Efficacy and Safety of a Steroid-Free Immunosuppressive Regimen after Liver Transplantation for Hepatocellular Carcinoma.

BACKGROUND/AIMS: We aimed to evaluate the efficacy and safety of an immunosuppressive regimen without steroids after liver transplantation (LT) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).
METHODS: Sixty-six HCC patients who underwent an immunosuppressive regimen without steroids after LT were enrolled in the steroid-free group. The preoperative characteristics and postoperative outcomes of these patients were compared with those of 132 HCC recipients who were placed on an immunosuppressive regimen using steroids (steroid group). The incidence of acute rejection, HBV recurrence, infection, and new-onset diabetes mellitus and the overall and tumor-free survival rates were compared between the two groups.
RESULTS: Differences were not observed in the 1-year (83.3% vs 97.0%, p=0.067), 3-year (65.4% vs 75.8%, p=0.067) or 5-year (56.3% vs 70.7%, p=0.067) patient survival rates or in the 1-year (62.1% vs 72.7%, p=0.067), 3-year (49.8% vs 63.6%, p=0.067) or 5-year (48.6% vs 63.6%, p=0.067) tumor-free survival rates between the two groups, respectively. In the steroid-free group, the patients who fulfilled the Milan criteria had higher overall and tumor-free survival rates than those in the steroid group (p<0.001). The prevalence of HBV recurrence (3.0% vs 13.6%, p=0.02) was significantly lower in the steroid-free group compared with the steroid group.
CONCLUSIONS: After LT, an immunosuppressive regimen without steroids could be a safe and feasible treatment for HBVrelated HCC patients, thus resulting in the reduction of HBV recurrence. Based on the observed survival rates, patients who fulfill the Milan criteria may derive benefits from teroidfree immunosuppression.

INTRODUCTION

Liver transplantation (LT) is the optimal therapy for patients with hepatocellular carcinoma (HCC) with cirrhosis because it treats both the tumor and the underlying liver disease.1–3 Unfortunately, HCC recurrence is reported to be as high as 40% after LT and remains the major cause of death.4,5

Since the first LT performed by Thomas Starzl in 1963, steroids have become the gold standard, together with calcineurin inhibitors, for immunosuppression after LT. However, long-term steroid use may facilitate the proliferation and spread of malignant cells.6 It has been reported that steroids play an important role in tumor recurrence after LT for HCC.7 In addition, the numerous side effects of steroids, such as infection, obesity, hypertension and diabetes mellitus, have urged the need to avoid or limit steroid usage.8–10

In the past decade, several studies have evaluated the feasibility of steroid-free protocols after LT. Most of these studies have focused on hepatitis C virus-related liver disease.11–20 However, hepatitis B virus (HBV) infection is the leading cause of liver cirrhosis and end-stage liver disease in China, and the possible role of a steroid-free protocol in HBV-related HCC recurrence has rarely been evaluated.

In this study, we evaluated the safety and efficacy of steroid-free immunosuppression in patients undergoing LT for HBV-related HCC.

MATERIALS AND METHODS

1. Patients

From April 2009 to June 2011, 66 HBV-related HCC patients who underwent LT at the First Affiliated Hospital, Zhejiang University School of Medicine were enrolled in the steroid-free group. All of the recipients in the steroid-free group received methylprednisolone (1,000 mg during the operation) and basiliximab (20 mg during the operation and another 20 mg at 4 days after LT). The steroid-free patients were matched at a 1:2 ratio, by sex, age, donor source, Model for End-stage Liver Disease score, body mass index, α-fetoprotein level and tumor characteristics, with control patients receiving the standard protocol as described previously (methylprednisolone, 1 g on the first day and prednisolone, 20 mg tapered to 0 mg within the first 3 months)21 from January 2006 to April 2009 (steroid group, n=132). All the recipients were administered tacrolimus after LT, with a target serum trough level of 10 to 12 ng/mL during the first month and 8 to 10 ng/mL from the second month. Mycophenolate mofetil was prescribed for 1 year at a dose of 0.5 to 1.0 g/day.

In the steroid-free group, 33 recipients fulfilled the Milan criteria22 (subgroup SF1) and 33 exceeded the Milan criteria (subgroup SF2). In the steroid group, 53 recipients fulfilled the Milan criteria (subgroup S1) and 79 exceeded the Milan criteria (subgroup S2).

All of these recipients received preoperative antiviral treatments, including monotherapy of nucleoside analogs (lamivudine or entecavir) and multiple therapies of nucleoside analogs (lamivudine plus adefovir) and had an HBV DNA-negative status prior to LT. The main patient clinical characteristics are summarized in Table 1. All patients received lamivudine combined with low-dose hepatitis B immune globulin therapy after LT, as described previously.23 Enhanced computed tomography images and abdominal ultrasonography were performed every 3 to 6 months for HCC recurrence surveillance.

Table 1

Clinical Characteristics of the Patients

Characteristic	Steroid group (n=132)	Steroid-free group (n=66)	p-value
Age, yr	49.55±0.75	50.23±1.02	0.599
Sex, male/female	117/15	62/4	0.232
BMI, kg/m2	22.47±0.23	22.60±0.30	0.751
Transplantation type			0.061
LDLT	27	5
DDLT	92	52
DCD	13	9
Cold ischemia time, hr
LDLT	1.31±0.265	1.21±0.095	0.872
DDLT	9.72±0.26	10.03±0.317	0.462
DCD	9.47±0.754	8.8±1.135	0.616
DM pre-LT	12 (9.1)	7 (10.6)	0.733
Hypertension pre-LT	13 (9.8)	4 (6.1)	0.370
MELD score	13.20±0.54	13.47±1.04	0.798
Child score	7.47±0.20	6.94±0.34	0.204
HBV-DNA			0.191
<103 copies/mL	65	39
≥103 copies/mL	67	27
Pretransplant AFP	3,986.31±1,219.74	2331.96±1784.25	0.461
Hangzhou criteria, fulfilling/exceeding	96/36	44/22	0.377
Milan criteria, fulfilling/exceeding	53/79	33/33	0.188
Tumor characteristics
Size of greatest tumor, cm	4.60±0.26	4.39±0.43	0.661
No. of tumors	3.89±0.77	1.88±0.18	0.066
Histological differentiation			0.181
Well differentiated	3	5
Moderately differentiated	64	28
Poorly differentiated	65	33
Vascular invasion	31	17	0.358
Tacrolimus levels post-LT, ng/mL
7 Day	7.59±0.55	6.95±0.48	0.384
14 Day	9.00±0.50	9.55±0.49	0.433
30 Day	7.76 ±0.37	6.20±0.29	0.001
60 Day	8.66±0.36	7.24±0.33	0.005
90 Day	7.43±0.42	7.20±0.67	0.756
120 Day	6.85±0.49	6.50±0.65	0.668

Data are presented as mean±SD or number (%).

BMI, body mass index; LDLT, liver living donor liver transplantation; DDLT, deceased donor liver transplantation; DCD, donation after cardiac death; DM, diabetes mellitus; LT, liver transplantation; MELD, Model for End-stage Liver Disease; HBV, hepatitis B virus; AFP, alpha fetoprotein.

Each organ donation and transplantation strictly followed the guidelines of the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine (approval number: 2013-12), the current regulation of the Chinese government and the Declaration of Helsinki 2004. Informed consent was obtained from all patients.

2. Data collection

HBV recurrence was defined by the reoccurrence of serum HBV surface antigens. Acute rejection (AR) was identified by liver biopsies according to the presence of at least two of the three following criteria according to the Banff classification: portal inflammatory infiltration, endothelialitis, and bile duct damage.24,25 The Milan criteria were defined as follows: patients with a single tumor ≤5 cm or no more than three tumors, each no larger than 3 cm.22 New-onset diabetes mellitus (NODM) was defined as a fasting glucose level of at least 7 mmol/L (126 mg/dL) or a nonfasting glucose level of at least 11.1 mmol/L (200 mg/dL) confirmed on at least two occasions or a need for anti-diabetic drugs persisting beyond the first month but within the first year after transplantation.21,26 Hypertension was defined as blood pressure greater than 140/90 mm Hg on two consecutive visits and/or the need for antihypertensive therapy. Hyperlipidemia was defined as serum triglycerides ≥150 mg/dL (1.69 mmol/L), serum cholesterol ≥200 mg/dL (5.17 mmol/L), or a need for pharmacological treatment 2 months after LT.27 Early renal dysfunction (ERD) was defined as serum creatinine ≥2 mg/dL and/or the need for renal replacement therapy in the first posttransplant week. Early allograft dysfunction (EAD) was defined by the presence of at least one of the following characteristics: total bilirubin >10 mg/dL, prothrombin time ≥17 seconds and hepatic encephalopathy from day 2 to day 7 posttransplantation.28

3. Statistical analysis

Quantitative variables are expressed as the mean±standard deviation or median and range depending on the distribution. Categorical variables are presented as values and percentages. Student t-test was used to compare quantitative variables. Chi-square test was used to compare categorical variables. The Kaplan–Meier method and log-rank test were used for survival analysis. SPSS version 11.0 (SPSS Inc., Chicago, IL, USA) was used to complete all the analyses, and a p-value less than 0.05 was considered to be statistically significant (two-tailed test).

RESULTS

1. Patient characteristics

There were no significant differences in age, sex, primary disease, comorbidity, or operation type between the two groups. The tumor characteristics, including size, number, histological differentiation, and vascular invasion did not differ significantly between the two groups (Table 1). Compared with the steroid group, the steroid-free group showed significantly lower serum alanine transaminase and aspartate transaminase during the first two post-transplant weeks (Fig. 1).

Fig. 1

(A–D) Comparison of post-liver transplantation liver and kidney function. Compared with the steroid group, the steroid-free group showed significantly lower serum alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) levels during the first 2 post-transplant weeks. The creatinine (Cr) level did not differ significantly between the two groups. *p<0.05, †p<0.01.

2. HCC recurrence and survival

There were no differences in the 1-year (83.3% vs 97.0%, p=0.067), 3-year (65.4% vs 75.8%, p=0.067), or 5-year (56.3% vs 70.7%, p=0.067) patient survival rates or the 1-year (62.1% vs 72.7%, p=0.067), 3-year (49.8% vs 63.6%, p=0.067), or 5-year (48.6% vs 63.6%, p=0.067) tumor-free survival rates between the steroid and steroid-free groups, respectively (Fig. 2). Subgroup SF1 showed the highest 1-year, 3-year, and 5-year overall (p<0.001) and tumor-free (p<0.001) survival rates in the four subgroups (Fig. 3A and B).

Fig. 2

Between-group comparison of the cumulative patient (A) and tumor-free (B) survival rates. Differences in the overall survival curves of patients in the steroid-free group and patients in the steroid group did not achieve statistical significance (1-year, 83.3% vs 97.0%; 3-year, 65.4% vs 75.8%; 5-year, 56.3% vs 70.7%, respectively; p=0.067 for all). Additionally, the difference in the tumor-free survival curves of patients in the steroid-free group and patients in the steroid group did not reach the level of statistical significance (1-year, 62.1% vs 72.7%; 3-year, 49.8% vs 63.6%; 5-year, 48.6% vs 63.6%, respectively; p=0.067 for all).

Fig. 3

Comparison of the cumulative patient (A) and tumor-free (B) survival between the patients who fulfilled and exceeded the Milan criteria. The patients who fulfilled the Milan criteria in the steroid-free group (subgroup SF1) had an improved prognosis compared with the following patients: patients who exceeded the Milan criteria in the steroid-free group (subgroup SF2), patients who fulfilled the Milan criteria in the steroid group (subgroup S1), and patients who exceeded the Milan criteria in the steroid group (subgroup S2).

3. HBV recurrence

Post-transplant HBV recurrence was observed among 10.1% of the patients (20/198). The mean interval for the development of HBV recurrence was 6.36±4.32 months (range, 2.5 to 15.5 months). The HBV recurrence rate was 13.6% in the steroid group (18/132), but only 3.0% in the steroid-free group (2/66) (p=0.020) (Table 2).

Table 2

Complications after Liver Transplantation

	Steroid group (n=132)	Steroid-free group (n=66)	p-value
Acute rejection	8 (6.1)	2 (3.0)	0.359
Hypertension	6 (4.5)	1 (1.5)	0.276
Infection
Viruses	3 (2.3)	2 (3.0)	0.742
Tuberculosis	1 (0.76)	1 (1.5)	0.615
Bacteria	6 (4.5)	6 (9.1)	0.206
Fungus	3 (2.3)	2 (3.0)	0.749
NODM	37 (28.0)	14 (21.2)	0.301
HBV recurrence	18 (13.6)	2 (3.0)	0.020
Hyperlipidemia	7 (5.3)	2 (3.0)	0.469
HCC recurrence	61 (46.2)	24 (36.4)	0.187
ERD	11 (8.3)	3 (4.5)	0.327
EAD	6 (4.5)	2 (3.0)	0.610

Data are presented as number (%).

NODM, new-onset diabetes mellitus; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; ERD, early renal dysfunction; EAD, early allograft dysfunction.

4. Other posttransplant complications

There were no significant differences in the incidences of AR (6.1% vs 3.0%, p=0.359), hyperlipidemia (5.3% vs 3.0%, p=0.469), viral infection (2.3% vs 3.0%, p=0.742), tuberculosis infection (0.76% vs 1.5%, p=0.615), bacterial infection (4.5% vs 9.1%, p=0.206), fungus infection (2.3% vs 3.0%, p=0.749), new-onset hypertension (4.5% vs 1.5%, p=0.276), ERD (8.3% vs 4.5%, p= 0.327) and EAD (4.5% vs 3.0%, p=0.610) between the steroid and steroid-free groups, respectively (Table 2).

DISCUSSION

Steroids have long been considered a linchpin in the prevention and treatment of rejection. However, there are well-known steroid-related adverse effects that result in significant morbidity, including hypertension, diabetes mellitus, hyperlipidemia and obesity, leading to increased cardiovascular risk and infectious complications. The adverse effects of long-term steroid use have stimulated interest in the feasibility of steroid-free maintenance immunosuppressive regimens.8–10 Our result demonstrated that a steroid-free protocol was safe in patients undergoing LT for HBV-related HCC. Liver function recovered well during the first 2 posttransplant weeks.

As we know, the prevention of rejection is the initial purpose of using steroids. Steroids inhibit prostaglandin synthesis, inhibiting interleukin 1 (IL-1) transcription and IL-1-dependent lymphocyte activation, stabilizing lysosomal membranes and reducing histamine and bradykinin release.29 Safety is the premise of a steroid-free immunosuppressive regimen. Early steroid avoidance in LT showed a high incidence of AR.30 An immunosuppressive regimen with a humanized IL-2 receptor (IL-2R) inhibitor has been shown to achieve very low rates of AR.15,31,32 In this study, we also used a humanized IL-2R inhibitor called basiliximab in the steroid-free group and detected no difference in the AR rate between the two groups, a finding that is consistent with that of many previous studies.33,34

One important finding of this study is that the incidence of HCC recurrence did not decrease significantly in the steroid-free immunosuppression recipients. We found that both the tumor-free survival and patient overall survival were similar in patients receiving steroid-free immunosuppression compared to those with a steroid protocol. A recent study in China revealed that HCC patients with steroid avoidance achieved a lower tumor recurrence rate after LT than those with steroids withdrawn after 14 days, 3 months or 6 months.20 The latter finding is inconsistent with the results of our study. At First Affiliated Hospital, the patients in the steroid-group received steroids for only 3 months; this situation could be viewed as an early steroid withdrawal protocol because steroids are administered for a longer time at many other centers.

The mechanism underlying how steroids contribute to tumor recurrence is not well established. Steroids may reduce the potency of the immune inflammatory response35,36 or inhibit neutrophil-mediated tumor cell apoptosis, thereby promoting the growth of tumor cells.37 In our study, the highest overall and tumor-free survival were observed in the patients fulfilling the Milan criteria using an immunosuppressive regimen without steroids. This phenomenon revealed that strictly selected HCC patients with steroid avoidance would achieve a good prognosis.

Another important finding is that steroid-free immunosuppression might reduce HBV recurrence. In China, a large proportion of LT patients have HBV-related liver disease. Nevertheless, 10% of LT patients with underlying HBV experience a recurrence.38,39 A study conducted in Korea obtained the outcome that HBV recurrence rates in patients who underwent the steroid-free protocol were much lower than those in patients who underwent the steroid protocol; analysis of the interaction between steroid pulse therapy and HBV recurrence revealed that the HBV has a corticosteroid receptor that promotes virus replication.40 Our study showed similar results. Steroid-free immunosuppression may help reduce the high HBV recurrence rate after LT.

In this study, there were no significant differences in hypertension, infection, NODM and hyperlipidemia between the two groups. The side effects of steroids have been observed in other studies. In one study, the incidence of early post-transplant diabetes mellitus (DM), hypertension and infection were lower in the steroid early avoidance recipients. No differences were found in the incidence of hypertension and hyperlipidemia between the steroid early avoidance and standard groups.19 In another study, the incidence of hypertension, hyperglycemia, and DM were significant lower in steroid-free recipients,20 demonstrating that the effect of steroids on metabolic complications are still controversial.

In summary, an immunosuppressive regimen without steroids could be safe and feasible for HBV-related HCC patients after LT with a reduction in HBV recurrence. It seems that patients fulfilling the Milan criteria may benefit more from steroid-free immunosuppression according to the survival rates found.