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Long-Term Follow-Up of 23 Operational Tolerant Liver Transplant Recipients

Tryphonopoulos, Panagiotis; Ruiz, Phillip; Weppler, Debbie; Nishida, Seigo; Levi, David M.; Moon, Jang; Tekin, Akin; Velez, Madeline; Neuman, Danielle Rachel; Island, Eddie; Selvaggi, Gennaro; Tzakis, Andreas G.

doi: 10.1097/TP.0b013e3182003db7
Clinical and Translational Research
Free

Introduction. This is a follow-up of a withdrawal study that we previously performed on 104 liver transplant patients in which immunosuppression was gradually withdrawn over a period of 3 years. Eighty-one patients were not able to be withdrawn (rejectors), and 23 patients were successfully weaned off immunosuppression (tolerants).

Methods. In this study, we present their follow-up after the end of the withdrawal study: we compared the results of the tolerant patients (n=23) with those of the rejectors (n=81). Follow-up was until February 2010.

Results. Operational tolerant patients were off immunosuppression for an average of 7.27±0.28 years. Patient survival in the tolerant and the rejector groups was 63.66% and 74.25%, respectively (P=not significant). A patient in the rejector group received two retransplants for chronic rejection. In the rejector group, 19 patients presented 26 rejection episodes: clinically suspected (n=19) and biopsy-proven mild (n=4), moderate (n=2), and severe (n=1) rejection episodes. A tolerant patient had a moderate rejection episode of 5.3 years after immunosuppression withdrawal. In the rejector group, five patients received a kidney transplant and four more are on dialysis versus a tolerant patient on dialysis. Freedom from rejection in the tolerant and rejector groups was 95% and 73%, respectively (P<0.05), and freedom from renal replacement treatment was 83.33% vs. 44.58%, respectively (P=not significant).

Conclusions. Long-term outcomes of operationally tolerant liver transplant patients are at least as good as those of control patients. Operational tolerance is not a permanent state, and continuous vigilance is required to detect rejection episodes.

Division of Transplantation, Department of Surgery, University of Miami School of Medicine, Highland Professional Building, Miami, FL.

The authors declare no conflict of interest.

Address correspondence to: Andreas G. Tzakis, M.D., Ph.D., Liver/GI Transplant Program, University of Miami School of Medicine, Highland Professional Building, 1801 NW 9th Avenue, 3rd Floor, Miami, FL 33136.

E-mail: atzakis@med.miami.edu

Received 30 July 2010. Revision requested 20 August 2010.

Accepted 5 October 2010.

The advances of immunosuppression and postoperative follow-up in liver transplantation have contributed to an improvement of the long-term survival of liver transplant recipients. According to the last data provided by United Network for Organ Sharing, patient survival after liver transplantation is 71.3% to 72.7% at 5 years posttransplant (1). However, complications related to immunosuppression affect both long-term survival and the quality of life of these patients. Infectious complications, organ toxicity, and neoplasms are related to long-term maintenance immunosuppression and negatively affect the outcome of solid-organ transplants (2). Successful attempts to discontinue immunosuppression posttransplantation have been reported both in kidney and liver transplant recipients (3–12).

In this study, we will focus on a group of patients who participated in an immunosuppression withdrawal study that we performed in our center about 10 years ago (3). In that study, we attempted to wean off immunosuppression in 104 adult liver transplant recipients with the following characteristics: no autoimmune disease, stable graft function for at least 1 year before enrollment, and at least 3 years posttransplantation. Forty-five of these patients had received donor bone marrow infusion at the time of their transplant. Immunosuppression was withdrawn gradually by one third every year until complete withdrawal by the end of the third year of the study. Patients who had a biopsy-proven rejection or presented with a persistent increase of their liver function tests requiring treatment (steroids, increase in maintenance immunosuppression) were removed from the study along with the noncompliant patients. Of 802 patients who received a primary liver allograft between July 1990 and March 1998, 104 were enrolled in the withdrawal study, and 23 of them were successfully weaned off immunosuppression.

Twenty-one of them had their immunosuppression withdrawn at the first attempt, and two additional patients who presented initially rejection during the immunosuppression withdrawal were successfully withdrawn off immunosuppression during a second attempt. In this study, we present a long-term follow-up of this cohort of patients on how the immunosuppression-free state has influenced the outcome of their transplant.

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MATERIALS AND METHODS

We allocated these patients into two groups: a tolerant group (group 1 [G1]) and a rejector group (group 2 [G2]). Group 1 included patients who were successfully weaned off immunosuppression (n=23). Twenty of them had their immunosuppression withdrawn at the first attempt according to the study protocol, an additional patient who did not comply with the study procedures weaned off immunosuppression earlier than planned, and two patients who initially rejected were successfully withdrawn during a second attempt. Group 2 included patients where complete withdrawal of immunosuppression was not possible because of rejection (n=74) or noncompliance with the study procedures (n=7). The study was approved by the Institutional Review Board of the University of Miami. Follow-up was observed until February 2010.

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Statistical Methods

Comparisons of mean values were performed using standard t tests. Comparisons of the incidence of acute rejection, retransplantation, and death were time adjusted through the use of Kaplan-Meier curves and log-rank tests. Percentages were compared using the chi-square test and the Fisher's exact probability test.

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RESULTS

A total of 104 patients were enrolled in the study. Twenty-three of them (21 men and 2 women) were off immunosuppression and were enrolled in the tolerant group (G1). The remaining 81 (48 men and 33 women) were receiving maintenance immunosuppression and constituted the rejector group (G2). Patient demographics and primary disease characteristics are depicted in Table 1.

TABLE 1

TABLE 1

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Patient Characteristics

Tolerant Group (G1)

Primary liver disease of these patients included hepatitis C (n=12), cryptogenic cirrhosis (n=4), hepatitis B (n=3), Laennec's cirrhosis (n=3), and A1A deficiency (n=1). Twelve of these patients had received donor bone marrow infusions at the time of the transplant. The patients were followed up for an average of 7.27±0.28 years after the end of the withdrawal study.

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Rejector Group (G2)

Primary liver disease of these patients included hepatitis C (n=35), cryptogenic cirrhosis (n=12), hepatitis B (n=13), Laennec's cirrhosis (n=13), fulminant hepatic failure (n=4), Budd Chiarri syndrome (n=2), neuroendocrine tumor (n=1), and trauma (n=1). Thirty-three of these patients had received donor bone marrow infusions at the time of the transplant. The patients were followed up for an average of 7.81±0.28 years after the end of the withdrawal study.

There were no significant differences in the primary liver disease of these patients: more than half of the cases were due to viral hepatitis, followed by Laennec's and cryptogenic cirrhosis. There was also no difference if the patients had received donor bone marrow infusions or not at the early posttransplant period (Table 1). There were no significant differences regarding donor and recipient age at the time of transplant, severity of liver disease pretransplant, and intraoperative characteristics (warm and cold ischemia time and intraoperative blood transfusions). The gender of the patients was statistically significant: Twenty-one of the 23 patients off immunosuppression were male. In six patients in G1 versus two patients in G2, there was a better human leukocyte antigen (HLA) match between donor and recipient (≥2 AB antigens; Table 2).

TABLE 2

TABLE 2

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Patient Survival

Patient and Graft Survival

With a follow-up until the end of February 2010, there were 6 mortalities in G1 and 19 mortalities in G2 (Fig. 1a). In G1 (tolerant group), deaths were because of cardiovascular causes (n=4) (myocardial infarct [n=2], cerebrovascular accident postablation for atrial flutter [n=1], and pulmonary embolism [n=1]). Other mortalities included biliary obstruction by biliary stone-sepsis (n=1) and chronic rejection-sepsis (n=1) (Table 3). Average age of these patients at the time of death was 69.44±3.26 years.

FIGURE 1.

FIGURE 1.

TABLE 3

TABLE 3

In G2 (rejector group), deaths were because of cardiovascular causes (n=5) (myocardial infarct [n=3], cerebrovascular accident [n=1], and pulmonary embolism [n=1]), recurrent hepatitis C virus (n=4), pneumonia-sepsis (n=4), dialysis-sepsis (n=1), bile leak-sepsis (n=1), colon necrosis-sepsis (n=1), and lung, colon, and esophageal cancer (n=1 each). One patient in the rejector group was retransplanted successfully twice during this time period because of chronic rejection (Fig. 1b). Until the end of the follow-up period, patient survival in the tolerant and the rejector group was 63.66% vs. 74.25% and graft survival was 63.66% vs. 72.96%, respectively. The average posttransplant survival of the 6 patients who died in the tolerant group versus the 19 patients who died in the rejector group was 13.84±0.62 vs. 10.42±0.75 years (P=.03), respectively, and the average postwithdrawal study survival of the same patients was 7.41±1.02 vs. 5.84±0.29 years, respectively.

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Rejection Episodes

After the end of the withdrawal study, in G2, 19 patients presented 19 clinically suspected and 7 biopsy-proven mild (n=4), moderate (n=2), or severe (n=1) rejection episodes. In G1, a tolerant patient presented a moderate rejection 5.3 years after immunosuppression withdrawal and was restarted on maintenance immunosuppression (Table 3). All episodes were treated successfully. Freedom from rejection in the tolerant and rejector groups was 95% and 72.98%, respectively (P<.05; Fig. 1c).

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Renal Function

There was a trend for serum creatinine to be lower in tolerant transplant patients (P=not significant). In the rejector group, since the end of the withdrawal study, five patients received a kidney transplant and four more are on dialysis (Table 3). In the tolerant group, until the end of the follow-up period, one patient was on dialysis. Freedom from renal replacement in the tolerant and rejector groups was 83.33% vs. 44.58%, respectively (P=not significant; Fig. 1d). Two additional patients, one in the rejector group and one in the tolerant group, presented with acute deterioration of their renal function during hospitalization for sepsis and received short courses of dialysis before they died. In addition, 13 patients had sirolimus (n=7) or mycophenolate mofetil (n=7) added to their immunosuppressive regimen, and 5 more were converted to sirolimus/mycophenolate mofetil immunosuppression for nephrotoxicity. The posttransplant time until the renal replacement treatment of the patient who had dialysis in the tolerant group versus the 9 patients who had kidney transplant or dialysis in the rejector group was 13.9 vs. 9.67±1.2 years, respectively, and the average postwithdrawal study time until the renal replacement treatment was 7.95 vs. 4.17±1.1 years, respectively.

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Neoplasms

In the rejector group, 12 patients presented neoplasms since the end of the withdrawal study including squamous cell skin carcinoma (n=5), melanoma, uterine, colon, esophageal, pharynx and lung cancer (n=1 each), and prostate cancer (n=3) (Table 3). In the tolerant group, four patients presented with squamous cell skin cancer (n=1), throat (n=1), kidney (n=1), and lung cancer (n=1).

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Maintenance Immunosuppression

In the rejector group, immunosuppression was gradually decreased but not discontinued. At the end of the follow-up period, patients were on an average of 2.5 mg of tacrolimus daily dose with 13 of them receiving 1 mg or less/day.

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DISCUSSION

In our previous report (3), we focused on the effect of donor bone marrow infusion during gradual immunosuppression withdrawal in a selected group of patients. A the time we published that report, poststudy follow-up was 1 to 3 years for the patients who attained operational tolerance and for those who had rejection episodes. In this study with an average poststudy follow-up of almost 8 years (7.3 years for the tolerant group and 7.8 years for the rejector group), we evaluated the outcomes regarding safety, benefits, if any, derived for the tolerant patients from the discontinuation of immunosuppression, and the extend to which operational tolerance can be maintained.

In our study population, gender is a variable that seems to have an association with operational tolerance. Our operationally tolerant group included 91% (21/23) male patients versus 59.25% (48/81) in the rejector group. We do not know whether this is because liver transplant recipients are most commonly male (61% of liver transplants) (1) or there are other gender-related factors such as sex-linked genes that could provide greater proportions of regulatory processes that allow for easier removal of immunosuppression.

Donor-recipient HLA matching seems to have also a correlation with operational tolerance and immunosuppression withdrawal in our study group. In six patients in the tolerant group (6/23, 26%), there was a better HLA match between donor and recipient (two or more common AB antigens) versus two patients in the rejector group (2/81, 2.5%), and this seemed to also improve the chances of removing immunosuppressive drugs from these patients. In theory, this is likely due to lower antidonor responses when there is greater HLA matching, and thus, the need for immunosuppression is less in these patients.

Both in the tolerant and the rejector group, approximately 50% of the patients had received donor bone marrow infusion at the time of the transplant procedure. This indicates that in our group of patients, bone marrow infusion had no obvious beneficial effect regarding immunosuppression withdrawal. We believe this is because despite the bone marrow infusion, robust levels of chimerism were never achieved in the peripheral blood. As we have previously published (3), peripheral blood chimeric levels were less than 1% in the patients who had received donor bone marrow infusions and 10 times less in the patients who did not receive any bone marrow at all. The origin of the scarce chimeric cells in the latter case was from the donor liver.

At this point, the critical variables that allow certain persons to be successfully withdrawn are not precisely known. There is an overall array of host factors that affect the ability to attain operational tolerance, likely interrelated in a complicated biologic algorithm that yet has to be clarified. Both groups, tolerants and rejectors, presented with morbidity and mortality complications during the follow-up period. Although there was no difference in patient survival (63.66% vs. 74.25% in the tolerant and rejector group respectively), our data suggest that mortalities in the rejector group occurred earlier than in the tolerant group. The average posttransplant survival of the 6 patients who died in the tolerant group versus the 19 patients who died in the rejector group was 13.84±0.62 vs. 10.42±0.75 years (P=0.03), respectively, and the average postwithdrawal study survival of the same patients was 7.41±1.02 vs. 5.84±0.29 years, respectively. The increased number of deaths because of cardiovascular reasons can also been explained from the advanced age of this group of patients at the time of death.

Renal function outcomes were not significantly different between the two groups; however, our data suggest that in our patient sample, intervention (dialysis or kidney transplant) for end-stage renal disease occurred earlier in the rejector group than in the tolerant group. The posttransplant time until the renal replacement treatment of the patient who had dialysis in the tolerant group versus the nine patients who had kidney transplant or dialysis in the rejector group was 13.9 vs. 9.67±1.2 years, respectively, and the average postwithdrawal study time until the renal replacement treatment was 7.95 vs. 4.17±1.1 years, respectively. Patients in the tolerant group were also exposed to immunosuppression treatment and its subsequent nephrotoxicity for a period of approximately 5 years from the time of the transplant until withdrawal was achieved. Many of the patients had also concomitant diseases such as hypertension or diabetes that aggravated even more their kidney function. Immunosuppression withdrawal may delay the progression of kidney disease but could not reverse it. This observation raises also the question of immunosuppression minimization and withdrawal as early as possible after liver transplantation to avoid long-term exposure and organ damage from calcineurin inhibitor toxicity.

There was no difference in the occurrence of neoplasms between the two groups. As with the other immunosuppression complications, the absence of significant differences might be due to the small sample size of our study and also because all patients were exposed to immunosuppression for a significant amount of time, more than a decade in the rejector group, approximately 5 years in the tolerant group. Neoplasms because of the immunosuppressed state might have been formed during that period of time and were clinically diagnosed later on.

Patients in the tolerant group presented with significantly less rejection episodes. More important, operational tolerance is not a permanent state; one patient presented with a moderate rejection after being immunosuppression free for more than 5 years. We do not know what was the triggering event that caused the rejection episode. The medical history and clinical examination of the patient at that time did not reveal any precipitating causes, no other coexisting viral diseases, or other diseases that could be responsible for an increased immune reactivity. This event brings up the safety issue of immunosuppression withdrawal. Vigilance and continuous follow-up are required to avoid catastrophes. Operationally tolerant patients have to continue their follow-up regularly, and the transplant team has to be always vigilant for a rejection episode. In our study, we implemented a rigorous follow-up of these patients to ensure their safety. One operationally tolerant patient who presented rejection was timely detected and treated successfully.

Not all patients were successful during their first attempt. Immunosuppression minimization and rejection or successful withdrawal is a two-way phenomenon, as we learned from the two patients who rejected during the initial immunosuppression withdrawal and were successful at their second attempt and from the patient who was tolerant for a period of 5 years before presenting a rejection episode.

It would be most beneficial for the safety of the patients to develop a screening assay to suggest which patient would be a good candidate for immunosuppression withdrawal and which one would most probably reject. Studies of operationally tolerant patients (8–10) have indicated that these patients share a tolerance genetic fingerprint. As in our series, we had patients who rejected initially before developing operational tolerance and vice versa, serial sampling and longitudinal study of this fingerprint overtime may elucidate mechanisms related to the tolerance state.

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CONCLUSIONS

In our long-term follow-up of operationally tolerant liver transplant patients, we did not observe any statistical significance in the outcomes, compared with a control group who failed to wean off immunosuppression. The safety of the patients was not compromised being in the tolerant state because of the rigorous follow-up for early detection and timely treatment of rejection episodes, and their outcomes were at least as good as the ones of the control group. The lack of a difference in the outcomes might be due to the small study sample and to the fact that tolerance was induced late posttransplantation. The tolerant patients were exposed to the deleterious effects of immunosuppression for a long period of time before withdrawal.

Both patients and transplant teams should always remember that tolerance, when achieved, is not a permanent state, and continuous vigilance and follow-up are required to detect rejection episodes. The mechanisms of operational tolerance are not yet elucidated. Multicenter longitudinal immunosuppression withdrawal trials might help us to better understand this phenomenon.

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REFERENCES

2. Ojo AO, Held PJ, Port FK, et al. Chronic renal failure after transplantation of a nonrenal organ. N Engl J Med 2003; 349: 931.
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11. Braud C, Baeten D, Giral M, et al. Immunosuppressive drug-free operational immune tolerance in human kidney transplant recipients: Part I. Blood gene expression statistical analysis. J Cell Biochem 2008; 103: 1681.
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Keywords:

Liver transplantation; Immunosuppression withdrawal; Tolerance

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