratio 1.7, P = 0.03) and cirrhosis (hazard ratio 2.5, P = 0.001) remained the only independent predictors of dose reduction or discontinuation of therapy.

Table 6.  Univariate analysis of association of baseline factors with dose modifications (dose reduction or discontinuation of therapy)
VariableRisk of dose modifications (%)P-value
  1. ULN, upper limit of normal; HBV, hepatitis B virus; IFN, interferon.

Age (years)
 <35270.19
 ≥3534
Sex
 Male290.27
 Female35
Weight
 <75 kg340.11
 ≥75 kg25
Race
 Caucasian280.07
 Asian/other37
ALT
 <4× ULN290.47
 ≥4× ULN32
log HBV-DNA
 <9360.06
 ≥926
Bilirubin
 <11280.57
 ≥1131
Platelets (×109/L)
 <200340.13
 ≥20025
Neutrophils (×109/L)
 <3390.003
 ≥324
Cirrhosis
 No290.0005
 Yes62
Previous IFN therapy
 Yes340.34
 No28
Previous lamivudine therapy
 Yes380.15
 No27

Discussion

The side-effects of conventional IFN therapy have been well documented in patients with chronic hepatitis B and C.8, 12, 14, 17, 18 The most common side-effects include influenza-like symptoms, fatigue, gastrointestinal symptoms (nausea, anorexia, weight loss), alopecia and neuropsychiatric symptoms (irritability, depression, insomnia). IFN also causes mild bone marrow depression with a temporary decrease in neutrophil, leucocyte and platelet counts. These side-effects have an impact on the quality of life, and can lead to dose reduction or treatment discontinuation. This report is the first on safety of PEG-IFN in patients with CHB. All patients reported one or more of the known adverse events of standard IFN. The adverse events reported in our patients are largely those expected of standard IFN, and no new events were reported. The frequency of the most common adverse events – flu-like syndrome (66%), headache (39%), fatigue (37%) and myalgia (28%) – was comparable with those previously reported in CHB patients treated with standard IFN in a dose of 4.5 MU14 or 10 MU t.i.w.17 for 16–32 weeks. Obviously, these comparisons with historical controls must be interpreted with caution, as the treatment duration in our study was longer and patient populations and therapy doses differ between studies.

Pegylated interferons have been reported to be safe and more effective in patients with chronic hepatitis C.8, 11, 12, 18, 19 In hepatitis C patients, the side-effect profile is the same as that of standard IFN, with some difference in frequencies between different doses and formulations. Haematological abnormalities (especially neutropenia) occur more frequently in chronic hepatitis C patients treated with pegylated interferons (PEG-IFN or peginterferon alpha-2a) than with standard IFN. The rate of dose reductions (22%) and therapy discontinuations (9%) in our study is comparable with the frequencies reported with pegylated interferons in patients with chronic hepatitis C8, 12, 13, 20 and with 24 weeks of peginterferon alpha-2a in patients with CHB.14 The proportion of patients withdrawn from therapy with pegylated interferons is similar to that of conventional IFN, but, when compared with standard IFN, the proportion of patients requiring dose reductions is higher with PEG-IFN in most hepatitis C studies.8, 11, 12, 18 In CHB patients, the frequency of dose reduction with peginterferon alpha-2a was higher than with conventional IFN (22–30% vs. 10%).14 The increased rate of dose reductions with pegylated interferons seems mainly because of the increased occurrence of neutropenia.

Until now it was unknown whether PEG-IFN-induced neutropenia and thrombocytopenia indeed lead to an increase of bacterial infections and bleeding complications respectively. In our study, neutropenia of <1.5 × 109/L significantly increased the risk of bacterial infections (Table 5). However, the number of bacterial infections was rather low and the infections were relatively mild. Only one serious infection (Salmonella gastroenteritis) occurred in a patient with neutropenia above grade I. The risk of bleeding complications was increased in patients with more severe thrombocytopenia, especially in patients with pre-existent cirrhosis and platelets dropping below 75 × 109 U/L. However, bleeding complications were generally mild (epistaxis). Only one potentially life-threatening bleeding complication (bleeding duodenal ulcer) occurred in a patient with mild liver fibrosis.

We also investigated the course of side-effects and adherence to therapy over time. Side-effects were most pronounced at the beginning of therapy and subsided over time. They were generally well tolerated, but flu-like symptoms, lethargy and depression in some cases necessitated dose reduction. Informing patients about the course of these adverse events and adequate treatment with paracetamol and specific serotonin reuptake inhibitors (SSRIs) may lead to an increased proportion of patients capable of completing the treatment. Neutropenia and thrombocytopenia are other frequent causes for dose reduction. Considering the mildness and rareness of complications of neutropenia in our patients, it might be worthwhile to investigate, in a randomized study, if it is safe to accept grade III neutropenia without dose reduction during PEG-IFN treatment for CHB. As, in our study particularly, cirrhotic patients were at an increased risk of thrombocytopenia and (minor) bleeding complications we recommend to monitor them closely and avoid a decrease in platelet count below 75 × 109/L. In a previous study in 217 cirrhotic patients with chronic hepatitis C treated with peginterferon alpha-2a, despite substantial decreases in neutrophil and platelet counts, episodes of infection and bleeding were mild and the treatment was reported to be safe for this patient population.21

In conclusion, prolonged treatment with PEG-IFN in patients with CHB and compensated liver disease is safe. Adding lamivudine to PEG-IFN did not affect PEG-IFN related side-effects. Haematological abnormalities during PEG-IFN treatment led to an increased risk of minor infections and bleeding complications. Cirrhosis and low neutrophil count at baseline are independent predictors of dose reduction or therapy discontinuation. In our opinion, one should closely monitor patients with cirrhosis when initiating PEG-IFN treatment.

Acknowledgements

The study was organized and sponsored by the Foundation for Liver Research (SLO), Rotterdam, The Netherlands. Financial support and study medication were provided by: Schering-Plough International, Kenilworth, NJ, USA; GlaxoSmithKline, Research and Development, Greenford, UK. Monitoring was coordinated by Denys Research Consultants bvba, De Haan, Belgium. Data collection and data management were done by Elke Verheij and Eva Leeuwenhoek, Clinical Research Bureau, Department of Gastroenterology & Hepatology, Erasmus MC, University Medical Center Rotterdam, The Netherlands. Dr Janssen is a Clinical Research Fellow from The Netherlands Organisation of Scientific Research (NWO).

Appendix

In addition to the authors, the HBV 99-01 Study Group includes the following investigators: The Netherlands: H.G.M. Niesters, P.E. Zondervan, B.E. Hansen (University Medical Center Rotterdam), B.C.M. Vroom (University Medical Center Utrecht) C.M.J. van Nieuwkerk (VU University Medical Center Amsterdam), R.A. de Vries (Rijnstate Hospital Arnhem), J. Jansen, J. Drenth, S.J. van den Hazel (University Medical Centre Radboud Nijmegen), J.W. den Ouden-Muller (St. Franciscus Hospital Rotterdam), A.C. Tan (Canisius Wilhelmina Hospital Nijmegen); Belgium: D.M. Adler (Hopital Erasme Brussels), P. Michielsen (University Hospital Antwerp), H. van Vlierberghe (University Hospital Gent), F. Nevens (University Hospital Leuven), J. Delwaide (Centre Hospitalier Universitaire Liège), J. Henrion (Hopital de Jolimont, Haine St. Paul); Germany: G. Gerken, S. Bein, U. Treichel (University Hospital Essen), J. Trojan (J.W. Goethe Universität Frankfurt), M.P. Manns, J. Hadem (Medizinische Hochschule Hannover), C. Niederau (St. Jozef Hospital Oberhausen); Denmark: M.R. Buhl, I.M. Hansen (Skejby Hospital, Arhus), K. Krogsgaard (Copenhagen University Hospital Hvidovre); Poland: J. Cianciara, J. Jablonska, J Kozlowska (Medical Academy of Warsaw), D. Prokopowicz, R. Flisiak (Medical Academy of Bialystok), T. Mach (Collegium Medicum UJ Kraków); Spain: M. Buti, A. Valdes, R Esteban (Hospital Valle Hebron, Barcelona), M. Rodriguez, M. Garcia Espiga (Hospital Central de Asturias, Oviedo); Italy: A. Andriulli, G. Stornaiulo, G.B. Gaeta (Ospe. Gesù e Maria, Napoli), G. Montalto, F. D'Antona (Università di Palermo); Greece: G.E. Kitis, P. Xiarchos Panagiotis (George Papanikolaou General Regional Hospital, Thessaloniki), N.C. Tassopoulos (West Attica Hospital Athens); Turkey: G. Ersöz (Ege University Faculty of Medicine Izmir), S. Karayalcin, C. Yurdayin, H. Bozkaya (Medical School Cebeci Kampusu Ankara), H. Simsek, Y. Balaban (Hacettepe University Faculty of Medicine Ankara), F. Tabak (Istanbul University Cerrahpasa Medical School, Istanbul); Israel: Y. Lurie (Sauraski Medical Center Tel-Aviv); Canada: J. Heathcote (Toronto Western Hospital, Toronto); S.V. Feinman (Mount Sinai Hospital Toronto); S. Greenbloom (General Hospital Etobicoke); Indonesia: D.A. Sulaiman (Ciptomangunkusomo Hospital Jakarta); Singapore: R. Guan (Mount Elizabeth Medical Center Singapore); Malaysia: I. Merican (Institute for Medical Research Kuala Lumpur).

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