Objectives: The use of induction therapy in liver transplant is debatable. We aimed to compare clinical outcomes of different induction protocols in liver transplant recipients.

Materials and Methods: This was a retrospective cohort analyses using the University of Washington Transplant Database from January 2005 to May 2012 for adult (≥ 18 y old) primary liver transplant patients. All patients received induction therapy. Maintenance immuno­suppressive agents were tacrolimus or tacrolimus-mycophenolate mofetil. Primary endpoints were acute cellular rejection, patient survival, and graft survival. In patients with chronic hepatitis C, the degree of histologic inflammation or fibrosis at 1 year was assessed. Cox proportional hazards models were constructed to evaluate variables associated with both patient and graft survival.

Results: We identified 595 patients: 322 patients received rabbit antithymocyte globulin and 273 received interleukin 2 receptor blocker. Acute cellular rejection was higher in patients who received interleukin 2 receptor blocker than in patients who received rabbit antithymocyte globulin (27% vs 18%; P < .03). Both patient survival at 1 year (95% vs 90%), 3 years (92% vs 87%), and 5 years (86% vs 80%) and graft survival at 1 year (93% vs 88%), 3 years (90% vs 86%), and 5 years (83% vs 78%) were superior with rabbit antithymocyte globulin than with the interleukin 2 receptor blocker (P < .002). In patients with hepatitis C virus, type of induction therapy did not have any effect on the timing of hepatitis C virus recurrence. At 1 year after transplant, 33.3% in the rabbit antithymocyte globulin group had grade 3/4 inflammation and 10.2% had stage 3/4 fibrosis, compared with 16.8% and 4.8% in the interleukin 2 receptor blocker group (P ≤ .002 and not significant). Female recipient, Model for End-Stage Liver Disease score, hepatocellular carcinoma, and high preoperative serum creatinine levels were associated with less favorable patient and graft survival.

Conclusions: Rabbit antithymocyte globulin is asso­ciated with lower rejection rate and improved patient and graft survival in liver transplant. Type of therapy affects the degree of histologic hepatitis C virus recurrence.

Key words : Liver transplantation, Induction therapy, Outcomes

Introduction

The ideal immunosuppressive protocol would have a very low incidence of rejection and minimal infectious, toxic, or cancer-related complications, with consequent improvement in graft acceptance and quality of life. Over the past several years, the use of antibody induction therapy has increased and currently 20% to 25% of liver transplant recipients receive this therapy.^1,2 Antibody therapy has been used in liver transplant to decrease the incidence of acute cellular rejection (ACR) and to treat steroid-resistant acute rejection, to minimize the use of maintenance immunosuppression, and to delay the introduction of calcineurin inhibitors in patients with renal dysfunction.³ Although the benefits of induction immunosuppression with antibody induction in terms of ACR and graft survival have been demonstrated in kidney transplant, there is scarce and controversial data in the liver transplant population. Studies have suggested that the use of induction agents is associated with a decreased incidence of ACR, no detrimental effect in post­transplant recurrence of hepatitis C virus (HCV), improvement in postoperative renal function, and improved patient and graft survival compared with noninduction therapy protocols.^3-5 However, it has also been shown that the use of rabbit antithymocyte globulin (rATG) as induction therapy or therapy of steroid-resistant ACR is associated with increased risk of aggressive HCV recurrence.^6-8

Since 2000, our standard antibody induction pro­tocol has included an interleukin 2 receptor (IL-2R) antibody. In the face of the increasing Model for End-Stage Liver Disease (MELD) scores of our recipient population and the use of more expanded criteria deceased donors, in 2005, rATG was introduced as an alternative agent as a way to spare the kidney, with a later introduction of calcineurin drugs and minimized immunosuppressive load in the short and long term. No study has yet assessed the clinical effects of rATG versus IL-2R antibodies as induction therapies in liver transplant, although 1 study reported good overall patient survival (96% at 17 mo) using rATG.9 We hypothesized that the alternative use of rATG versus IL-2R antibody would be associated with a decreased rate of ACR and improved patient and graft survival rates. We designed this study to compare clinical outcomes of these 2 induction protocols in liver transplant recipients.

Materials and Methods

Protocol, study design, and patient population
We performed a retrospective case series study of all liver transplant recipients who received transplants between January 1, 2005, and May 31, 2012, at the University of Washington Medical Center, using clinical data drawn from the electronic medical record transplant database of the University of Washington (MAX database). The MAX database contains all pretransplant demographic and clinical characteristics, transplant surgery information, and posttransplant care, covering all patients who undergo transplant at our center.

For the present study, inclusion was restricted to adult patients (≥ 18 y of age) who received a primary liver transplant; those who required retransplant during the same admission had the second transplant censored. Due to the low number of patients who required this intervention, their impact on the final outcomes was expected to be negligible. Patients with multiorgan transplants were also excluded from the analyses.

Immunosuppression protocol and treatment of rejection
Two immunosuppression antibody induction regimens were available and in use at our institution during the study period. Interleukin 2 receptor blocker (Simulect, Novartis, Basel, Switzerland) was given at a dose of 20 mg at the day of transplant and on postoperative day 4; rATG (Thymoglobulin, Genzyme, Cambridge, MA), introduced in 2005 as an alternative induction agent, was given at 1.5 mg/kg/day, with a total of 3 doses in the initial 2 to 5 days after surgery. Rabbit antithymocyte globulin induction was the protocol of choice unless the peritransplant conditions of the recipient prompted the surgeon to opt for IL-2R induction; such conditions included persistent postoperative hemodynamic instability (requiring ≥ 2 vasopressor support > 6 h after transplant), persistent cardiac arrhythmias, or history of pulmonary hypertension.

Steroids were used during the initial 3 days after surgery as premedication for rATG and then discontinued. Tacrolimus monotherapy was used for maintenance therapy in most of our patients with trough levels ranging from 10 to 12 ng/mL during the first 3 months, tapering down to final goal of 3 to 5 ng/mL at 1 year posttransplant. In those patients receiving rATG, tacrolimus was started on post­operative day 4; for patients receiving IL-2R blocker, it was started on postoperative day 1. In patients with renal dysfunction, mycophenolic acid (1,000 mg orally twice per day) was started on postoperative day 1.

All patients received standard prophylaxis for infections after transplant: ganciclovir or valganciclovir for patients at risk for Cytomegalovirus disease, trimethoprim/sulfamethoxazole or pentamidine for Pneumocystis jiroveci prophylaxis, and clotrimazole or fluconazole for Candida prophylaxis. Mild and moderate episodes of biopsy-proven ACR were treated with bolus methylprednisolone: 1000 mg at 1 dose, 500 mg at 1 dose, and 250 mg at 1 dose followed by prednisone taper. Severe episodes of ACR and steroid-resistant rejection were treated with 5 boluses of rATG 1 mg/kg/day. Percutaneous liver biopsy was performed when persistent testing results demonstrated a 1.5-fold increase of serum aspartate aminotransferase or alanine aminotransferase above the upper limit of normal; ACR was defined by the 1997 Banff criteria.¹⁰

All patients were followed until graft loss, loss to follow-up (documented by clinic coordinators), or death. We defined graft loss as a record of a subsequent liver transplant in the MAX database or death. Our clinical coordinators conducted patient follow-up at least annually and maintained records for both loss to follow-up (lack of contact greater than 1 y) and mortality.

Groups, endpoints, and covariate definitions
Eligible liver transplant recipients were categorized into 2 groups based on their induction therapy: rATG versus IL-2R blocker. The primary outcomes of this study were patient and graft survival. Secondary outcomes included ACR, posttransplant lympho­proliferative disorder, posttransplant serum creatinine levels, need for future kidney transplant, and hepatitis C recurrence. Rejection was defined according to Banff definitions¹⁰ as reported in the pathology section of the MAX database. Recurrence of hepatitis C infection was defined histologically based on the identification of apoptotic bodies and portal inflammation in 2 consecutives biopsies and further graded and staged using the Batts-Ludwig classification system.¹¹ Our center utilizes serial protocol biopsies in HCV-positive patients at 3, 6, and 12 months and annually thereafter.

Statistical analyses
Patient characteristics and outcomes were sum­marized using frequency distributions for categorical variables and means and standard deviations for continuous variables. Statistical significance was assessed using P values from t tests for continuous variables or the chi-squared test for categorical variables. Patient and graft survival rates by induction group were estimated using the Kaplan-Meier estimator. The log-rank test was used to determine whether there was a significant difference in the curves. Cox proportional hazards models were constructed to adjust for confounding factors and to evaluate variables associated with patient and graft survival.

All analyses were performed with SAS 9.3 (SAS Institute Inc., Cary, NC, USA). The Human Subjects Division at the University of Washington approved this study (#47930).

Results

Pretransplant clinical characteristics of the study cohort
During the study period, 630 patients underwent liver transplant at the University of Washington Medical Center. Of those, 595 met the eligibility criteria for the study, which included 322 in the rATG group and 273 in the IL-2R blocker group. The overall median follow-up was 4.7 years: 4.2 years for the rATG group and 5.6 years for the IL-2R blocker group. The demographics of the cohort are shown in Table 1. Groups were comparable with age at transplant, gender, MELD score, preoperative serum creatinine level, international normalized ratio and bilirubin, cause of end-stage liver disease, recipient of deceased donation, previous abdominal surgery, ascites, transjugular intrahepatic portosystemic shunt, spontaneous bacterial peritonitis, and cold ischemia time. Patients who received rATG were more likely to have hepatocellular carcinoma (45% vs 37.5%; P = .04) and less likely to be on hemodialysis (7% vs 13%; P = .004) at the time of transplant.

Posttransplant outcomes of interest
Patients in the rATG group had better patient survival (95%, 92%, and 90% vs 87%, 86%, and 80% at 1, 3, and 5 years) and graft survival (93%, 90%, and 88% vs 86%, 83%, and 78%) than those in the IL-2R blocker group. P values from unadjusted log-rank test were 0.02 for both patient and graft survival (Figures 1 and 2). After adjustments were made for recipient’s age, sex, preoperative serum creatinine level, international normalized ratio, bilirubin, hepa­tocellular carcinoma, HCV, MELD score, previous abdominal surgery, ascites, portal vein thrombosis, spontaneous bacterial peritonitis, transjugular intrahepatic portosystemic shunt, and cold ischemia time, the use of rATG was associated with significantly better patient survival (hazard ratio 0.61; 95% confidence interval, 0.38-0.97) and better graft survival with borderline significance (hazard ratio 0.64; 95% confidence interval, 0.41-1.01). Female recipients, increased MELD score, hepato­cellular carcinoma, and high preoperative serum creatinine levels were associated with worse patient and graft survival.

The overall incidence of ACR was significantly higher in the IL-2R blocker group than in the rATG group (27% vs 18%; P ≤ .03); 32 patients (10%) in the rATG group had steroid-resistant ACR compared with 38 patients (14%) in the IL-2R blocker group (P = not significant). In the rATG group, only 3 patients (1%) developed posttransplant lymphoproliferative disorder, compared with 11 patients (4%) in the other group (P ≤ .03). Both induction groups experienced a decrease in posttransplant serum creatinine levels in the first 2 years after liver transplant, with the rATG group having a lower level at all time points (Figure 3). Kidney transplant was required in 7 patients (2%) who received rATG compared with 12 patients (4%) in the IL-2R blocker group (P = not significant). In patients who received hemodialysis before transplant, a kidney transplant was required in 5 patients (23%) who received rATG, compared with 8 patients (27%) in the IL-2R blocker group (P = not significant). For patients who received a liver transplant due to HCV, the type of induction therapy did not have any significant effect on the timing of recurrence of HCV infection (P = not significant; Figure 4). At 1 year after transplant, 33.3% in the rATG group had grade 3/4 inflammation and 10.2% had stage 3/4 fibrosis, compared with 16.8% and 4.8% in the IL-2R blocker group (P ≤ .002 and not significant; Table 3).

Discussion

Prior studies have shown that the use of antibody induction is associated with a decreased incidence of acute rejection, no detrimental effect in post­transplant recurrence of HCV, improvement in postoperative renal function, and excellent patient and graft survival compared with that shown with noninduction therapy protocols. However, its use in liver transplant remains controversial.^3-5

When our program transitioned to rATG from the IL-2R blocker, we hypothesized that this change in induction protocol would be associated with a decreased rate of ACR and perhaps improved patient and graft survival. This study demonstrates that the use of rATG as induction therapy in liver transplant is associated with a significant drop in the rate of acute rejection and improved patient and graft survival compared with IL-2R blockers.

Acute cellular rejection in relation to rATG has been previously studied under different regimens of induction of immunosuppression. Retrospective data from a single center cohort study found that induction therapy with rATG reduced 1-year ACR rate from 31.8% with standard therapy to 14.5%.12 Two randomized, single center studies evaluating use of rATG induction therapy showed similar frequency of episodes of ACR compared with the control groups.^13,14

Contrary to the literature, which states that the use of antibody induction therapy in patients with HCV is associated with early recurrence and rapid progression of HCV after liver transplant,6-8 our study demonstrated that neither the recurrence rate nor the histologic stage is associated with the use of or type of antibody induction therapy, and it compares well with the recurrence rate seen in patients who do not receive any induction therapy. Horton and associates had previously demonstrated that the use of rATG had no significant effect on either overall graft survival or patient survival in HCV-positive recipients.⁵ Eason and associates in a randomized clinical trial evaluating steroid-free induction reported that 9 of 18 patients (50%) randomized to rATG and 12 of 17 (71%) assigned to the standard therapy have both biochemical and histologic evidence of HCV, showing a trend for lower frequency of HCV recurrence in the patients who received rATG.15 More recently, McCashland and associates randomized HCV-positive patients to receive rATG versus no induction therapy during liver transplant. They were able to demonstrate that the rate of HCV recurrence was lower in the rATG group than in the control group.¹⁶ In accordance with our findings, they also demonstrated that the HCV-free survival was not significantly different between both groups. Unlike this study, we found that the rate of advanced inflammation at 1 year was worse with the use of rATG. This suggests that, although the type of induction therapy does not affect the recurrence rate of HCV, when it happens, it is more severe after the use of rATG. With the introduction of new drug therapies for treatment of HCV, this effect of rATG on HCV recurrence will probably be irrelevant in our future practice.

Another major concern surrounding the use of induction therapy is the increased incidence of lymphoproliferative disorders, which might restrict its indications to specific at-risk groups (African American, retransplant, presensitization).¹⁷ In this study, we showed that the rate of lymphoproliferative disease was lower with the use of rATG than with the IL-2R blocker. Furthermore, of those patients who developed posttransplant lymphoproliferative disease, all but 1 patient in each group were positive for Epstein-Barr virus at the time of transplant. Among the risks factors associated with post­transplant lymphoproliferative disease after liver transplant, Narkewicz and associates18 found that ACR is associated with increased risk of post­transplant lymphoproliferative disease, likely due to the increased amount of immunosuppression that has to be administered. With the use of rATG, we demonstrated a significantly decreased rate of ACR, which could explain the lower incidence of posttransplant lymphoproliferative disease in this group. Our rate of posttransplant lymphoproliferative disease is comparable to what is published in orthotopic liver transplant patients (1% to 3%).19

One of the major goals with the use of induction therapy is to decrease the renal dysfunction associated with calcineurin inhibitors by delaying or minimizing its doses posttransplant. Glomerular filtration rate at 1 year is predictive of late renal dysfunction,²⁰ and an increased creatinine level 1 month after transplant is associated with an increased risk of developing chronic renal failure.²¹ In this study, both induction groups experienced a decrease in posttransplant serum creatinine in the first 2 years after liver transplant, with the rATG group having a lower level at all time points. Several retrospective reviews have demonstrated the successful use of rATG induction with delayed introduction of calcineurin inhibitors. These reviews have shown improved renal function and similar or less frequent ACR compared with standard calcineurin inhibitor regimens alone. Soliman and associates reported a single center review of 262 patients after orthotopic liver transplant who received rATG induction for 3 days with delayed, standard-dose of calcineurin inhibitors compared with 129 patients treated with immediate, standard-dose calcineurin inhibitors. Serum creatinine (P < .01) and glomerular filtration rate (P < 0.02) were significantly improved at 1 year in the rATG group.¹²

Similar to previous reports, we have shown that the need for further kidney transplant is fairly low, and this is not associated with the choice of induction agent in the short-term.²² In those patients who were on hemodialysis before transplant, we found a higher rate of use of IL-2R blocker instead of rATG. Although this seems to be a paradox with the main principle of using rATG, we are usually forced in our practice to use IL-2R blocker due to its lower adverse effect profile in this compromised patient population.

Regarding the Cox regression analyses of factors predicting patient and graft survival, we found that female recipients, MELD score, and the presence of hepatocellular carcinoma have negative effects on both patient and graft survival. As previously published by Busuttil and associates,²³ malignant disease and higher MELD at the time of the transplant (sicker patients) are associated with worse long-term patient survival in a multivariate analysis of 3200 orthotopic liver transplants. Rabbit antithymocyte globulin was an independent predictor of patient survival. Mangus and associates retrospectively assessed their outcomes of using different regimens of rATG in 1000 orthotopic liver transplants and likewise found that the use of rATG-based induction immunosuppression can be safely used in adult orthotopic liver transplant recipients with excellent survival and low rejection rates.²⁴

Our study has several limitations. First are the limitations inherent to retrospective and single center studies. Rabbit antithymocyte globulin was instituted in our program in 2005 in the face of a less expensive treatment (namely, IL-2R blockers), with the goal of introducing a flexible protocol that would address an expanding quality criteria standard for both donor and recipient and the goal of improving clinical outcomes. During this transition, the choice between 1 type of induction therapy over the other was based on clinical conditions at the time of transplant. Over time, rATG has been used on a more regular basis due to our observation of a lower rate of rejection and currently is the default induction agent at our center. However, during this time, there were no changes in the standard surgical technique or organ procurement/preservation, anesthesia technique, or perioperative management. We believe that the ability to apply these 2 immunosuppressive drugs in a stable program has led to important observations. Second, since the opening of our liver transplant program in 1990, we have always used some type of induction therapy; therefore, we do not have an induction therapy-free control group for comparison, except for what is published in the literature. Our rationale for using induction therapy in all liver transplant recipients is based on our practice of minimizing the maintenance immunosuppression therapy and therefore the well-known complications associated with its long-term use.

Despite these limitations, the observed effect of differential immunosuppression induction with reduced rejection rate and prolonged patient and graft survival stimulates novel and compelling hypotheses yet to be explored. Our center’s experience suggests that the use of rATG is associated with a lower incidence of acute rejection episodes compared with IL-2R blockers and has a positive effect on both patient and graft survival. This is the largest reported series of transplant patients comparing the use of IL-2R blocker with rATG. The use of rATG should be considered in patients before liver transplant. Larger multicenter studies with prolonged follow-up are necessary to confirm our findings and fully understand the long-term effects of rATG in patients undergoing liver transplant.

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