Author Affiliations: Department of Surgery, the University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong SAR, China (Au KP, Chan SC, Chok KSH, Sharr WW, Dai WC, Sin SL, Wong TCL and Lo CM)

Corresponding Author: See-Ching Chan, MD, Department of Surgery, the University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong SAR, China (Tel: +852-97565959; Email: seechingchan@gmail.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60391-5

Published online July 2, 2015.

 

 

Contributors: CSC proposed the study. AKP and CSC performed the research, collected and analyzed the data. All authors contributed to the interpretation of the results and writing the manuscript. AKP is the guarantor.

Funding: None.

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: With improvements in survival, liver transplant recipients now suffer more morbidity from long-term immunosuppression. Considerations were given to develop individualized immunosuppression based on their risk of rejection.

 

METHOD: We retrospectively analyzed the data of 788 liver transplants performed during the period from October 1991 to December 2011 to study the relationship between acute cellular rejection (ACR) and various clinical factors.

 

RESULTS: Multivariate analysis showed that older age (P=0.04, OR=0.982), chronic hepatitis B virus infection (P=0.005, OR= 0.574), living donor liver transplantation (P=0.02, OR=0.648) and use of interleukin-2 receptor antagonist on induction (P<0.001, OR=0.401) were associated with fewer ACRs. Patients with fulminant liver failure (P=0.004, OR=4.05) were more likely to develop moderate to severe grade ACR.

 

CONCLUSIONS: Liver transplant recipients with older age, chronic hepatitis B virus infection, living donor liver transplantation and use of interleukin-2 receptor antagonist on induction have fewer ACR. Patients transplanted for fulminant liver failure are at higher risk of moderate to severe grade ACR. These results provide theoretical framework for developing individualized immunosuppression.

 

(Hepatobiliary Pancreat Dis Int 2015;14:367-373)

 

KEY WORDS: liver transplantation; acute rejection; immunosuppression

Acute cellular rejection (ACR) affects 24%-80%^[1] of liver transplant recipients and is an important cause of graft dysfunction. The gold standard of diagnosing ACR is liver biopsy. Snover et al^[2] described the histological hallmark which comprised: 1) mixed but predominantly mononuclear portal inflammation; 2) sub-endothelial inflammation; and 3) lymphocytic cholangitis. The Banff schema^[3] graded ACR histologically depending on the extent of inflammatory infiltrates. Severe or persistent ACR could lead to irreversible loss of bile ducts and vasculatures, resulting in chronic rejection and eventually graft loss.

 

Calcineurin inhibitors, antimetabolites and corticosteroids comprise most of the current maintenance immunosuppressive regimens and are culprits of various medical complications.^[4] With the improvement of survival, long-term immunosuppression now emerges as the major cause of morbidity in liver transplant recipients. Hypertension and diabetes are 3 and 6 times more common in liver transplant recipients than in normal population.^[5] Chronic renal failure develops in 18% of transplant recipients in 5 years.^[6] De novo malignancies occur in 30% of transplant recipients after 10 years.^[7]

 

In contrast, consequences of ACR might not be as devastating. Chronic rejection is relatively uncommon in liver transplantation and is responsible for less than 5% of late graft loss.^[8] Concerns are raised on balancing risk of immunosuppression with risk of rejection. Efforts are made on minimizing immunosuppression and achieving immune tolerance.^[9] Risk profiles of transplant recipients might vary, and stratification of patients could allow safe reduction of immunosuppression in low risk patients. In this study, we investigated the clinical factors affecting rejection rate after liver transplantation.

 

 

From October 1991 to December 2011, 809 liver transplantations were performed in Queen Mary Hospital, the University of Hong Kong Medical Center, Hong Kong SAR, China. Twenty-one of those were retransplants and they were excluded from this study. Liver functions were monitored regularly after operation. Detecting a graft dysfunction would initiate a workup with serological tests and imaging. A liver biopsy was performed when other apparent causes of graft malfunction were excluded. ACR was diagnosed and graded with the Banff schema.^[3]

 

Review of the transplant database identified recipients with biopsy proven ACRs. They were categorized according to the histological grade of rejection, allowing a separate analysis for those with moderate to severe ACRs. Corresponding rejection rates in the first year after transplant were determined. Patient data were analyzed retrospectively to study the relationship between ACR and various clinical factors. Demographic characteristics included patient age and gender. The clinical conditions warranting liver transplantation were classified into cirrhosis with life-threatening complications, hepatocellular carcinoma, acute flare of chronic hepatitis, fulminant liver failure and others. Commonly listed life-threatening complications of cirrhosis were variceal bleeding, hepatic encephalopathy and spontaneous bacterial peritonitis. Etiology of liver disease was categorized into chronic hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, alcoholic liver disease, primary biliary cirrhosis (PBC), autoimmune hepatitis, cryptogenic liver disease, drug-induced liver failure, Wilson disease and others. Preoperative levels of serum creatinine, bilirubin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were included. Comparisons were made between living donor and deceased donor liver transplantation. The effect of various induction immunosuppressants was studied, including cyclosporin, tarcolimus, mycophenolate mofetil (MMF) and interleukin-2 (IL-2) receptor antagonist. The cyclosporin based induction immunosuppression was used in the early study period, and was gradually replaced by tarcolimus since 1996. MMF was added since 2001. From 2002 onwards, our refined regimen comprised IL-2 receptor antagonist, MMF and a reduced dose of tarcolimus.

 

Continuous normally distributed variables were expressed in mean±standard deviation. Patient age was also expressed in two age groups for comparison. Univariate analysis was performed with binary logistic regression. Significant factors affecting rejection rates were got into a multivariate analysis using multiple logistic regressions. Kaplan-Meier curves were plotted to visualize rejection-free survivals. All statistical calculations were performed with SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistical significance was defined by a P value less than 0.05.

 

 

788 liver transplantations were included into this study. The most common indication was cirrhosis with life-threatening complications (51.3%), whereas the most common etiology was chronic HBV infection (70.2%). During a median follow-up time of 80.1 months, 157 recipients suffered at least one episode of ACR, and 148 (94.3%) of which occurred in the first year. Rejection-free survival was shown on a Kaplan-Meier curve (Fig. 1). In the same period, there were 40 moderate to severe rejections. The first year incidence of all ACRs and moderate to severe ACR was 18.8% and 5.1%, respectively.

 

Univariate analysis was performed (Table 1). When ACR of all grades were considered, older age was a protective factor (P<0.001, OR=0.969, 95% CI: 0.953-0.986). When divided into two age groups by the median age of 50, the older group had a lower overall rejection rate (13.7% vs 24.2%, P<0.001, OR=0.498, 95% CI: 0.345-0.719). Patients with fulminant liver failure had more ACRs (P=0.004, OR=1.28, 95% CI: 1.08-1.51). Chronic HBV carriers were at lower risk of ACR (P<0.001, OR=0.496, 95% CI: 0.343-0.718), whereas patients with cryptogenic liver disease (P=0.03, OR=1.18, 95% CI: 1.03-1.37) and Wilson disease (P=0.009, OR=1.22, 95% CI: 1.05-1.41) were more likely to develop ACR. Patients who received liver grafts from living donors were less likely to develop ACR (15.6% vs 23.4%, P=0.006, OR=0.603, 95% CI: 0.421-0.864). The earlier cyclosporin-based induction immunosuppression was associated with higher rejection rates (P<0.001, OR=3.62, 95% CI: 1.94-6.78). In the later part of the study, the use of tarcolimus (P<0.001, OR=0.215, 95% CI: 0.155-0.403), MMF (P<0.001, OR=0.386, 95% CI: 0.265-0.562) and IL-2 receptor antagonist (P<0.001, OR=0.348, 95% CI: 0.237-0.511) on induction was associated with fewer rejections.

 

Upon multivariate analysis (Table 2), older recipient age (P=0.04, OR=0.982, 95% CI: 0.964-0.999), chronic HBV infection (P=0.005, OR=0.574, 95% CI: 0.389-0.845), living donor liver transplantation (P=0.02, OR=0.648, 95% CI: 0.446-0.939) and induction with IL-2 receptor antagonist (P<0.001, OR=0.401, 95% CI: 0.269-0.598) were independently associated with lower risk of developing ACR. The corresponding rejection-free survivals were plotted as Kaplan-Meier curves (Figs. 2-5).

 

Logistic regression was repeated for moderate to severe ACRs (Table 1). Univariate analysis revealed that fulminant liver failure (P=0.004, OR=1.42, 95% CI: 1.12-1.80) and cryptogenic liver disease (P=0.046, OR=1.25, 95% CI: 1.00-1.56) were risk factors of moderate to severe rejections. Multivariate analysis (Table 3) confirmed that fulminant liver failure was the only independent predictor of moderate to severe ACR (P=0.004, OR=4.05, 95% CI: 1.58-10.40). A Kaplan-Meier survival curve was provided (Fig. 6).

 

 

This study aimed to identify clinical risk factors of ACR, to provide a theoretical basis for individualized immunosuppressive therapy. Using the 1-year rejection rate, we demonstrated that older age was associated with fewer ACRs, which was consistent with other centers' experiences.^[10-12] Rejection rate was lower in patients older than 50 years of age. Aging is associated with the decline in cell-mediated immunity, particularly in T-cell function.^[13] ACR is predominantly cell-mediated,^[14] and T-cells play a crucial role. CD4⁺ T-helper cells recognize foreign allograft antigens and secrete IL-2, stimulating T-cell clonal expansion and differentiation, eventually evoking rejection effector mechanisms via activating CD8⁺ cytotoxic T-cells.^[14] Older liver graft recipients have lower CD8⁺ T-cell count,^[11] reflecting a relative immune tolerance. In elderly renal transplant recipients, lower rejection rates were also observed,^[15] but acute rejection per se had more effect on graft survival,^[15] comparing to younger recipients. The more forgiving liver grafts might allow reduced immunosuppression in older recipients without compromising graft survivals.

 

IL-2 is an essential pathway in T-cell activation targeted by immunosuppression. IL-2 receptor antagonist was introduced for kidney transplantation in 1998. It consists of monoclonal antibodies binding to the CD25⁺ ligand of IL-2 receptor.^[16] Competitive blockage of IL-2 signaling inhibits T-cell clonal expansion and differentiation, resulting in suppression of cell-mediated immunity. We started using basiliximab, a chimeric murine-human antibody, as an induction immunosuppressant since 2002. The new regimen reduced rejection rate from 33.0% to 14.6%. In addition to lowering rejection rates, the new immunosuppressive protocol, with omission of maintenance steroid and reduced dose of calcineurin inhibitor, reduced the incidence of diabetes and cytomegalovirus antigenemia.^[17]

 

Chronic HBV carriers had the lowest rejection rate among all etiologies of liver diseases. Multivariate analysis revealed that chronic HBV infection remained a protective factor which was consistent with previous studies.^{[18, 19]} HBV cirrhotic patients were analyzed separately from those with fulminant liver failure secondary to acute HBV infection, as they represent two distinct immune situations. The hepatitis phase of acute HBV infection is induced by intact immune response for viral clearance resulting in hepatocyte damage. On the contrary, chronic HBV infection is always associated with a viral induced immune tolerance. Most chronic HBV infections result from vertical transmission during the perinatal period. Transplacental transfer of maternal HBeAg induces a specific unresponsiveness of T-helper cells,^[20] leading to viral tolerance and persistence of HBV virus. This viral induced deficiency in cell-mediated immunity persists after liver transplantation,^{[21, 22]} resulting in suppressed allogeneic response.

 

HCV infection was associated with more ACR (21.3%). McTaggart et al^[23] reported HCV infection as an independent risk factor for ACR, and hypothesized a relation to recurrent HCV infection. It was suggested that HCV reactivation stimulates immune response against both HCV and liver allograft. Part of this relation could be due to a biopsy bias,^[24] as HCV reactivation produces biochemical abnormalities and triggers more biopsies. The bias is further reinforced by the histological similarities between recurrent HCV and ACR.

 

Our initial experiences^[25] in the 1990s indicated that rejection rates were similar between living donor and deceased donor liver transplantation. With more experiences and larger patient samples we observed that living donor liver transplantation was indeed associated with fewer ACRs than deceased donor liver transplantation. This is in concordance with observations in renal transplantation^[26] and is attributed to several factors. Liver grafts from healthy living donors are free from the stress induced by extensive physiological shifts in brain dead donors. In addition, the living donor operation is well coordinated with the simultaneous recipient operation, and there is a shorter cold ischemic time. However, the theoretical genetic advantage in living donor liver transplantation with fewer HLA mismatches has not been proven,^[27] unlike in renal transplantation.^[28] This is probably explained by liver graft being less immunogenic. Maluf et al^[29] observed a lower rejection rate in living donor liver transplantation, and reduced the immunosuppression in living donor liver graft recipients in the later part of their series. Steroid was tapered off earlier and dose of calcineurin inhibitor was reduced, thus resulting in fewer septic complications and improved survival.

 

Histological grades of ACR have prognostic implications. While mild ACRs seldom have long-term consequences, moderate to severe ACRs were more likely to progress to graft failure.^[30] Multivariate analysis confirmed that fulminant liver failure remained the only significant predictive factor of the event. 36.6% of fulminant liver failure patients receiving a liver graft developed ACR within the first year, 40% of them belonged to moderate to severe grade. This finding is less discussed in the current literature. Common causes of fulminant liver failure included drug-induced (36.6%) and cryptogenic liver disease (36.6%). Exclusion of etiological factors in multivariate analysis indicates that the tendency towards higher grade ACR probably relates more to the acuteness of the disease course. Fulminant liver failure is defined by the rapid development of liver dysfunction with hepatic encephalopathy in a patient without known liver disease.^[31] Hepatocyte damage is immune mediated, and elevated serum concentrations of inflammatory mediators, including IL-1, IL-6 and tumor necrosis factor -α, have been reported.^[32] The resultant hepatic necrosis releases more toxic products to keep the immune respond aroused. The rapid clinical course reflects a fulminant immune response, which might persist to reject the liver allograft after liver transplantation. As higher grade rejection and graft survival are concerned, this finding prompts more investigation towards stronger immunosuppression for patients transplanted for fulminant liver failure. 

 

The limitation of this study is its retrospective nature and some analyses could be open to bias. Protocolled biopsies were not performed in our center thus this study did not include subclinical rejection. The protective effect of IL-2 receptor antagonist could be confounded by a time factor, as IL-2 receptor antagonist was used in all liver transplant operations after 2002. It was difficult to analyze the therapeutic effect of maintenance immunosuppression as patients often switch from one immunosuppressant to other. Nonetheless, in this study we identified several clinical factors affecting ACR, and their implications could be answered by future prospective studies with protocolled biopsies.

 

This study comprised a multivariate analysis looking into the relationship between various clinical factors and ACR. The study showed that older recipient age, chronic HBV infection, living donor liver transplantation and induction with IL-2 receptor antagonist were associated with fewer ACRs, while patients with fulminant liver failure had more moderate to severe ACRs. These results provide theoretical framework for developing individualized immunosuppression.

 

 

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