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Seventy-two long-surviving liver transplant recipients were evaluated prospectively, including a baseline allograft biopsy for weaning off of immunosuppression. Thirteen were removed from candidacy because of chronic rejection (n=4), hepatitis (n=2), patient anxiety (n=5), or lack of cooperation by the local physician (n=2). The other 59, aged 12–68 years, had stepwise drug weaning with weekly or biweekly monitoring of liver function tests. Their original diagnoses were PBC (n=9), HCC (n=1), Wilson’s disease (n=4), hepatitides (n=15), Laennec’s cirrhosis (n=1), biliary atresia (n=16), cystic fibrosis (n=1), hemochromatosis (n=1), hepatic trauma (n=1), alpha-1-antitrypsin deficiency (n=9), and secondary biliary cirrhosis (n=1). Most of the patients had complications of long-term immunosuppression, of which the most significant were renal dysfunction (n=8), squamous cell carcinoma (n=2) or verruca vulgaris of skin (n=9), osteoporosis and/or arthritis (n=12), obesity (n=3), hypertension (n=11), and opportunistic infections (n=2). When azathioprine was a third drug, it was stopped first. Otherwise, weaning began with prednisone, using the results of corticotropin stimulation testing as a guide. If adrenal insufficiency was diagnosed, patients reduced to <5 mg/day prednisone were considered off of steroids. The baseline agents (azathioprine, cyclosporine, or FK506) were then gradually reduced in monthly decrements. Complete weaning was accomplished in 16 patients (27.1%) with 3–19 months drug-free follow-up, is progressing in 28 (47.4%), and failed in 15 (25.4%) without graft losses or demonstrable loss of graft function from the rejections. This and our previous experience with self-weaned and other patients off of immunosuppression indicate that a significant percentage of appropriately selected long-surviving liver recipients can unknowingly achieve drug-free graft acceptance. Such attempts should not be contemplated until 5–10 years posttransplantation and then only with careful case selection, close monitoring, and prompt reinstitution of immunosuppression when necessary.
Lifetime immunosuppression has been a presumed necessity after clinical whole-organ transplantation. However, we have suggested elsewhere that liver allograft acceptance without a need for maintenance immunosuppression may have been accidentally achieved more often than realized (1). The recently proposed concept that donor leukocyte migration and long-term microchimerism is the basis of allograft acceptance (2) would account for the slow evolution of the self-sustaining drug-free tolerance that has been most frequently seen in, but not confined to, liver recipients (3).
The possibility of drug weaning had previously been demonstrated by 6 noncompliant liver recipients who were found in April 1992 to have successfully discontinued all medications from 5 to 13 years previously (1), and by 5 more with EBV-associated B cell lymphomas whose drugs had been stopped 6 months to 8 years posttransplantation with subsequent drug-free survival for 0.5 to 2.8 years (4). The present prospective weaning study did not include these 11 earlier recipients, all of whom continue to be well with 1½ to 2½ more years of follow-up.
We describe here a prospective trial of drug weaning of 59 more long-surviving liver recipients, all of whom had complications of chronic immunosuppression. All patients reported with complete drug discontinuance have been observed subsequently for 6.5 to 22.5 months.
Between June 1992 and March 1994, 72 patients were evaluated for the weaning protocol. Candidacy criteria were: (1) ≥5 yr post-transplantation; (2) ≥ yr without episode of rejection; (3) history of medical compliance; (4) evidence of complications related to chronic immunosuppressive therapy; (5) primary physician cooperation; and (6) baseline liver biopsy showing no evidence of rejection or severe hepatic disease. Thirteen of the 72 patients were withdrawn from the candidacy list because of biopsy evidence of acute or chronic rejection (n=4), severe hepatitis on biopsy (n=2), patient anxiety (n=5), and refusal of cooperation by primary physician (n=2). The remaining 59 included 20 who were 12–20 years old when weaning was started, and 39 who were 21–68.
The drugs regimens from which weaning occurred were: azathioprine and prednisone (AZA/PRED) (n=10), cyclosporine and prednisone (CsA/PRED) (n=19), cyclosporine and azathioprine (CsA/AZA) (n=3), cyclosporine, azathioprine, and prednisone (CsA/AZA/PRED) (n=12), cyclosporine alone (CsA) (n=9) and tacrolimus (FK506, Prograf) (n=6). The 6 patients on tacrolimus had been converted from other regimens earlier in their course.
If AZA was part of triple therapy, it was weaned before beginning prednisone reduction, but if it was the principal immunosuppressant it was weaned in the same way as the other baseline drugs (see below). Patients still receiving prednisone were begun with a 25–50% steroid reduction. In the event of hepatoceliular enzyme elevation, no further changes were made until the enzymes stabilized. Further decrements in prednisone were at one-month intervals. Corticotropin stimulation testing was used to detect adrenal cortical insufficiency before complete steroid withdrawal. Corticotropin (.25 mg) was administered intravenously after obtaining a baseline serum cortisol level. Serum cortisol was then measured at 30 and 60 min post–corticotropin administration (5). Patients with normal adrenocortical function responded to stimulation with >7 µg/dl increase in serum cortisol.
Reduction of the baseline immunosuppressive agents, CsA, AZA, and tacrolimus (FK506, Prograf) also were considered every month. CsA was reduced by 10–25%/month until blood levels were <50 ng/ml for 3 months and then by 50%/month until cessation. A similar protocol was used for FK506.
Baseline enzyme measurements of AST, ALT, GGTP, and serum bilirubin were performed weekly or biweekly between the monthly decision points, as well as CsA and FK506 trough levels. Enzyme elevations or patient symptoms triggered an increased frequency of liver function tests, and a judgment about restoration of immunosuppression. In reaching a decision, the preweaning biopsy was invaluable for comparison with subsequent ones.
Microchimerism studies had been performed previously on 13 of the 59 patients, all of whom had donor cells or DNA demonstrated in peripheral tissues, or blood by the detection of Y chromosome in female patient recipients of male donor organs, or by the detection of donor specific HLA alleles—using cytostaining and polymerase chain reaction (PCR) techniques (1, 2). Because chimerism was invariably demonstrable in all of our long-surviving liver and kidney allograft recipients, it was assumed to be present in the rest of the cases of the present study and these tests were not performed.
Twenty-six patients were studied. To assess the development of donor-specific hypo reactivity, their peripheral blood mononuclear cells (PBMC) were tested in standard mixed lymphocyte reaction (MLR) against a panel of homozygous typing ceils (HTC) as previously described (6). These stimulator cells were selected based on their homozygosity for HLA-DR and in each assay HTCs for 7 different Dw specificities were included, with 2–3 HTCs per specificity. Each responder was tested with HTCs that identified “self” specificities, HTCs that defined the donor DR type, and unrelated control third party stimulators. The results were expressed as double normalized values (DNVs). Immune responsiveness was evaluated through mitogen induced proliferation. Isolated mononuclear cells from patients heparinized blood were cultured. Proliferation (i.e., responsiveness) was assessed by 3H-thymidine uptake after induction with phytohemagglutinin (PHA) and concanavalin A (Con A).
Failure of Weaning. After a follow-up of 6.5 to 22.5 months (mean 15 months) 15 (25.1%) of the 59 patients have had a diagnosis of rejection (Table 1). Three of the 15 were treated without pathologic documentation of rejection and were subsequently placed back on the weaning protocol. There were no graft losses, or examples of jaundice. All liver functions eventually returned to their preweaning values. The enzyme data and summarized in Figure 1.
Rejection in the 12 biopsy-confirmed cases (AJD) was diagnosed by the presence of portal triad cellular infiltrates causing duct damage, duct loss, or central venulitis. Two patients had histologic classification of moderate-to-severe rejection, and the 2 were converted to FK506. The other 10 rejections were classified as minimal-to-mild. When rejection was diagnosed, it was treated with 1 g methyl prednisolone and a 6-day prednisone cycle, starting at 200 mg on the first day and ending at 10 mg/day. Two patients were converted from CsA to FK506. One of these patients, who also required additional pulse steroid therapy, developed herpes keratitis that responded to acyclovir therapy and reduction of immunosuppression. This was the only serious complication in the trial.
Sixteen patients (27.1%) are off medications, including 2 kept on <5mg of prednisone due to an unresponsive corticotropin stimulation test. The other 28 (47.4%) are still at various phases of weaning.
Twenty patients, including some fully weaned and still being weaned, had enzyme elevations that spontaneously returned to normal despite further weaning or drug discontinuance (Fig. 2). The enzyme patterns were similar to those in the weaning failure group (compare with Figure 1). In those cases in which biopsies were obtained, the histopathologic findings were nonspecific, including the lobular reactivity or inflammation that is not diagnostic of rejection (7) (Table 2).
The most consistent improvement was the involution of verrucous warts. One patient with squamous cell carcinoma of the skin had dramatic improvement with virtual disappearance of lesions 6 months after completion of weaning. Preexisting hypertension and renal dysfunction were not affected (Table 3).
There was a high rate of weaning failure in patients previously treated with triple-or double-drug cyclosporine-based immunosuppression. Good results were obtained if prior treatment was with cyclosporine or with azathioprine/prednisone (Table 4).
Seven of the 13 patients proved in April–June 1992 to have donor leukocyte chimerism (1) have since been successfully weaned. Two more are still in the process, and 5 were returned to immunosuppression either because of biopsy evidence of rejection (n=4) or unconfirmed suspicion of this diagnosis (n=1).
Results from samples drawn before or close to the onset of weaning were equivocal in 10 of the 26 patients due to low reactivity to all panels or inadequate sampling, making the test impossible to interpret. Of the other 16.8 were reactive to the whole panel. Five have had episodes of rejection and 3 are still weaning (Table 5).
Eight patients were hyporeactive for donor-specific DR antigen only. Five of this group are off medications, 1 is still being weaned, and 2 failed weaning (Table 5).
The rationale for this trial was based in part on the previous observation in early 1992 that 15% of our liver recipients followed >10 years had discontinued all medications 5 to 13 years previously without developing rejection (1). With 2½ more years of follow-up, these 6 recipients, who for the most part had reached a drug-free status by noncompliance, are still well with 2½ more years follow-up. A further collection of 5 children whose physician-directed drug discontinuance .5 to 8 years posttransplantation was prompted by the development of Epstein-Barr–associated B cell lymphomas (4) continue to be stable and now have been drug-free for from 1 or to 3.3 years.
The prospective weaning trial herein reported has confirmed our suspicion that many long-term survivors after liver transplantation no longer need chronic immunosuppression at the prescribed levels, if at all. One-fourth of the patients entered have been able to discontinue immunosuppression altogether, half are still weaning without complications, and only a quarter have failed the effort. No hepatic grafts have been lost and none has suffered measurable damage. The only significant complication was in a patient who developed readily treated herpes keratitis. In 2 examples of histopathologically severe rejection, a safety net was provided by the “rescue” capabilities of FK506 (8) to which the patients were converted.
The need for close and continuance medical surveillance was evident. All of the rejections were signaled without the occurrence of jaundice by rises in the transaminase levels and in the canalicular tests of which the GGTP is the most specific. Confirmatory allograft biopsies were of the utmost importance for management decisions because enzyme rises during weaning were seen with equal frequency in allografts with and without evidence of rejection. There was no clear explanation for the transient enzyme increases in patients whose weaning was not interrupted. One possibility was that there was an unapparent and self-resolving rejection. Another might have been the loss during drug withdrawal of the hepatotrophic effects of either cyclosporine (9) or FK506 (10). Two of these nonrejecting patients have had waxing and waning of the enzyme activity during further weaning or drug stoppage.
Although it is too soon to be confident about the safety of the weaned patients, the demonstration of allograft stability in the previously reported drug-free patients for as long as 15 years is reassuring. In the presently reported prospective study, the peak risk appeared to have been passed by the sixth month. Early dividends of discontinuance of immunosuppression have already been seen, most frequently the involution of benign as well as malignant skin lesions. Disappointingly, there has been no improvement in preexisting arterial hypertension or renal dysfunction.
The subgroup of 13 patients with previously documented chimerism was of special interest. We have emphasized frequently our belief that this chimerism is a cardinal requirement for allograft acceptance and for the potential eventual evolution of donor specific nonreactivity (tolerance) but that it is not synonymous with either outcome. The proposition as originally stated was that “Clinical success—tolerance or graft acceptance—means that a characteristic lymphoid and dendritic cell chimerism has been introduced, which may be stable either without further treatment or only when continued immunosuppression is provided; an unstable graft and its migrated cells may either be rejected or cause GVHD” (2). The heterogeneity of results from weaning in these 13 cases conformed entirely with this central hypothesis. Although 9 of the 13 liver recipients have been able to stop drugs (n=7) or are successfully weaning (n=2), the onset of rejection required resumption of immunosuppression in the other 4.
As discussed in detail elsewhere (11), acceptance of whole-organ allografts and the achievement of a drug-free state are in fact mirror image events to those occurring after successful bone marrow transplantation in cytoablated recipients. The fundamental difference is that the trace leukocyte population in the David/Goliath cell relationship following whole-organ transplantation is donor rather than recipient, with the obvious implication that the principal risk is rejection rather than GVHD. Using this two-way paradigm of transplantation immunology, it is easy to understand the lengthy periods of immunosuppressive coverage that are usually necessary before a drug-free state can evolve. When bone marrow transplantation is from MHC-matched donors, continuous immunosuppression is frequently required for a year or more, and when there are 1 to 3 HLA allele mismatches, a period of 5 years or longer is needed before drugs can be stopped.
The liver transplant recipient has delineated an even more protracted time frame for solid-organ recipients of mismatched whole-cadaver organs (12). The risk in liver recipients of weaning attempts too early, too abruptly, or without frequent testing of allograft function have been described by Sanborn et al. (13) in a series of patients weaned from a cyclosporine-based triple-drug therapy. Moderate-to-severe rejection was frequently encountered in this series of 12 cases, leading to secondary complications after renewal of high-dose immunosuppression and eventually 2 deaths. The difficulty of weaning from cyclosporine when it was part of a triple-drug regimen also was noted in our experience.
Although this study was concerned only with liver allografts, we believe that the same trends can be found with other whole organs if these are systematically looked for. Five of our long-term recipients of living-related kidneys (not twins) have been off of immunosuppression for 1, 1.7, 14, 28, and 29 years (3). However, the lower density of chimerism in kidney and heart recipients undoubtedly makes weaning more hazardous than in the recipient of the inherently tolerant liver, and consequently this has been attempted only when there are immunosuppression-associated life-threatening complications (14).
The inability to accurately predict which patients can be successfully weaned means that all such attempts are on a trial-and-error basis, particularly when the donor leukocytes are unavailable for prognostic in vitro testing. Because we did not have donor lymphoid tissue in any of the liver weaning cases, we attempted to test recipient responsiveness to cells homozygous to the donor DR antigens as described by Reinsmoen et al. (6). A trend was noted of antidonor responsiveness in the rejection group and nonresponsiveness in patients successfully weaned, but this was imprecise. It has been pointed out before that even in vitro testing with donor cells does not correlate well with tolerance in animals (15–17) and humans (18, 19). This fundamental limitation of in vitro testing, rather than inherent imperfections in the surrogate technique of homozygous typing, shows that more predictive methods need to be developed.
1Presented at the 20th Annual Meeting of the American Society of Transplant Surgeons, May 18–20, 1994, Chicago, IL.
2Supported by Research Grants from the Veterans Administration and by Project Grant DK 29961 from the National Institutes of Health, Bethesda, MD.