Rabbit Anti-Thymocyte Globulin in Liver Transplantation: All That Glitters is Not Gold, but 1000 Patients are So Many to Dazzle

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1 LIVER TRANSPLANTATION 18: , 2012 EDITORIAL Rabbit Anti-Thymocyte Globulin in Liver Transplantation: All That Glitters is Not Gold, but 1000 Patients are So Many to Dazzle Marina Berenguer 1,2,3 and Jose-Antonio Pons 4,5,6 1 Liver Transplantation and Hepatology Unit, La Fe Hospital, Valencia, Spain; 2 Network Center for Biomedical Research in Hepatic and Digestive Diseases, Valencia, Spain; 3 Faculty of Medicine, University of Valencia, Valencia, Spain; 4 Virgen de la Arrixaca Hospital, Murcia, Spain; 5 Network Center for Biomedical Research in Hepatic and Digestive Diseases, Murcia, Spain; and 6 Faculty of Medicine, University of Murcia, Murcia, Spain Received February 10, 2012; accepted March 11, See Article on Page 786 With increasing survival rates (73%-74% and 68%- 69% on average at 5 years for patient and graft survival, respectively), liver transplantation (LT) has become the standard of care for patients with endstage liver disease. The development of effective immunosuppressive drugs and particularly calcineurin inhibitors (CNIs) has been essential to this success, 1,2 and despite known limitations, they continue to be the first-line therapy in organ transplantation. In 2008, almost 98% of LT recipients in the United States received CNIs at hospital discharge. 3 With the increasing number of LT procedures and the longer follow-up of survivors as well as the recent increase in the number of suboptimal candidates, 4 a greater awareness of these limitations (particularly nephrotoxicity) has rapidly evolved. 5-7 Two major approaches have been assessed to minimize morbidity and mortality. The first strategy, which has mostly been orchestrated by pharmaceutical companies, is the development of new agents that lack CNI-related side effects, such as belatacept and sotrastaurin. Although the drugs are good in that respect, they have not fulfilled expectations in terms of overall survival benefits. 8,9 Whether these agents will be potent enough to allow CNI avoidance is unclear. The second strategy is the improvement of current immunosuppression protocols in order to minimize CNI exposure without compromising survival. The reduction of CNI doses (with or without delays) and the use of nonnephrotoxic agents [eg, mycophenolate mofetil (MMF), mammalian target of rapamycin inhibitors, and antibody induction] have been investigated 6,7,10,11 with acceptable (though not striking) results. In this issue of Liver Transplantation, Mangus et al. 12 present their experience with rabbit anti-thymocyte globulin (ratg) based immunosuppression induction in 1013 LT recipients followed for a median of 63 months. The authors compare 3 immunosuppression eras in which ratg induction (3 doses for a total of 6 mg/kg) was used according to different protocols together with tacrolimus (TAC) monotherapy (started on postoperative day 2). In the first era ( , n ¼ 166), the first dose of ratg was administered perioperatively; in the second era ( , n ¼ 259), the first ratg dose was delayed until 48 hours after transplantation; and in the third era Abbreviations: AZA, azathioprine; BAS, basiliximab; CNI, calcineurin inhibitor; CY, cyclosporine; DAC, daclizumab; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; LT, liver transplantation; MELD, Model for End-Stage Liver Disease; MMF, mycophenolate mofetil; ND, not determined; ratg, rabbit anti-thymocyte globulin; ST, steroid; TAC, tacrolimus; UNOS, United Network for Organ Sharing. The Network Center for Biomedical Research in Hepatic and Digestive Diseases is funded by the Carlos III Institute of Health. Address reprint requests to Marina Berenguer, M.D., Ph.D., Liver Transplantation and Hepatology Unit, La Fe Hospital, C/Bulevar sn, Valencia, Spain Telephone: þ ; Fax: ; mbhaym@teleline.es DOI /lt View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI /lt. Published on behalf of the American Association for the Study of Liver Diseases VC 2012 American Association for the Study of Liver Diseases.

2 756 BERENGUER AND PONS LIVER TRANSPLANTATION, July 2012 ( , n ¼ 588), ratg was also delayed and was combined with a single dose of rituximab 72 hours after transplantation. The TAC trough levels were 7 to 10 ng/ml in the first 3 months and 6 to 8 ng/ml thereafter. Less than 5% of the patients required additional immunosuppressive agents during their follow-up, one-third of the hepatocellular carcinoma (HCC) cases were outside the Milan criteria, and posttransplant protocol biopsy was performed at 4 and 12 months. The results were similar to data from large databases: patients who underwent transplantation more recently differed from transplant patients in earlier eras, with increases in the number of patients with HCC, in the frequency of combined kidney-liver transplants, and in the number of grafts obtained from donation after cardiac death donors. The main findings of the study were as follows: 1. The adjusted 5-year survival rates did not differ between the 3 protocol groups in the overall transplant population, among hepatitis C virus (HCV) positive patients, or among patients with HCC. 2. The 3- and 5-year survival rates reached 75% to 80% and 67% to 75%, respectively. 3. The factors associated with death included the Model for End-Stage Liver Disease (MELD) score, the donor and recipient ages, HCV, and donation after cardiac death; the type of immunosuppression protocol did not affect survival rates. 4. The 1-year rates of HCV recurrence (ie, progression to stage F2; 7%-15%) and HCC recurrence (0%-11%) did not differ between the groups. 5. Complications such as rejection within 1 year (1%-2%), lymphoproliferative disorders (<1%), and cytomegalovirus (2%-3%) or fungal infections (3%-5%) occurred at similar rates. 6. Renal function after transplantation did not differ between the groups. From this study, we might be tempted to conclude that ratg with delayed and reduced TAC with or without rituximab is the optimal immunosuppressive cocktail for the LT population. A more careful analysis of the data, however, reveals that all that glitters is not gold. Indeed, although the survival and HCV and HCC recurrence rates compare favorably with data reported from other series and transplant registries, 3,13-16 the patients included in this series might be considered low-risk recipients for negative outcomes. For instance, the median donor age, the strongest factor for determining recurrent HCV, 14 was only 40 years and ranged from 35 years in the first era to 43 years in the last one. Furthermore, although the MELD score appears to be a weak predictor of posttransplant outcomes in most studies, 4 some series have reported increasing posttransplant mortality with increasing MELD scores, 17,18 and in this study, the median MELD score was relatively low (approximately 16); this may have contributed at least partially to the results. In terms of HCC recurrence, approximately one third of the patients were outside the Milan criteria, but no additional data were provided about the expanded criteria used. Because of the large number of determinants of HCC recurrence, 15,19 a 1-year HCC recurrence rate of 0% to 11% and an overall recurrence rate at any time of 11% to 29% do not differ significantly from the results of reported series. Finally, in terms of renal function, which is one of the major endpoints in most CNI minimization studies, 7 it is difficult to draw conclusions from this study because a clear definition of chronic kidney disease is lacking. Information about other potentially relevant endpoints that are usually evaluated in CNI minimization studies (eg, arterial hypertension and diabetes) is not reported. Despite these limitations, the authors need to be congratulated for this effort to report one of the largest series using ratg, a drug belonging to a group of immunosuppressive T cell depleting agents that also includes muromonab-cd3 (OKT3), horse anti-thymocyte globulin (Atgam), and alemtuzumab. Atgam, which was used in the earliest LT series, was insufficient to prevent rejection and was replaced with the advent of CNIs. OKT3 was associated with aggressive HCV recurrence, posttransplant lymphoproliferative disorders, and severe infections. Despite a renewed interest in recent years in using induction regimens to minimize CNI doses, 20 there is still not much information, particularly from randomized studies. 21,22 An improvement in renal function without a negative effect on recurrent HCV is the major goal. Overall, published data suggest that the delayed initiation of CNIs with ratg is associated with similar survival rates, improvements in renal function, and a lower incidence of early acute rejection in comparison with no antibody use and early CNI initiation. 25,27 Unfortunately, in the only randomized study based on ratg, these endpoints were not analyzed 21 because the purpose of the study was to demonstrate the feasibility of complete steroid (ST) avoidance without compromising rejection and survival. According to the United Network for Organ Sharing (UNOS) database (452 of 16,898 patients received ratg, and 1758 received ratg and STs), Uemura et al. 28 showed that ratg induction, when it was preferentially used in patients with renal dysfunction, was associated with improvements in renal function, and there were no differences between the ratg group and the groups receiving STs (n ¼ 14,005) or daclizumab (DAC) alone (n ¼ 683) at 6 and 12 months. In the present study by Mangus et al., 12 there was no control group, and the reduction of the glomerular filtration rate (averaging 14 and 18 ml/minute) was similar to that reported in other studies using ratg. On the basis of all the studies, it appears that ratg induction is an attractive approach for patients with existing renal dysfunction, whose numbers are expected to continue rising in the future. However, other strategies, including the use of reduced-dose TAC with MMF 11 and antibody induction therapy with interleukin-2 receptor antibodies and delayed and reduced-dose TAC, 10 have demonstrated similar results. Our ability to

3 TABLE 1. HCV in HCV-Infected LT Recipients Given ratg or Anti Interleukin-2 Receptor Monoclonal Antibodies Induction Study Therapy Study Type Patients: Study/ Control (n/n) Follow-Up (Months) Protocol Liver Biopsy Criteria Rate: Induction/ Control (%/%) Significant Difference in Severe Criteria Severe Rate: Induction/ Control (%/%) Significant Difference in Severe Nair ratg/sts Prospective, et al. 22 randomized, and De Ruvo ratg/sts Prospective and et al. 29 un Belli et al. 30 ratg/cy/tac/ MMF/AZA 33/ No Inflammation at 3 months 22/30 12 No Signs of hepatitis and HCV-RNA+ 74/75 No Fibrosis stage 3 54/60 No Ishak staging Retrospective 141 (ratg) Yes ND ND ND Ishak fibrosis stage > 3 Kamar et al. 31 ratg/anti-cd25 Retrospective 16/15 6 No Virological/biological/liver biopsy Tector et al. 24 ratg Prospective and un Bajjoka 57/no controls 13 No Hepatitis activity index et al. 27 cal/liver biopsy ratg/sts Retrospective 63/11 12 No Virological/biological/liver Llado et al. 33 BAS and no STs/ BAS and STs Calmus BAS, STs, CY, and et al. 34 AZA Filipponi BAS and no STs/ et al. 35 BAS and STs Neuhaus BAS and no STs/ et al. 36 BAS and STs Klintmalm DAC and no STs/ et al. 37 TAC and STs/TAC, MMF, and STs Nelson DAC, STs, and et al. 38 MMF/TAC and STs Prospective, randomized, and 43/46 24 Yes Scheuer histological grading 50/42 (1-2 years) 0/0 (>4 years) ND (staging: 0.85/1.54) 25 (all groups) No No Multivariate analysis: less aggressive course with ratg 56/80 No ND ND ND 32 ND 5 40/64 No ND ND ND 97/97 No Fibrosis stage 3 Prospective 31 6 No ND 48 ND ND Prospective, randomized, double-blinded, and Prospective, randomized, double-blinded, and Prospective and randomized Prospective and un 66/74 12 Yes Ishak histological grade 8 64/ endpoints: first biopsy-confirmed rejection episode, HCV recurrence, death, and graft loss 143/77/72 24 Yes Batts-Ludwig system: inflammation grade 3 and/ or fibrosis stage Yes Knodell modification of Ishak scoring system 22/31 (2 years) 37/41 No ND ND ND 73/88 Yes ND ND ND 29/34/34 No Inflammation grade 3or fibrosis stage 2 92/60 Yes Fibrosis stage 3 No 68/69/75 No 45/26 Yes

4 758 BERENGUER AND PONS LIVER TRANSPLANTATION, July 2012 decide on one or another of these strategies should ideally come from well-conducted prospective, randomized studies comparing these different options. In the absence of such studies, center experience together with candidate characteristics, donor quality, and transplant complications will likely provide the clues for choosing the best immunosuppression strategy. Along this line, one factor that may influence the decision to use one strategy or another is the presence of HCV. The effects of current regimens on HCV disease remain unclear. 14 The outcomes of patients infected with HCV and receiving induction therapy with ratg have been reported in single-center analyses 22,29-31 and in population-based data sets from UNOS. 28,32 The major limitations of published studies using induction therapy are their retrospective nature, the relatively small numbers of patients, and the lack of protocol biopsy samples (Table 1). Two UNOSbased analyses of the impact of induction therapy have provided conflicting results. In the first study, in which Moonka et al. 32 analyzed the outcomes of LT with antibody induction therapy and with no induction, induction significantly improved survival for HCV patients and non-hcv patients. When patients receiving individual agents were compared to patients receiving no induction, the survival rate was higher with all induction agents, but the difference in HCV patients was significant only for anti-thymocyte globulin and DAC. In the second UNOS study, an ratg-st group with HCV had a significantly lower survival rate than a group receiving STs alone. 28 In the present series, 12 the 5-year survival rate for HCV patients was 65% (similar to rates reported in other series); this suggests no deleterious effects of induction therapy with ratg on recurrent HCV. As mentioned previously, however, we would expect better survival rates with the inclusion of low-risk patients. A potential additional advantage associated with induction therapy is the promotion of a tolerogenic environment. Over the past 15 years, several investigators 39 have championed the hypothesis that the administration of reduced doses of conventional immunosuppressive drugs under the cover of lymphocyte-depleting antibodies in the early posttransplant period will maximize the intrinsic tolerogenic properties of the graft, facilitate immunological engagement between the allograft and the recipient s immune system, and ultimately lead to a state in which normal graft function is maintained with no or minimal doses of immunosuppression (tolerance or prope tolerance). In various published studies, complete immunosuppression withdrawal was attempted according to preestablished protocols. In some of these series, withdrawal was initiated after pretreatment with tolerogenic molecules (mainly ratg) with the aim of eliminating clones of immune cells involved in rejection before they could come into contact with the donor antigens. The hypothesis was that once transplantation was performed, immune restoration would occur in the setting of the adaptation of donor antigens to the recipient s environment; this situation would be further facilitated by the minimization of immunosuppression. Following this hypothesis, Starzl et al. 40 in 2003 published their favorable experience with 82 patients who were awaiting kidney, liver, pancreas, or intestinal transplantation and were pretreated with ratg. Eighteen months after transplantation, there was no immunosuppression-related mortality; 43 of the 72 recipients with surviving grafts were on spaced TAC doses, but complete withdrawal was not achieved. Unfortunately, these initial results have not been confirmed in more recent studies, including the first randomized LT trial to compare a prope tolerance strategy [ie, the administration of anti-thymocyte globulin (Fresenius) and lowdose TAC] to a standard regimen (full-dose TAC and STs). 41 In this study, the tolerance regimen was associated with a high rejection rate and a failure to achieve very low TAC doses. In conclusion, more than two decades have passed since LT became an established treatment for patients with end-stage liver disease, and we are still struggling to decide the optimal immunosuppression therapy. The low figures for acute and/or chronic rejection in recent studies suggest that current immunosuppression strategies are in fact excellent for preventing allograft loss due to rejection. On the other hand, the increasing prevalence of posttransplant complications associated with lifelong immunosuppression, such as nephrotoxicity and malignancies, is a concern. In the absence of robust evidence based on prospective clinical trials, we are left with studiessuchasthisonebymangusetal., 12 in which a single-center experience with ratg is reported with relatively good results in terms of survival and posttransplant toxicity. With so many centers and so many individual experiences with different immunosuppression strategies, studies such as this one that cover a long period of time, use a rarely evaluated strategy, and include a large number of patients are welcome; however, it is unlikely that the results from this study will substantially modify the protocols in place. We will most likely adopt an interferon-free regimen against HCV before a CNI-free immunosuppression regimen. REFERENCES 1. Starzl TE, Fung JJ. Themes of liver transplantation. Hepatology 2010;51: Thuluvath PJ, Guidinger MK, Fung JJ, Johnson LB, Rayhill SC, Pelletier SJ. 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