Key words : End-stage liver disease, Immunosuppression therapy, Liver transplantation

Introduction

Liver transplant (LT) is now universally recognized as the only treatment option for end-stage liver disease. Advances in surgical technique, perioperative mana-gement, and immunosuppression therapy have resulted in excellent outcomes for graft and patient survival in the short-term.^1,2

Despite recent advances in LT that have resulted in significantly increased rate of patient survival, postoperative rejection remains a persistent problem. Also, a small number of patients experience chronic rejection (CR) due to immune-mediated insults to the parenchyma that cause damage to vessels and bile ducts. Chronic rejection is diagnosed by clinical, laboratory, histological, and radiological criteria; among these, histologic recognition of CR is a critical step in the diagnosis, and the Banff Working Group is the most widely accepted histology criteria for CR. Generally, CR manifests as a broad spectrum of symptoms such as fatigue, abdominal pain, and fever. The laboratory data may appear unremarkable until presentation of liver failure, with presentation of jaundice, coagulopathy, and malnutrition.^3,4

The incidence of CR after LT tends to decrease over time (from 15%-20% to 2%-5%) after 5 years, which may be the result of the introduction of new powerful immunosuppression regimens, such as tacrolimus-based therapy.³ However, the important point is that CR can only be successfully controlled in the early stages; otherwise, retransplant may be required to avoid irreversible injury, including graft loss, liver failure, and death. In addition, CR is more common in patients with multiple episodes and/or more severity of acute rejection, as well as patients who undergo retransplant to treat autoimmune disease. There are probably some other risk factors that may play a role in CR, such as female recipients paired with male donors, higher donor age, low serum level or nonadherence of immunosup-pression, longer cold ischemia time, and genetically unrelated donors.^5-7

Identification of CR risk factors in transplant recipients is critical for understanding the etiology of CR and may facilitate prevention of further injury and graft loss.⁸ Therefore, we investigated the characteristics of LT recipients who had experienced CR and the associated risk factors versus a control group without CR.

Materials and Methods

In this retrospective case-cohort study, we reviewed the medical reports of 3022 LT recipients at Abu-Ali Sina Hospital (Shiraz Organ Transplant Center) between April 2011 and March 2018. Adult patients who had undergone LT >3 years previously were enrolled. Of these patients, 80 had experienced CR. Also, 98 LT recipients who did not have CR were included as a control group. The control group was selected randomly from all patients.

Data were collected by retrospective medical chart review and included demographic charac-teristics of recipients, as well as donor, type of graft, underlying liver disease, duration between LT and CR diagnosis, frequency of acute rejection, use of immunosuppression therapy, and clinical and para-clinical laboratory data. The study protocol was previously approved by the Ethical Committee of Shiraz University of Medical Sciences (No. IR.SUMS.MED.REC.1400.002).

Immunosuppressive regimen
Except for patients with pretransplant diagnoses of autoimmune liver disease, all patients were treated with standard immunosuppression therapy of tapered corticosteroids within 6 to 12 months after LT. For the first 24 to 36 months, all patients were given tacrolimus and mycophenolate mofetil. When the liver function was stable, immunosup-pression was reduced. Patients were weaned from mycophenolate mofetil, and tacrolimus was continued as monotherapy in the second or third year. In our protocol for patients with autoimmune liver disease, we taper immunosuppression but never discontinue prednisolone, and all patients receive at least 5 mg prednisolone daily after 2 years.⁹

Diagnosis and management of rejection
All LT recipients were monitored daily by liver function tests for the first 3 weeks after LT, followed by monthly checks until the end of the first posttransplant year. Changes in each parameter were important to the transplant team, but the final confirmation of rejection was based on changes in alanine aminotransferase (ALT) and patient symptoms such as fever, purities, and jaundice.

During patients’ follow up, if there were ALT elevations, but less than 3 times of previous patient’s liver enzyme, no further reduction in immunosup-pression drugs was ordered by the physician. In these patients, liver function tests were repeated 7 to 14 days later. If the ALT level rose to 3 times or more above the patient’s previous liver enzyme finding, then liver biopsy was performed to confirm allograft rejection; also, immunosuppression was increased until the results of biopsy were available.

We used the updated Banff criteria for the diagnosis and staging of CR. Damage to intrahepatic vessels and bile ducts was seen in CR.^4,10,11

For acute rejections, we used pulse therapy with methylprednisolone; in the case of humoral rejection, plasmapheresis or intravenous immunoglobulin was applied. We used antithymocyte globulins if the rejection course did not resolve. When CR is suspected, we modify the immunosuppression maintenance regimen based on clinical status of patients and a liver function test.

Statistical analyses
The Shapiro-Wilk test was used to evaluate the normal distribution. For continuous and categorical variables, data are presented as mean values (±SD) and number (%), respectively. The chi-square test and t test were used for comparisons between the 2 groups. The Kaplan-Meier method with log-rank test and the Cox proportional hazard regression model were performed for survival analysis. In this model, CR was considered as an event. We used the forward condition method for variable selection and entered the clinically significant We used SPSS software (version 16.0) for statistical analyses. P < .05 was considered statistically significant.

Results

Demographic and baseline data
There were 3022 patients enrolled in this registry-based cohort study. The age of the patients ranged from 18 to 74 years, with a mean age of 43.25 ± 13.6 years. The range of follow-up time was 4 to 10 years, with a median of 7 years. All patients as a group constituted a 20 709 person-year. Of these cases, 80 patients had CR episodes; thus, the incidence rate of CR was 3.86 per 1000 person-year. Overall, these 80 patients were selected as the case group who had experienced CR, and 98 patients selected randomly from remaining patients were considered as the control group without CR.

The mean age of recipients with CR was 37.02 ± 14.06 years versus 43.57 ± 13.81 years in the control group. The ages of recipients and the donors were significantly lower in the CR group versus the control group (P = .003 and P = .049, respectively).

The most common underlying reason for LT in the CR group was autoimmune hepatitis overlapped with primary biliary cirrhosis (n = 20; 25.0%) followed by viral hepatocellular carcinoma (n = 17; 21.3%) and primary sclerosing cholangitis (n = 16; 20.0%). In the control group, the most common etiologies were viral hepatocellular carcinoma (n = 31; 31.6%). The difference between the 2 groups regarding the underlying disease was significant (P = .006).

Our results showed that most recipients in the CR group were men (n = 59; 73.8%) (significantly different vs the control group; P = .030). Also, sex concordance was higher in the CR group (61.3%) versus the control group (41.8%), and the difference was significant (P = .01). There were no male donors in the control group; however, most donors in the CR group were men (59 or 80.8%), and the difference between the 2 groups was significant (P < .001). All organs were from deceased donors.

A total of 71 patients (88.8%) in the CR group had experienced at least 1 acute rejection episode; however, only 39 patients (39.8%) in the control group had experienced at least 1 episode of acute rejection. The difference between the 2 groups for acute rejection frequency was significant (P < .001). Also, cold ischemia time was longer in patients from the CR group versus the control group (P = .031).

We observed no significant differences between the 2 study groups in the Model for End-Stage Liver Disease (MELD) score (P = .905), type of organ donation (P = .855), recipient and donor blood group (both P > .05), warm ischemia time (P = .214), and hepatic arterial thrombosis (P = .091). Demographic and baseline data of both groups are listed in (Table 1).

Drug consumption and laboratory data
The CR group showed a statistically higher percentage of immunosuppression use versus the control group. Use of drugs such as prednisolone (P < .001), tacrolimus (P = .008), mycophenolate mofetil (P < .001), sirolimus (P < .001), everolimus (P = .005), and cyclosporine (P < .001) was significantly different between the CR group and the control group. In addition, previous and recent laboratory data as well as changes in these data were compared between the 2 groups. The levels of aspartate aminotransferase and ALT and the changes in these data from previous results were significantly different between the 2 groups, with lower levels in the control group. Also, levels and changes of alkaline phosphatase and total bilirubin were significantly different between the 2 groups. Details of drug consumption and laboratory data in both groups are listed in (Table 2).

Outcomes
Although the frequency of death was higher in the CR group versus the control group (32.5% vs 23.5%, respectively), this was not significant (P = .180). Also, there was no statistically significant difference in patient survival between the 2 groups (log rank) (Figure 1). The data for multi-year survival versus rejection are displayed in (Figure 2). The median rejection time was 2.90 years after LT. In addition, hospital readmission was more common in the CR group (P < .001). As a result of disease progression, retransplant was more frequent in the CR group, but the difference was not significant (P = .142).Significant demographic, baseline, and laboratory data were analyzed with the Cox proportional hazard regression model. Model results showed that CR was significantly associated with recipient sex (hazard ratio 3.2, 95% CI 1.77-6.08; P < .001) and sex concordance between the recipient and donor (hazard ratio 2.93, 95% CI 1.67-5.13; P < .001). The results of the Cox proportional hazard regression model on CR are summarized in (Table 3).

Discussion

Identification of risk factors associated with CR in LT recipients is crucial for introduction of preventive interventions and strategies.⁸ In this retrospective cohort study of 3022 patients, we investigated the characteristics of LT recipients who had experienced CR and the risk factors for CR.

The most common underlying reason for LT in the CR group was autoimmune liver disease. Although the difference between the 2 groups regarding the underlying disease was significant, the underlying disease was not shown to be an independent risk factor. Previous studies have reported some risk factors for CR, including autoimmune liver disease, nonadherence with immunosuppression regimens, cyclosporine-based immunosuppression therapy (versus tacrolimus-based regimens), donor-recipient sex mismatch, recipient and donor age, cold ischemia time, living versus deceased donor LT, and number and severity of acute rejection episodes.^4,7,12

Our results showed that recipients and donors were significantly younger in the CR group than in the control group. In addition, significantly more patients with CR were men versus that shown in the control group. Also, sex concordance was more prevalent in the CR group, with a significant difference between groups.

Although there were no male donors in the control group (without CR), most donors in the CR group were men; the difference between the 2 groups was significant. Our study also showed that male LT recipients from female donors were the most prevalent group that had not experienced CR. It should be noted that we did not match the case group with the control group for sex and age of the patients; therefore, conclusions drawn from these differences should be done with clinical judgment and precaution.

With improvements in immunosuppression regi-mens, the incidence of CR has dropped dramatically in recent decades. Following LT, adequate immuno-suppression therapy is defined as a regimen that maintains a viable graft and a healthy patient while balancing the risk of rejection and the risk of immunosuppression-related adverse side effects. Nonetheless, various immunosuppression strategies are presently in use, which are given depending on a variety of variables such as the recipient’s renal function, cardiovascular disease, risk of infections and tumors, and recurrence of underlying liver disease. However, there is no unique definition for the optimal immunosuppression regimen; rather, therapy should be tailored to each recipient individually.⁴ According to our results, there was a statistically significant difference between the 2 groups for use of prednisolone, tacrolimus, mycophenolate mofetil, sirolimus, everolimus, and cyclosporine . We observed that patients in the CR group were more likely to receive immunosuppression and more likely to receive higher doses of immunosuppression. These results may be because of changes in treatment based on laboratory data before pathology confirmation of CR, and so we cannot conclude that there is a relationship between immunosuppression regimen and CR.

Tacrolimus based regimens prevented rejection in our patients, but we did not find any protective effect for tacrolimus in our study by multivariate analysis. However, as mentioned earlier, tacrolimus has proved to be a potent immunosuppressive agent in LT for prevention of CR. The conversion from cyclosporine to tacrolimus is associated with a 70% response rate and graft survival.^4,5,7 Nonetheless, immunosuppressive drugs have 2 dimensions: improved survival rate and known adverse side effects. Calcineurin inhibitor drugs such as cyclosporine and tacrolimus have been chosen for avoidance of rejection.^13,14

In our study, most patients with CR (88.8%) had at least 1 episode of acute rejection; however, nearly 40% of patients without CR had at least 1 episode of acute rejection, and the difference in acute rejection frequency between the 2 groups was significant. However, frequency of at least 1 occurrence of acute rejection was reported as up to 35%. In previous studies, a history of repeated acute rejection episodes has been identified as a risk factor associated with the development of CR.^12,15,16 Dogan and colleagues did not find acute rejection as cause of graft failure; also, time of acute (early or late) rejection did not have any effect on graft survival.¹⁷ The entity of CR is not well understood. There may be a sophisticated and multidimensional explanation for occurrence of CR, and perhaps CR is caused by diverse immunological processes.¹⁶ The differences in reported acute rejection may be due to differences in the immuno-suppression protocols, different criteria for acute rejection, and various follow-up times.

Although the frequency of death was higher in the CR group versus the control group in this study, this difference was not significant. Also, there was no significant difference in patient survival between the 2 groups. Rana and colleagues recognized rejection as reason for death among 1.7% of patients, which suggests that CR is a very uncommon cause of death with regard to long-term complications of immunosuppression.¹⁸

Strengths and limitations
This study is the first study in Shiraz Organ Transplant Center to investigate the factors as-sociated with CR, the incidence rate, and mortality. In addition, compared with previous studies on smaller populations with shorter follow-up periods, our study was conducted with larger control and rejection groups with a longer average duration of follow-up (7 years). However, the weaknesses of our study include the retrospective design and the lack of some data due to incomplete follow-ups.

Conclusions

We observed that sex and sex concordance were independent factors associated with CR. Most patients with CR responded to medical treatment, and the rate of graft loss was low among our recipients.

References:

1. Jadlowiec CC, Taner T. Liver transplantation: current status and challenges. World J Gastroenterol. 2016;22(18):4438-4445. doi:10.3748/wjg.v22.i18.4438
   CrossRef - PubMed
2. Adam R, Karam V, Cailliez V, et al. 2018 Annual Report of the European Liver Transplant Registry (ELTR): 50-year evolution of liver transplantation. Transpl Int. 2018;31(12):1293-1317. doi:10.1111/tri.13358
   CrossRef - PubMed
3. Demetris AJ, Bellamy C, Hubscher SG, et al. 2016 Comprehensive update of the Banff Working Group on Liver Allograft Pathology: introduction of antibody-mediated rejection. Am J Transplant. 2016;16(10):2816-2835. doi:10.1111/ajt.13909
   CrossRef - PubMed
4. Angelico R, Sensi B, Manzia TM, et al. Chronic rejection after liver transplantation: opening the Pandora’s box. World J Gastroenterol. 2021;27(45):7771-7783. doi:10.3748/wjg.v27.i45.7771
   CrossRef - PubMed
5. Ali MA, Elshobari MM, Salah T, et al. Impact of donor-recipient genetic relationship on outcome of living donor liver transplantation. Liver Transpl. 2017;23(1):43-49. doi:10.1002/lt.24599
   CrossRef - PubMed
6. Choudhary NS, Saigal S, Gautam D, et al. 4. Outcome of living donor liver transplantation between genetically related and unrelated donors: experience of 1372 adult living donor liver transplantations from a single centre. J Clin Exp Hepatol. 2018;8(Suppl 1):S86. doi:10.1016/j.jceh.2018.06.430f
   CrossRef - PubMed
7. Choudhary NS, Saigal S, Bansal RK, Saraf N, Gautam D, Soin AS. Acute and chronic rejection after liver transplantation: what a clinician needs to know. J Clin Exp Hepatol. 2017;7(4):358-366. doi:10.1016/j.jceh.2017.10.003
   CrossRef - PubMed
8. Gupta P, Hart J, Cronin D, Kelly S, Millis JM, Brady L. Risk factors for chronic rejection after pediatric liver transplantation. Transplantation. 2001;72(6):1098-1102. doi:10.1097/00007890-200109270-00020
   CrossRef - PubMed
9. Shamsaeefar A, Nikoupour H, Kazemi K, et al. Comparison of the characteristics of adult liver transplant recipients with prope (almost) tolerance and full immunosuppression regimen. Transpl Immunol. 2021;68:101440. doi:10.1016/j.trim.2021.101440
   CrossRef - PubMed
10. Neil DA, Hubscher SG. Current views on rejection pathology in liver transplantation. Transpl Int. 2010;23(10):971-983. doi:10.1111/j.1432-2277.2010.01143.x
    CrossRef - PubMed
11. Krenzien F, Keshi E, Splith K, et al. Diagnostic biomarkers to diagnose acute allograft rejection after liver transplantation: systematic review and meta-analysis of diagnostic accuracy studies. Front Immunol. 2019;10:758. doi:10.3389/fimmu.2019.00758
    CrossRef - PubMed
12. Tannuri AC, Lima F, Mello ES, Tanigawa RY, Tannuri U. Prognostic factors for the evolution and reversibility of chronic rejection in pediatric liver transplantation. Clinics (Sao Paulo). 2016;71(4):216-220. doi:10.6061/clinics/2016(04)07
    CrossRef - PubMed
13. Tasdogan BE, Ma M, Simsek C, Saberi B, Gurakar A. Update on immunosuppression in liver transplantation. Euroasian J Hepatogastroenterol. 2019;9(2):96-101. doi:10.5005/jp-journals-10018-1301
    CrossRef - PubMed
14. De Simone P, Carrai P, Coletti L, Ghinolfi D, Petruccelli S, Filipponi F. Modification of immunosuppressive therapy as risk factor for complications after liver transplantation. Best Pract Res Clin Gastroenterol. 2017;31(2):199-209. doi:10.1016/j.bpg.2017.03.001
    CrossRef - PubMed
15. Jang JK, Kim KW, Choi SH, et al. CT of acute rejection after liver transplantation: a matched case-control study. Eur Radiol. 2019;29(7):3736-3745. doi:10.1007/s00330-018-5971-4
    CrossRef - PubMed
16. Ronca V, Wootton G, Milani C, Cain O. The immunological basis of liver allograft rejection. Front Immunol. 2020;11:2155. doi:10.3389/fimmu.2020.02155
    CrossRef - PubMed
17. Dogan N, Husing-Kabar A, Schmidt HH, Cicinnati VR, Beckebaum S, Kabar I. Acute allograft rejection in liver transplant recipients: incidence, risk factors, treatment success, and impact on graft failure. J Int Med Res. 2018;46(9):3979-3990. doi:10.1177/0300060518785543
    CrossRef - PubMed
18. Rana A, Ackah RL, Webb GJ, et al. No gains in long-term survival after liver transplantation over the past three decades. Ann Surg. 2019;269(1):20-27. doi:10.1097/SLA.0000000000002650
    CrossRef - PubMed